| United States Patent Application | 20090275649 |
| Kind Code | A1 |
| Qin; Ning ; et al. | November 5, 2009 |
It has now been discovered that certain cannabinoids specifically activate TRPV2 channel activity. Based on the discovery, novel compositions and methods for screening, identifying and characterizing compounds that increase or decrease the biological activity of a TRPV2.
| Inventors: | Qin; Ning; (Blue Bell, PA) ; Liu; Yi; (Jenkintown, PA) ; Flores; Christopher M.; (Lansdale, PA) ; Hutchinson; Tasha; (Green Lane, PA) ; Neeper; Michael P.; (Collegeville, PA) |
| Correspondence Address: |
PHILIP S. JOHNSON;JOHNSON & JOHNSON
ONE JOHNSON & JOHNSON PLAZA
NEW BRUNSWICK
NJ
08933-7003
US
|
| Serial No.: | 499891 |
| Series Code: | 12 |
| Filed: | July 9, 2009 |
| Current U.S. Class: | 514/454; 435/252.3; 435/254.11; 435/29; 435/320.1; 435/325; 435/69.1; 435/7.21; 514/568; 514/729; 530/350; 536/23.1 |
| Class at Publication: | 514/454; 435/7.21; 435/29; 435/69.1; 435/325; 435/252.3; 435/254.11; 435/320.1; 514/568; 514/729; 530/350; 536/23.1 |
| International Class: | A61K 31/352 20060101 A61K031/352; G01N 33/53 20060101 G01N033/53; C12Q 1/02 20060101 C12Q001/02; C12P 21/02 20060101 C12P021/02; C12N 5/10 20060101 C12N005/10; C12N 1/21 20060101 C12N001/21; C12N 1/15 20060101 C12N001/15; C12N 15/63 20060101 C12N015/63; A61K 31/192 20060101 A61K031/192; A61K 31/05 20060101 A61K031/05; C07K 14/00 20060101 C07K014/00; C07H 21/00 20060101 C07H021/00 |
1-18. (canceled)
19. A method for identifying a compound that increases the biological activity of TRPV2, comprising the steps ofa. obtaining atomic coordinates defining a three-dimensional structure of a complex comprising a TRPV2 interacting with a cannabinoid that is capable of activating the TRPV2;b. elucidating a structural relationship between the TRPV2 and the interacting cannabinoid;c. designing a structural analog of the cannabinoid based on the structural relationship;d. synthesizing the structural analog;e. determining the extent to which the structural analog alters the biological activity of the TRPV2, thereby identifying the compound that increases the biological activity of TRPV2.
20. The method of claim 19, wherein the biological activity of the TRPV2 is determined as calcium-influx into a cell expressing the TRPV2.
21. The method of claim 19, wherein the biological activity of the TRPV2 is measured by a method of patch clamp.
22. The method of claim 19, wherein the biological activity of the TRPV2 is measured by a CA mobilization assay.
23. The method of claim 19, wherein the biological activity of the TRPV2 is determined as its binding affinity to the cannabinoid that is capable of activating the TRPV2 activity.
24. A method of increasing the biological activity of a TRPV2, comprising the step of contacting the TRPV2 with a cannabinoid that is capable of activating the TRPV2 activity.
25. The method of claim 24, wherein the TRPV2 is associated with an isolated membrane.
26. The method of claim 24, wherein the TRPV2 is present in a cell.
27. The method of claim 26, wherein the cell is a neuron.
28. The method of claim 24, wherein the cannabinoid is selected from the group consisting of .DELTA..sup.9-tetrahydrocannabinol, 11-hydroxy-.DELTA..sup.9-tetrahydrocannabinol, cannabinol, cannabidiol, O-1821, nabilone, CP55940, 2-AG, and HU210, HU211, HU308, and HU331.
29. A method for stimulating noxious thermo-sensation in a subject, comprising administering to the subject a pharmaceutical composition comprising an effective amount of cannabinoid that is capable of activating the TRPV2 activity, thereby stimulating the noxious thermo-sensation in the subject.
30. The method of claim 29, wherein the subject is a human.
31. The method of claim 29, wherein the cannabinoid is selected from the group consisting of .DELTA..sup.9-tetrahydrocannabinol, 11-hydroxy-.DELTA..sup.9-tetrahydrocannabinol, cannabinol, cannabidiol, O-1821, nabilone, CP55940, 2-AG, and HU210, HU211, HU308, and HU331.
32. An isolated polypeptide consisting essentially of an amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17.
33. An isolated nucleic acid molecule that encodes a polypeptide consisting essentially of an amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17.
34. The isolated nucleic acid molecule of claim 33 consisting essentially of a nucleotide sequence of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28.
35. An expression vector comprising nucleotide sequence that encodes a polypeptide consisting essentially of an amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17.
36. A recombinant cell comprising an expression vector of claim 35.
37. A method of producing a polypeptide consisting essentially of an amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17, comprising the step of growing a cell of claim 36 under a condition whereby the polypeptide is produced by the cell.
CROSS REFERENCE TO RELATED APPLICATIONS
[0001]This application claims priority to Application No. 60/731,686 filed on Oct. 31, 2005 and Application No. 60/782,656 filed on Mar. 15, 2006, the entire contents of which are incorporated by reference herein.
FIELD OF THE INVENTION
[0002]The present invention relates to the regulation of thermal receptor ion channel proteins. In particular, the present invention relates to compositions and methods for screening, identifying and characterizing compounds that increase or decrease the biological activity of a TRPV2.
BACKGROUND
[0003]In mammals, the sensation of pain triggered by thermal, mechanical or chemical stimuli is a useful warning and protective system. Considerable efforts have been put into elucidating the biochemical mechanisms involved in the detection, transduction and transmission of hot and cold sensations in neuronal tissues. Thermal stimuli activate specialized receptors located on sensory neurons, such as those deriving from the dorsal root ganglion (DRG) and the trigeminal ganglion (TG). When these stimuli are in the noxious range (i.e., very hot or cold), they activate a certain subset of thermal receptors on a sub-population of sensory neurons called nociceptors (pain-sensing neurons). Upon activation, the thermal receptors (e.g., ion channels) transduce the noxious stimulus into an electrical signal that is propagated along the sensory neuron to the spinal cord, where it is relayed to the brain, ultimately leading to the perception of pain. Accordingly, these thermal receptors represent highly promising targets for developing drugs for the treatment of various painful conditions.
[0004]Several temperature-activated receptors have been identified with wide ranging temperature sensitivities from noxious heat to noxious cold. These temperature-activated receptors belong to the transient receptor potential (TRP) family of non-selective cation channels, which in C. elegans and D. melanogaster are involved in mechano- and osmoregulation. Several of these temperature-activated receptors, including TRPV1 and TRPV2, are implicated in noxious heat sensation (Caterina et al., 1997, Nature, 389: 816; and Caterina et al., 1999, Nature 398: 436). TRPV1, the most extensively characterized member of the thermo-TRP family, is activated by moderate heat (.about.43.degree. C.), capsaicin, protons and certain endocannabinoids, such as anandamide and 2-AG. It is well accepted that TRPV1 contributes to acute thermal nociception and hyperalgesia after injury (Clapham, Nature. 2003, 426(6966): 517-24).
[0005]TRPV2, also termed VRL-1, has been proposed as a sensor of noxious temperatures (>52.degree. C.), which presumably mediates "first" pain, i.e. the rapid, acute, and sharp pain evoked by noxious stimuli (Caterina et al., 1999, supra; Story et al., Cell, 2003, 112:819-829, and references therein). TRPV2 is structurally most closely related to TRPV1 (.about.50% sequence identity at the protein level). TRPV2 is expressed in medium- to large-diameter neurons of sensory ganglia, as well as at lower levels in brain, spinal cord, spleen and lung. Furthermore, TRPV2 is upregulated in sympathetic postganglionic neurons following injury, suggesting a potential role for TRPV2 in sympathetically mediated pain (Gaudet et al., Brain Res. 2004, 1017(1-2):155-62). Thus, modulation of TRPV2 may potentially have many therapeutic applications.
[0006]Despite great interest in TRPV2 modulation, a system for screening, identifying and characterizing TRPV2 modulators has yet to be developed. This is in part due to the lack of known, and in particular, selective TRPV2 agonists, as well as the technical difficulty of assaying these channels in a high temperature environment. In general, TRPV2 does not respond to known TRPV1 agonists (Benham et al., 2003, Cell Calcium 33:479-487). However, a recent study reported that 2-aminoethoxydiphenyl borate (2-APB), a non-selective TRP modulator, was able to activate TRPV1, TRPV2, and TRPV3 (Hu et al (2004), J. Biol. Chem., 279: 35741-8), although TRPV2 activation by 2-APB was not observed by others (Chung et al. (2004), J Neurosci. 24: 5177-82).
[0007]In an effort to overcome the above-mentioned challenges, the present invention provides novel compositions and methods for screening, identifying and characterizing TRPV2 agonists.
SUMMARY
[0008]It has now been discovered that certain cannabinoids specifically activate TRPV2 channel activity.
[0009]In one general aspect, the present invention provides a method for identifying a compound that decreases the biological activity of TRPV2, comprising the steps of: a) contacting a TRPV2 polypeptide with a cannabinoid that is capable of activating TRPV2 activity under a condition in which the TRPV2 is activated by the cannabinoid; b) contacting the TRPV2 polypeptide with a test compound; c) measuring the biological activity of the TRPV2 in the presence of both the cannabinoid and the test compound; d) repeating step a); e) measuring the biological activity of the TRPV2 in the presence of the cannabinoid but not the test compound; and f) comparing the TRPV2 activity measured from step c) with that from step e); thereby identifying the compound that decreases the biological activity of TRPV2 when the TRPV2 activity measured from step c) is less than that from step e).
[0010]In another general aspect, the present invention provides a method for identifying a compound that increases the biological activity of TRPV2, comprising the steps of: a) obtaining atomic coordinates defining a three-dimensional structure of a complex comprising a TRPV2 interacting with a cannabinoid that is capable of activating the TRPV2; b) elucidating a structural relationship between the TRPV2 and the interacting cannabinoid; c) designing a structural analog of the cannabinoid based on the structural relationship; d) synthesizing the structural analog; and e) determining the extent to which the structural analog alters the biological activity of the TRPV2, thereby identifying the compound that increases the biological activity of TRPV2.
[0011]Another general aspect of the present invention is a method for increasing the biological activity of a TRPV2, comprising the step of contacting the TRPV2 with a cannabinoid that is capable of activating the TRPV2 activity.
[0012]The present invention further provides a method for stimulating noxious thermo-sensation in a subject, comprising administering to the subject a pharmaceutical composition comprising an effective amount of a cannabinoid that is capable of activating the TRPV2 activity, thereby stimulating the noxious thermo-sensation in the subject.
[0013]Other aspects, features and advantages of the invention will be apparent from the following disclosure, including the detailed description of the invention and its preferred embodiments and the appended claims.
DESCRIPTION OF THE FIGURES
[0014]FIG. 1 shows the subclasses of cannabinoids present in Cannabis (Thakur et al., Life Sci. 2005 Oct. 17, Epub ahead of print).
[0015]FIG. 2 shows non-enzymatic formation of .DELTA..sup.9-THC from its precursor (Thakur et al., supra).
[0016]FIG. 3 shows the structures of two representative endocannabinoid (Thakur et al., supra).
[0017]FIG. 4 shows concentration-dependent activation of rat TRPV2 by .DELTA..sup.9-THC in a FLIPR assay.
[0018]FIG. 5 illustrates activation of both rat and human TRPV2 by .DELTA..sup.9-THC and subsequent block of the .DELTA..sup.9-THC-activated currents by ruthenium red from whole-cell patch clamp studies.
[0019]FIG. 6 shows .DELTA..sup.9-THC activated deletion mutants of TRPV2 recombinantly expressed from HEK293 cells: (A) the N-terminal deletion mutants; and (B) the C-terminal deletion mutants.
[0020]FIG. 7 illustrates the activation of the human and rat TRPV2 chimera recombinantly expressed from HEK293 cells: (A) the chimera was expressed individually from the cells; and (B) the complementary effect of RRH and HRR when they were co-expressed from the cells.
DETAILED DESCRIPTION
[0021]All publications cited herein are hereby incorporated by reference. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains.
[0022]As used herein, the terms "comprising", "containing", "having" and "including" are used in their open, non-limiting sense.
[0023]The following are abbreviations that are at times used in this specification:
[0024]2-AG=2-arachidonylglycerol
[0025]AEA=anandamide=N-arachidonoylethanolamine
[0026]bp=base pair
[0027]cDNA=complementary DNA
[0028]Ca.sup.2+=calcium
[0029].DELTA..sup.9-THC=Delta-9-tetrahydrocannabinol
[0030]DRG=dorsal root ganglion
[0031]FLIPR=fluorescence imaging plate reader
[0032]kb=kilobase; 1000 base pairs
[0033]PAGE=polyacrylamide gel electrophoresis
[0034]PCR=polymerase chain reaction
[0035]SDS=sodium dodecyl sulfate
[0036]TG=trigeminal ganglion
[0037]TRPV2=transient receptor potential cation channel, subfamily V, member 2
[0038]As used herein, the term "biological activity of a TRPV2" refers to an activity exerted by the TRPV2 protein as determined in vivo or in vitro, according to standard techniques. Such an activity can be a direct activity such as the ability of a TRPV2 to bind to a ligand, such as a cannabinoid or an analog thereof. The activity can be the conductivity of an ion channel formed by the TRPV2. The activity can also be functional changes of cell physiology, such as calcium mobilization or nociceptive response of the cell. The biological activity of a TRPV2 can be an indirect activity, such as a signal transduction activity mediated by TRPV2 via its interaction with one or more than one additional protein or other molecule(s).
[0039]"Binding affinity" refers to the ability of two or more molecular entities to bind or interact with each other. The binding can be from the formation of one or more chemical bonds that results in continual and stable proximity of the two interacting entities. The binding can also be based solely on physical affinities, which can be equally effective in co-localizing the two interacting entities. Examples of physical affinities and chemical bonds include but are not limited to, forces caused by electrical charge differences, hydrophobicity, hydrogen bonds, van der Waals force, ionic force, covalent linkages, and combinations thereof. The state of proximity between the interacting entities can be transient or permanent, reversible or irreversible. In any event, it is in contrast to and distinguishable from contact caused by natural random movement of two entities.
[0040]"Cannabinoid" includes any of various compounds that activate a cannabinoid receptor or a structural analog of the compounds.
[0041]In one embodiment, "cannabinoid" includes herbal cannabinoids, a class of compounds that were originally extracted from the plant Cannabis sativa L or a metabolite thereof. Cannabis sativa L. is one of the oldest known medicinal plants and has been extensively studied with respect to its phytochemistry. The plant biosynthesizes a total of 483 identified chemical entities belonging to different chemical classes (ElSohly, 2002, In: F. Grotenhermen and E. Russo, Editors, Cannabis and Cannabinoids, Haworth Press, Binghamton (2002), pp. 27-36.), of which the cannabinoids are the most distinctive class of compounds, known to exist only in this plant. There are 66 known plant-derived cannabinoids (Thakur et al., Life Sci. 2005 Oct. 17, Epub ahead of print). The most prevalent of which are the tetrahydrocannabinols (THCs), the cannabidiols (CBDs), and the cannabinols (CBNs). The next most abundant cannabinoids are the cannabigerols (CBGs), the cannabichromenes (CBCs), and cannabinodiols (CBNDs).
[0042]FIG. 1 shows the representative structures of subclasses of cannabinoids present in Cannabis sativa. Most cannabinoids contain 21 carbon atoms, but there are some variations in the length of the C-3 side chain attached to the aromatic ring. In the most common homologues, the n-pentyl side chain is replaced with an n-propyl (De Zeeuw et al., Science. 1972, 175:778-779); and Vree et al., Journal of Pharmacy and Pharmacology. 1972, 24:7-12). These analogues are named using the suffix "varin" and are designated as THCV, CBDV, or CBNV, as examples. Cannabinoids with one (Vree et al., 1972, supra) and four (Smith, 1997, Journal of Forensic Sciences 42 (1997), pp. 610-618) carbons also exist but are minor components. Classical cannabinoids (CCs) are ABC tricyclic terpenoid compounds bearing a benzopyran moiety and are insoluble in water but soluble in lipids, alcohols, and other non-polar organic solvents (Thakur et al., 2005, supra). These phenolic derivatives are more water-soluble as their phenolate salts formed under strong alkaline conditions.
[0043]One particular example of "cannabinoid" is Delta-9-tetrahydrocannabinol (Delta-9-THC, .DELTA..sup.9-THC), the key psychoactive ingredient of cannabis (marijuana) (Gaoni and Mechoulam 1964, Journal of the American Chemical Society 86 (1964), pp. 1646-1647). As illustrated in FIG. 2, .DELTA..sup.9-THC is formed by the decarboxylation of its non-psychoactive precursor .DELTA..sup.9-THCA by the action of light or heat during storage or smoking or under alkaline conditions. .DELTA..sup.9-THCA is biosynthesized by a well-established pathway involving the action of several specific enzymes.
[0044]It was discovered that .DELTA..sup.9-THC interacts with the two known cannabinoid (CB) receptors, CB1 (Devane et al., 1988, Molecular Pharmacology 34 (1988), pp. 605-613; Gerard et al., 1990, Nucleic Acids Research 18 (1990), p. 7142; Gerard et al., 1991, Biochemical Journal 279 (1991), pp. 129-134; and Matsuda et al., 1990, Nature 346 (1990), pp. 561-564.) and CB2 (Munro et al., 1993, Nature 365 (1993), pp. 61-65). Both cannabinoid receptors belong to the super family of G-protein coupled receptors, and produce a broad spectrum of physiological effects (Grotenhermen, 2002, In: R. Grotenhermen and E. Russo, Editors, Cannabis and Cannabinoids, Haworth Press, Binghamton (2002), pp. 123-142) including antiemetic, appetite enhancing, analgesic, and lowering of intraocular pressure. The discovery of specific cannabinoid receptors inside animal ultimately led to the search and identification of endocannabinoid.
[0045]Thus, the term "cannabinoid" also includes endocannabinoid. The term "endocannabinoid" refers to a ligand to a cannabinoid receptor, wherein said ligand is endogenously produced by in the bodies of an animal. Exemplary endocannabinoids include, but are not limited to, N-arachidonoylethanolamine (AEA, anandamide) and 2-arachidonylglycerol (2-AG), the structures of which are shown in FIG. 3. Anandamide was shown to bind to the CB1 receptor with modest affinity (K.sub.i=61 nM), have low affinity for the CB2 receptor (K.sub.i=1930 nM) (Lin et al., 1998, Journal of Medicinal Chemistry 41 (1998), pp. 5353-5361), and behave as a partial agonist in the biochemical and pharmacological tests used to characterize cannabinoid activity. It was reported that anandamide can also bind to and activate TRPV1 (Di Marzo et al., Prostaglandins Leukot Essent Fatty Acids 2002; 66: 377-91). 2-AG binds weakly to both CB1 (K.sub.i=472 nM) and CB2 (K.sub.i=1400 nM) receptors (Mechoulam et al., 1995, Biochemical Pharmacology 50 (1995), pp. 83-90). 2-AG was isolated from intestinal and brain tissues and is present in the brain at concentrations approximately 170-fold higher than AEA (3) (Stella et al., 1997, Nature 388 (1997), pp. 773-778).
[0046]In yet another embodiment, the term "cannabinoid" covers the synthetic cannabinoids are produced by chemical synthesis and do not occur naturally. The synthetic cannabinoids can be synthesized based on the structure of herbal cannabinoids or endocannabinoid. Synthetic cannabinoids are particularly useful in experiments to determine the relationship between the structure and activity of cannabinoid compounds, by making systematic, incremental modifications of cannabinoid molecule. Exemplary synthetic cannabinoids includes dronabinol (synthetic THC), nabilone, and any other synthetic compounds that activate a cannabinoid receptor or a structural analog of the compounds.
[0047]A "cannabinoid that is capable of activating the TRPV2 activity" refers to any cannabinoid that is capable of binding to a TRPV2 channel and, in the absence of other stimulation, exhibits at least a 10% increase in the conductivity of the TRPV2 channel compared to the baseline. A person skilled in the art can experimentally determine whether a cannabinoid is capable of activating the TRPV2 activity. In some embodiments, "cannabinoid that is capable of activating the TRPV2 activity" is a cannabinoid which, upon binding to a TRPV2 channel, results in at least a 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% increase in the conductivity of the channel compared to the baseline. "Cannabinoid that is capable of activating the TRPV2 activity" includes, but is not limited to, .DELTA..sup.9-tetrahydrocannabinol, cannabinol, cannabidiol nabilone, CP55940, HU210, and 2-AG. Interestingly, the other endocannabinoid tested, anandamide, showed no or minimal activation effect on TRPV2 (Table 2, Example 4 infra).
[0048]A "cannabinoid receptor" or a "CB receptor" each refers to a protein that functions as a specific receptor for a cannabinoid. The "CB receptor" can be a CB1 receptor or a CB2 receptor.
[0049]The CB1 receptor has been detected primarily in brain, specifically in the basal ganglia and in the limbic system, including the hippocampus. They are also found in other tissues such as the cerebellum and in both male and female reproductive systems. CB1 receptors appear to be responsible for the euphoric and anticonvulsive effects of cannabis. A CB1 can (1) have greater than about 70% amino acid sequence identity to a human CB1 receptor depicted in GenBank protein ID: NP.sub.--057167 (the longer isoform of human CB1 receptor) or NP.sub.--149421 (the shorter isoform of human CB1 receptor); or (2) bind to antibodies, e.g., polyclonal or monoclonal antibodies, raised against the human CB1 receptor depicted in GenBank protein ID NP.sub.--057167 or NP.sub.--149421. In some embodiments, the CB1 receptor has greater than about 75, 80, 85, 90, or 95 percent amino acid sequence identity to the human CB1 receptor depicted in GenBank protein ID NP.sub.--057167 or NP.sub.--149421. The CB1 receptor includes orthologs of the CB1 receptors in animals including human, rat, mouse, pig, dog and monkey, etc. The CB1 receptor also includes structural and functional polymorphisms of the CB1 receptor. "Polymorphism" refers to a set of genetic variants at a particular genetic locus among individuals in a population. The CB1 receptor includes the structural and functional polymorphisms of the CB1 receptor from human (GenBank protein ID NP.sub.--057167 or NP.sub.--149421), rat (GenBank protein ID: NP.sub.--036916), or mouse (GenBank protein ID: NP.sub.--031752), or etc.
[0050]The CB2 receptor has been detected almost exclusively in the immune system, with the greatest density in the peripheral blood cells. CB2 receptors appear to be responsible for the anti-inflammatory and possible other therapeutic. A CB2 can (1) have greater than about 70% amino acid sequence identity to a human CB2 receptor depicted in GenBank protein ID: NP.sub.--001832; or (2) bind to antibodies, e.g., polyclonal or monoclonal antibodies, raised against the human CB2 receptor depicted in GenBank protein ID NP.sub.--001832. In some embodiments, the CB2 receptor has greater than about 75, 80, 85, 90, or 95 percent amino acid sequence identity to the human CB2 receptor depicted in GenBank protein ID NP.sub.--001832. The CB2 receptor includes orthologs of the CB2 receptors in animals including human, rat, mouse, pig, dog and monkey, etc. The CB2 receptor also includes structural and functional polymorphisms of the CB2 receptor. The CB2 receptor includes the structural and functional polymorphisms of the CB2 receptor from human (GenBank protein ID NP.sub.--001832), rat (GenBank protein ID: NP.sub.--065418), mouse (GenBank protein ID: NP.sub.--034054), or etc.
[0051]A "cell" refers to at least one cell or a plurality of cells appropriate for the sensitivity of the detection method. The cell can be present in a cultivated cell culture. The cell can also be present in its natural environment, such as a biological tissue or fluid. Cells suitable for the present invention may be bacterial, but are preferably eukaryotic, and are most preferably mammalian.
[0052]A "compound that increases the conductivity of a TRPV2 channel" includes any compound that results in increased passage of ions through the TRPV2 channel. In one embodiment, such a compound is an agonist for the TRPV2 channel that binds to the TRPV2 channel to increase its conductivity. Such a compound triggers, initiates, propagates, or otherwise enhances the channel conductivity. In another embodiment, such a compound is a positive allosteric modulator, which interacts with the TRPV2 channel at allosteric sites different from the agonist-binding site, and potentiates the response of the channel to an agonist.
[0053]A "compound that decreases the conductivity of a TRPV2 channel" includes any compound that results in decreased passage of ions through the TRPV2 channel. In one embodiment, such a compound is an antagonist for the TRPV2 channel that binds to the TRPV2 channel to counter, decrease or limit the action of an agonist in either a competitive or non-competitive fashion. In another embodiment, such a compound is a negative allosteric modulator, which interacts with the TRPV2 channel at allosteric sites different from the agonist or antagonist-binding site, and decreases the response of the channel to an agonist. In yet another embodiment, such a compound is an inverse agonist that binds to the TRPV2 channel and decreases the conductivity of the channel in the absence of any other compound, such as an agonist.
[0054]"Nucleotide sequence" refers to the arrangement of either deoxyribonucleotide or ribonucleotide residues in a polymer in either single- or double-stranded form. Nucleic acid sequences can be composed of natural nucleotides of the following bases: thymidine, adenine, cytosine, guanine, and uracil; abbreviated T, A, C, G, and U, respectively, and/or synthetic analogs of the natural nucleotides.
[0055]An "isolated" nucleic acid molecule is one that is substantially separated from at least one of the other nucleic acid molecules present in the natural source of the nucleic acid, or is substantially free of at least one of the chemical precursors or other chemicals when the nucleic acid molecule is chemically synthesized. An "isolated" nucleic acid molecule can also be, for example, a nucleic acid molecule that is substantially free of at least one of the nucleotide sequences that naturally flank the nucleic acid molecule at its 5' and 3' ends in the genomic DNA of the organism from which the nucleic acid is derived. A nucleic acid molecule is "substantially separated from" or "substantially free of" other nucleic acid molecule(s) or other chemical(s) in preparations of the nucleic acid molecule when there is less than about 30%, 20%, 10%, or 5% (by dry weight) of the other nucleic acid molecule(s) or the other chemical(s) (also referred to herein as a "contaminating nucleic acid molecule" or a "contaminating chemical").
[0056]Isolated nucleic acid molecules include, without limitation, separate nucleic acid molecules (e.g., cDNA or genomic DNA fragments produced by PCR or restriction endonuclease treatment) independent of other sequences, as well as nucleic acid molecules that are incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid molecule can include a nucleic acid molecule that is part of a hybrid or fusion nucleic acid molecule. An isolated nucleic acid molecule can be a nucleic acid sequence that is: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) synthesized by, for example, chemical synthesis; (iii) recombinantly produced by cloning; or (iv) purified, as by cleavage and electrophoretic or chromatographic separation.
[0057]The term "oligonucleotide" or "oligo" refers to a single-stranded DNA or RNA sequence of a relatively short length, for example, less than 100 residues long. For many methods, oligonucleotides of about 16-25 nucleotides in length are useful, although longer oligonucleotides of greater than about 25 nucleotides may sometimes be utilized. Some oligonucleotides can be used as "primers" for the synthesis of complimentary nucleic acid strands. For example, DNA primers can hybridize to a complimentary nucleic acid sequence to prime the synthesis of a complimentary DNA strand in reactions using DNA polymerases. Oligonucleotides are also useful for hybridization in several methods of nucleic acid detection, for example, in Northern blotting or in situ hybridization.
[0058]The terms "polypeptide," "protein," and "peptide" are used herein interchangeably to refer to amino acid chains in which the amino acid residues are linked by peptide bonds or modified peptide bonds. The amino acid chains can be of any length of greater than two amino acids. Unless otherwise specified, the terms "polypeptide," "protein," and "peptide" also encompass various modified forms thereof. Such modified forms may be naturally occurring modified forms or chemically modified forms. Examples of modified forms include, but are not limited to, glycosylated forms, phosphorylated forms, myristoylated forms, palmitoylated forms, ribosylated forms, acetylated forms, ubiquitinated forms, etc. Modifications also include intra-molecular crosslinking and covalent attachment to various moieties such as lipids, flavin, biotin, polyethylene glycol or derivatives thereof, etc. In addition, modifications may also include cyclization, branching and cross-linking. Further, amino acids other than the conventional twenty amino acids encoded by the codons of genes may also be included in a polypeptide.
[0059]An "isolated protein" is one that is substantially separated from at least one of the other proteins present in the natural source of the protein, or is substantially free of at least one of the chemical precursors or other chemicals when the protein is chemically synthesized. A protein is "substantially separated from" or "substantially free of" other protein(s) or other chemical(s) in preparations of the protein when there is less than about 30%, 20%, 10%, or 5% (by dry weight) of the other protein(s) or the other chemical(s) (also referred to herein as a "contaminating protein" or a "contaminating chemical").
[0060]Isolated proteins can have several different physical forms. The isolated protein can exist as a full-length nascent or unprocessed polypeptide, or as a partially processed polypeptide or as a combination of processed polypeptides. The full-length nascent polypeptide can be postranslationally modified by specific proteolytic cleavage events that result in the formation of fragments of the full-length nascent polypeptide. A fragment, or physical association of fragments can have the biological activity associated with the full-length polypeptide; however, the degree of biological activity associated with individual fragments can vary.
[0061]An isolated polypeptide can be a non-naturally occurring polypeptide. For example, an "isolated polypeptide" can be a "hybrid polypeptide." An "isolated polypeptide" can also be a polypeptide derived from a naturally occurring polypeptide by additions or deletions or substitutions of amino acids. An isolated polypeptide can also be a "purified polypeptide" which is used herein to mean a specified polypeptide in a substantially homogeneous preparation substantially free of other cellular components, other polypeptides, viral materials, or culture medium, or when the polypeptide is chemically synthesized, chemical precursors or by-products associated with the chemical synthesis. A "purified polypeptide" can be obtained from natural or recombinant host cells by standard purification techniques, or by chemical synthesis, as will be apparent to skilled artisans.
[0062]"Recombinant" refers to a nucleic acid, a protein encoded by a nucleic acid, a cell, or a viral particle, that has been modified using molecular biology techniques to something other than its natural state. For example, recombinant cells can contain nucleotide sequence that is not found within the native (non-recombinant) form of the cell or can express native genes that are otherwise abnormally, under-expressed, or not expressed at all. Recombinant cells can also contain genes found in the native form of the cell wherein the genes are modified and re-introduced into the cell by artificial means. The term also encompasses cells that contain an endogenous nucleic acid that has been modified without removing the nucleic acid from the cell; such modifications include those obtained, for example, by gene replacement, and site-specific mutation.
[0063]A "recombinant host cell" is a cell that has had introduced into it a recombinant DNA sequence. Recombinant DNA sequence can be introduced into host cells using any suitable method including, for example, electroporation, calcium phosphate precipitation, microinjection, transformation, biolistics and viral infection. Recombinant DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. For example, the recombinant DNA can be maintained on an episomal element, such as a plasmid. Alternatively, with respect to a stably transformed or transfected cell, the recombinant DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the stably transformed or transfected cell to establish cell lines or clones comprised of a population of daughter cells containing the exogenous DNA. Recombinant host cells may be prokaryotic or eukaryotic, including bacteria such as E. coli, fungal cells such as yeast, mammalian cells such as cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells such as Drosophila- and silkworm-derived cell lines. It is further understood that the term "recombinant host cell" refers not only to the particular subject cell, but also to the progeny or potential progeny of such a cell. Because certain modifications can occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
[0064]"Sequence identity or similarity", as known in the art, is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. As used herein, "identity", in the context of the relationship between two or more nucleic acid sequences or two or more polypeptide sequences, refers to the percentage of nucleotide or amino acid residues, respectively, that are the same when the sequences are optimally aligned and analyzed. For purposes of comparing a queried sequence against, for example, the amino acid sequence SEQ ID NO:2, the queried sequence is optimally aligned with SEQ ID NO: 2 and the best local alignment over the entire length of SEQ ID NO:2 is obtained.
[0065]Analysis can be carried out manually or using sequence comparison algorithms. For sequence comparison, typically one sequence acts as a reference sequence, to which a queried sequence is compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, sub-sequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
[0066]Optimal alignment of sequences for comparison can be conducted, for example, by using the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol., 48:443 (1970). Software for performing Needleman & Wunsch analyses is publicly available through the Institut Pasteur (France) Biological Software website: http://bioweb.pasteur.fr/seqanal/interfaces/needle.html. The NEEDLE program uses the Needleman-Wunsch global alignment algorithm to find the optimum alignment (including gaps) of two sequences when considering their entire length. The identity is calculated along with the percentage of identical matches between the two sequences over the reported aligned region, including any gaps in the length. Similarity scores are also provided wherein the similarity is calculated as the percentage of matches between the two sequences over the reported aligned region, including any gaps in the length. Standard comparisons utilize the EBLOSUM62 matrix for protein sequences and the EDNAFULL matrix for nucleotide sequences. The gap open penalty is the score taken away when a gap is created; the default setting using the gap open penalty is 10.0. For gap extension, a penalty is added to the standard gap penalty for each base or residue in the gap; the default setting is 0.5.
[0067]Hybridization can also be used as a test to indicate that two polynucleotides are substantially identical to each other. Polynucleotides that share a high degree of identity will hybridize to each other under stringent hybridization conditions. "Stringent hybridization conditions" has the meaning known in the art, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989). An exemplary stringent hybridization condition comprises hybridization in 6.times. sodium chloride/sodium citrate (SSC) at about 45.degree. C., followed by one or more washes in 0.2.times.SSC and 0.1% SDS at 50-65.degree. C., depending upon the length over which the hybridizing polynucleotides share complementarity.
[0068]A "TRPV2", "transient receptor potential cation channel, subfamily V, member 2", "VRL", "VRL1", "VRL-1", or "vanilloid receptor-like protein 1" each refers to a protein that forms an ion channel, the TRPV2 channel, that can be activated by high temperature and/or low osmolarity, and transduces heat responses in sensory ganglia. The TRPV2 channel can also be activated by certain compounds. An activated TRPV2 channel gates the influx of Ca.sup.2+ and other cations (e.g., Na.sup.+) through the channel, resulting in membrane depolarization. A TRPV2 protein can (1) have greater than about 70% amino acid sequence identity to a human TRPV2 (hTRPV2) protein depicted in SEQ ID NO: 2 (GenBank protein ID: NP.sub.--057197); or (2) bind to antibodies, e.g., polyclonal or monoclonal antibodies, raised against a hTRPV2 protein depicted in SEQ ID NO: 2. In some embodiments, the TRPV2 has greater than about 75, 80, 85, 90, or 95 percent amino acid sequence identity to SEQ ID NO: 2. TRPV2 includes orthologs of the TRPV2 in animals including human, rat, mouse, pig, dog and monkey, etc. TRPV2 also includes structural and functional polymorphisms of the TRPV2. TRPV2 includes the structural and functional polymorphisms of the TRPV2 from human, rat (GenBank protein ID: NP.sub.--058903, SEQ ID NO:4), mouse (GenBank protein ID: NP.sub.--035836, SEQ ID NO:6), or etc. For example, it was found that addition of a hemagglutinin A (HA) epitope tag to the end of the rat TRPV2 C-terminus did not alter the channel properties; and that deletion mutants of rat TRPV2-HA lacking the N-terminal 20, 32, and 65 and C-terminal 11, 23, or 32 amino acid residues of rat TRPV2 were still active in their responses to an elevated temperature of about 53.degree. C., lowered osmolarity, .DELTA.9-THC or 2-APB. Therefore, TRPV2 also includes deletion or modifications of the wild-type TRPV2 that maintains the biological activity of the TRPV2, such as the deletion mutants of rat TRPV2 consisting of the amino acid sequence of SEQ ID NOs: 7-14. Furthermore, TRPV2 also includes chimeras between TRPV2 of different animals. For example, it was found that chimeras (SEQ ID NO: 16) between rat and human TRPV2, named RHR (i.e. Rat 1-392/Human 391-646/Rat 647-761), was also active in its response to an elevation of temperature of about 53.degree. C., .DELTA..sup.9-THC and to 2-APB. In addition, TRPV2 further includes an active ion channel formed by the combination of two or more TRPV2 subunits, which by themselves are inactive or less active. For example, TRPV2 can be an active ion channel formed by the co-expression of the chimera RRH (Rat 1-392/Rat 393-646/Human 647-764) and HRR (Human 1-390/Rat 393-646/Rat 647-761).
[0069]"TRPV2 activation temperature" is the temperature at which a TRPV2 channel, in the absence of other stimulation, exhibits at least a 10% increase in its conductivity compared to the baseline. A person skilled in the art can experimentally determine the activation temperature for a TRPV2 channel. In some embodiments, "TRPV2 activation temperature" is the temperature at which a TRPV2 channel exhibits at least a 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% increase in its conductivity compared to the baseline. "TRPV2 activation temperature" is typically greater than of about 52.degree. C. In some embodiments, the TRPV2 activation temperature is about 52.degree. C.-55.degree. C. or 55.degree. C.-60.degree. C.
[0070]"TRPV2 non-activation temperature" is the temperature that falls outside of the range for a "TRPV2 activation temperature". An exemplary TRPV2 non-activation temperature is room temperature (about 22.degree. C.) or any temperature that is below about 52.degree. C.
[0071]"Vector" refers to a nucleic acid molecule into which a heterologous nucleic acid can be or is inserted. Some vectors can be introduced into a host cell allowing for replication of the vector or for expression of a protein that is encoded by the vector or construct. Vectors typically have selectable markers, for example, genes that encode proteins allowing for drug resistance, origins of replication sequences, and multiple cloning sites that allow for insertion of a heterologous sequence. Vectors are typically plasmid-based and are designated by a lower case "p" followed by a combination of letters and/or numbers. Starting plasmids disclosed herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids by application of procedures known in the art. Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well-known and readily available to those of skill in the art. Moreover, those of skill readily may construct any number of other plasmids suitable for use in the invention. The properties, construction and use of such plasmids, as well as other vectors, in the present invention will be readily apparent to those of skill from the present disclosure.
[0072]"Sequence" means the linear order in which monomers occur in a polymer, for example, the order of amino acids in a polypeptide or the order of nucleotides in a polynucleotide.
[0073]In practicing the present invention, many conventional techniques in molecular biology, microbiology and recombinant DNA are used. These techniques are well-known and are explained in, for example, Current Protocols in Molecular Biology, Vols. I, II, and III, F. M. Ausubel, ed. (1997); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001).
[0074]It was discovered in the present invention that a group of cannabinoids are capable of activating the TRPV2 but not TRPV1 activity. Thus, the present invention provides new methods for regulating the biological activity of TRPV2 and new methods for identifying compounds that regulate biological activity of TRPV2.
[0075]In one embodiment, the TRPV2 used in the present invention is present in a cell. The cell can express TRPV2 endogeneously or recombinantly. One exemplary endogeneous cell for TRPV2 is a dorsal root ganglia (DRG) neuron or trigeminal neurons. Other examples of endogeneous cell for TRPV2 include, but are not limited to, intestine intrinsic neurons, vascular smooth muscle cells, and human hepatoblastoma (HepG2).
[0076]It will be apparent to skilled artisans that any recombinant expression methods may be used in the present invention for purposes of expressing the TRPV2. Generally, a nucleic acid encoding TRPV2 can be introduced into a suitable host cell. Exemplary nucleic acid molecules that can be used in the present invention include cDNA that encodes for the full length TRPV2 from human (SEQ ID: 1, GenBank accession No: NM.sub.--016113), mouse (SEQ ID NO:5, GenBank accession No: NM.sub.--011706), or rat (SEQ ID NO:3 GenBank accession No: NM.sub.--017207).
[0077]Typically, the nucleic acids, preferably in the form of DNA, are incorporated into a vector to form expression vectors capable of directing the production of the interacting protein member(s) once introduced into a host cell. Many types of vectors can be used for the present invention. Methods for the construction of an expression vector for purposes of this invention should be apparent to skilled artisans apprised of the present disclosure. (See generally, Current Protocols in Molecular Biology, Vol. 2, Ed. Ausubel, et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13, 1988; Glover, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3, 1986; Bitter, et al., in Methods in Enzymology 153:516-544 (1987); The Molecular Biology of the Yeast Saccharomyces, Eds. Strathern et al., Cold Spring Harbor Press, Vols. I and II, 1982; and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989.)
[0078]Generally, the expression vectors include an expression cassette having a promoter operably linked to a DNA encoding an interacting protein member. The promoter can be a native promoter, i.e., the promoter found in naturally occurring cells to be responsible for the expression of the interacting protein member in the cells. Alternatively, the expression cassette can be a chimeric one, i.e., having a heterologous promoter that is not the native promoter responsible for the expression of the interacting protein member in naturally occurring cells. The expression vector may further include an origin of DNA replication for the replication of the vectors in host cells. Preferably, the expression vectors also include a replication origin for the amplification of the vectors in, e.g., E. coli, and selection marker(s) for selecting and maintaining only those host cells harboring the expression vectors.
[0079]The thus constructed expression vectors can be introduced into the host cells by any techniques known in the art, e.g., by direct DNA transformation, microinjection, electroporation, viral infection, lipofection, gene gun, and the like. The expression of the protein of interest may be transient or stable. The expression vectors can be maintained in host cells in an extrachromosomal state, i.e., as self-replicating plasmids or viruses. Alternatively, the expression vectors can be integrated into chromosomes of the host cells by conventional techniques such as selection of stable cell lines or site-specific recombination. In stable cell lines, at least the expression cassette portion of the expression vector is integrated into a chromosome of the host cells.
[0080]The vector construct can be designed to be suitable for expression in various host cells, including but not limited to bacteria, yeast cells, plant cells, insect cells, and mammalian and human cells. Methods for preparing expression vectors for expression in different host cells should be apparent to a skilled artisan. As described in the Example 1, infra, rat and human TRPV2 has been successfully expressed in HEK293.
[0081]Homologues and fragments of TRPV2 can also be easily expressed using the recombinant methods described above. For example, to express a protein fragment, the DNA fragment incorporated into the expression vector can be selected such that it only encodes the protein fragment. Likewise, a specific hybrid protein can be expressed using a recombinant DNA encoding the hybrid protein. Similarly, a homologue protein may be expressed from a DNA sequence encoding the homologue protein. A homologue-encoding DNA sequence may be obtained by manipulating the native protein-encoding sequence using recombinant DNA techniques. For this purpose, random or site-directed mutagenesis can be conducted using techniques generally known in the art. To make protein derivatives, for example, the amino acid sequence of a native interacting protein member may be changed in predetermined manners by site-directed DNA mutagenesis to create or remove consensus sequences.
[0082]In other embodiments, the TRPV2 is provided in a cell membrane. The membrane preparation can be isolated from a native host cell, for example, a DRG or TG cell, which expresses TRPV2 on its cell surface. The membrane preparation can also be isolated from a recombinant host cell, for example, a CHO, HEK293, or COS cell, which expresses a TRPV2 recombinantly on its cell surface. The membrane preparation can be further prepared from the biological membranes, such as the tissue membrane, plasma membrane, cell membrane, or internal organelle membrane expressing the TRPV2 channels. Methods are known to those skilled in the art for isolation and preparation of biological membrane preparations. For example, such a method can include the steps of mechanical or enzymatic disruption of the tissue or cells, centrifugation to separate the membranes from other components, and resuspending the membrane fragments or vesicles in suitable buffer solution. Alternatively, the membrane-containing preparation can also be derived from artificial membranes. Purified TRPV2 protein can be reconstituted into lipid bilayers to form artificial membrane vesicles (see Chen et al., 1996, J. Gen. Physiol. 108:237-250). Such type of membrane vesicle can be very specific to the channel of interest, avoiding the problem of contamination from other channels. For example, such artificial membranes can include an electrode to which is tethered a lipid membrane containing ion channels and forming ion reservoirs. Methods are known to those skilled in the art to prepare artificial membrane vesicles.
[0083]In some preferred embodiments, membranes can be broken under controlled conditions, yielding portions of cell membranes and/or membrane vesicles. Cell membrane portions and/or vesicles can, in some embodiments, provide an easier format for the inventive assays and methods, since cell lysis and/or shear is not as much of a concern during the assay. Cell membranes can be derived from tissues and/or cultured cells. Such methods of breaking cell membranes and stabilizing them are known in the art. Methods of treating tissues to obtain cell membranes are known in the art.
[0084]Preferably, human TRPV2 is used in the assays of the invention. Optionally, TRPV2 orthologs from other species such as rat or mouse, preferably a mammalian species, are used in assays of the invention.
[0085]The compound identification methods can be performed using conventional laboratory formats or in assays adapted for high throughput. The term "high throughput" refers to an assay design that allows easy screening of multiple samples simultaneously and/or in rapid succession, and can include the capacity for robotic manipulation. Another desired feature of high throughput assays is an assay design that is optimized to reduce reagent usage, or minimize the number of manipulations in order to achieve the analysis desired. Examples of assay formats include 96-well or 384-well plates, levitating droplets, and "lab on a chip" microchannel chips used for liquid handling experiments. It is well known by those in the art that as miniaturization of plastic molds and liquid handling devices are advanced, or as improved assay devices are designed, greater numbers of samples can be processed using the design of the present invention.
[0086]Any test compounds may be screened in the screening assays of the present invention to select modulators of the protein complex of the invention. By the term "selecting" or "select" compounds it is intended to encompass both (a) choosing compounds from a group previously unknown to be modulators of a protein complex or interacting protein members thereof, and (b) testing compounds that are known to be capable of binding, or modulating the functions and activities of, a protein complex or interacting protein members thereof. Both types of compounds are generally referred to herein as "test compounds" or "candidate compound". The candidate compounds encompass numerous chemical classes, including but not limited to, small organic or inorganic compounds, natural or synthetic molecules, such as antibodies, proteins or fragments thereof, antisense nucleotides, interfering RNA (iRNA) and ribozymes, and derivatives, mimetics and analogs thereof. Preferably, they are small organic compounds, i.e., those having a molecular weight of no greater than 10,000 daltons, more preferably less than 5,000 daltons. Preferably, the test compounds are provided in library formats known in the art, e.g., in chemically synthesized libraries (See generally, Gordan et al. J. Med. Chem., 37:1385-1401 (1994)), recombinantly expressed libraries (e.g., phage display libraries), and in vitro translation-based libraries (e.g., ribosome display libraries).
[0087]Candidate compounds comprise functional chemical groups necessary for structural interactions with polypeptides, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups and more preferably at least three of the functional chemical groups. The candidate compounds can comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more of the above-identified functional groups. Candidate compounds also can be biomolecules such as peptides, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the above, or combinations thereof and the like. Where the compound is a nucleic acid, the compound typically is a DNA or RNA molecule, although modified nucleic acids having non-natural bonds or subunits are also contemplated.
[0088]Candidate compounds are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, synthetic organic combinatorial libraries, phage display libraries of random peptides, and the like. Candidate compounds can also be obtained using any of the numerous approaches in combinatorial library methods known in the art, including biological libraries; spatially addressable parallel solid phase or solution phase libraries: synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection (Lam (1997) Anticancer Drug Des. 12: 145). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural and synthetically produced libraries and compounds can be readily modified through conventional chemical, physical, and biochemical means.
[0089]Further, known pharmacological agents can be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidation, etc. to produce structural analogs of the agents. Candidate compounds can be selected randomly or can be based on existing compounds that bind to and/or modulate the function of TRPV2 activity. Therefore, a source of candidate agents is one or more than one library of molecules based on one or more than one known compound that increases or decreases TRPV2 channel conductivity in which the structure of the compound is changed at one or more positions of the molecule to contain more or fewer chemical moieties or different chemical moieties. The structural changes made to the molecules in creating the libraries of analog activators/inhibitors can be directed, random, or a combination of both directed and random substitutions and/or additions. One of ordinary skill in the art in the preparation of combinatorial libraries can readily prepare such libraries based on the existing compounds.
[0090]A variety of other reagents also can be included in the mixture. These include reagents such as salts, buffers, neutral proteins (e.g., albumin), detergents, etc. that can be used to facilitate optimal protein-protein and/or protein-nucleic acid binding. Such a reagent can also reduce non-specific or background interactions of the reaction components. Other reagents that improve the efficiency of the assay such as nuclease inhibitors, antimicrobial agents, and the like can also be used.
[0091]Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: Zuckermann et al. (1994). J. Med. Chem. 37:2678. Libraries of compounds can be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. No. 5,571,698), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or phage (see e.g., Scott and Smith (1990) Science 249:386-390).
[0092]The selected compounds can be tested for their ability to decrease the channel conductivity of the TRPV2, or for their ability to decrease the binding activity of the TRPV2 to a cannabinoid that is capable of activating the TRPV2. During the test, the test compound can be added to the TRPV2 prior to, after, or simultaneously with cannabinoid that is capable of activating the TRPV2. In addition, the compounds can be tested in an animal model for pain, or inflammation, etc.
[0093]Generally, a control assay is performed in which the above screening assay is conducted in the absence of the test compound. The result of this assay is then compared with that obtained in the presence of the test compound.
[0094]The test compounds may be screened in an in vitro assay to identify compounds capable of binding to a TRPV2. For this purpose, a test compound is contacted with TRPV2 under conditions and for a time sufficient to allow specific interaction between the test compound and the TRPV2 to occur, thereby resulting in the binding of the compound to the TRPV2, and the formation of a complex. Subsequently, the binding event is detected.
[0095]In one particular embodiment, the TRPV2 is immobilized on a solid support (such as a protein microchip) or on a cell surface or a membrane. For example, the protein complex can be immobilized directly onto a microchip substrate such as glass slides or onto multi-well plates using non-neutralizing antibodies, i.e., antibodies capable of binding to the complex but do not substantially affect its biological activities. A cannabinoid labeled with a detectable marker is contacted with the immobilized TRPV2. Test compounds can be contacted with the immobilized TRPV2 protein to allow binding to occur under standard binding assay conditions. To identify compound binding to a TRPV2, one can measure the detectable marker associated with TRPV2 or disassociated from the TRPV2. A test compound that binds competitively with the labeled cannabinoid to the TRPV2 will result in less binding of TRPV2 to the cannabinoid, thus less labeling associated with TRPV2.
[0096]In one embodiment, the test compound can be further evaluated for its ability to increase or decrease the ion conductivity of a TRPV2 channel. Known to those skilled in the art are methods for measuring a TRPV2 channel conductivity, for example, via the cellular depolarization/hyperpolarization or an increase in intracellular calcium ion levels. The level of intracellular calcium can be assessed using a calcium ion-sensitive fluorescent indicator, such as a calcium ion-sensitive fluorescent dye. Suitable calcium ion-sensitive fluorescent dyes include, for example, quin-2 (see, e.g., Tsien et al., J. Cell BioL, 94:325, 1982), fura-2 (see, e.g., Grynkiewicz et al., J. BioL Chem., 260:3440, 1985), fluo-3 (see, e.g., Kao et al., J. BioL-43 Chem., 264:8179, 1989) and rhod-2 (see, e.g., Tsien et al., J. BioL Chem., Abstract 89a, 1987). Suitable calcium ion-sensitive fluorescent dyes are commercially available from, for example, Invitrogen (Molecular Probes Products, Eugene, Oreg.). Cellular fluorescence can also be monitored using a fluorometer or a flow cytometer having a fluorescence lamp and detector. FLIPR assay has been used routinely in measuring the ion conductivity.
[0097]The TRPV2 cation channels function to transport not only divalent cations, for example, Ca.sup.2+, but also monovalent cations, for example, Na.sup.+ or K.sup.+. Therefore, assays for determining changes in the transport of monovalent cation can also be performed to measure the conductivity of a TRPV2 channel. Na.sup.+- and K.sup.+-sensitive dyes are known in the art and commercially available from, for example, Invitrogen (Molecular Probes Products, Eugene, Oreg.).
[0098]The conductivity of a TRPV2 channel can also be measured by electrophysiologic techniques such as patch clamp. Patch clamp techniques are routinely used for studying electrical activities in cells, cell membranes, and isolated tissues. It involves forming an electrically tight, high-resistance seal between a micropipette and the plasma membrane. The current flowing through individual ion channels within the plasma membrane can then be measured. Different variants on the techniques allow different surfaces of the plasma membrane to be exposed to the bathing medium. The four most common variants include cell-attached, inside-out, outside-out and whole-cell patch clamp.
[0099]A patch-clamp method is commonly used with a voltage clamp that controls the voltage across the membrane and measures current flow. For example, in the case of whole-cell patch clamp, during the voltage clamp process, a microelectrode is inserted into a cell and current injected through the electrode so as to hold the cell membrane potential at some predefined level. A patch-clamp method can also be used in the current-clamp configuration, in which the current is controlled and the membrane potential is measured.
[0100]In another embodiment, the test compound can be further evaluated by administering it to a live animal. This can be useful to establish efficacy, toxicity and other pharmacological parameters important for establishing dosing regimens. For example, the compound can be administered to a dog to examine various pharmacological aspects of the compound in the dog. The dog testing can be particularly advantageous for identifying and establishing dosing regimens in humans, because dogs, particularly large breeds, are closer in weight to humans as compared to rats or mice and therefore provide a more suitable animal model for estimating human dosing.
[0101]The compound can also be administered to animals to assess the ability of the compound to alter nociceptive processes. Various animal models of pain exist. For example, the rat spinal nerve ligation (SNL) model of nerve injury is a model of neuropathic pain (Kim and Chung, Pain, 50:355-363, 1992).
[0102]Other suitable animal models of pain can be utilized in connection with the teachings herein. Commonly studied rodent models of neuropathic pain include the chronic constriction injury (CCI) or the Bennett model; neuroma or axotomy model; and the partial sciatic transection or Seltzer model (Shir et al., Neurosci. Lett., 115:62-67, 1990). Exemplary neuropathic pain models include several traumatic nerve injury preparations (Bennett et al, Pain 33: 87-107, 1988; Decosterd et al., Pain 87: 149-58, 2000; Kim et al, Pain 50: 355-363, 1992; Shir et al., Neurosci Lett 115: 62-7, 1990), neuroinflammation models (Chacur et al., Pain 94: 231-44, 2001; Milligan et al., Brain Res 861: 105-16, 2000), diabetic neuropathy (Calcutt et al., Br J Pharmacol 122: 1478-82, 1997), virus-induced neuropathy (Fleetwood-Walker et al., J Gen Virol 80: 2433-6, 1999), vincristine neuropathy (Aley et al., Neuroscience 73: 259-65, 1996; Nozaki-Taguchi et al., Pain 93: 69-76, 2001), and paclitaxel neuropathy (Cavaletti et al., Exp Neurol 133: 64-72, 1995), as well as acute nociceptive models and inflammatory models (Brennan, T. J. et al. Pain 64:493, 1996; D'Amour, F. E. and Smith, D. L. J Pharmacol 72: 74-79, 1941; Eddy, N. B. et al. J Pharmacol Exp Ther 98:121, 1950; Haffner, F. Dtsch Med Wochenschr 55:731, 1929; Hargreaves, K. et al. Pain 32: 77-88, 1988; Hunskaar, S. et al. J Neurosci Meth 14:69, 1985; Randall, L. O. and Selitto, J. J. Arch. Int. Pharmacodyn 111: 409-419, 1957; Siegmund, E. et al. Proc Soc Exp Bio Med 95:729, 1957).
[0103]The discovery that certain cannabinoids activate TRPV2 also provides new methods for identifying additional compounds that increase the biological activity of TRPV2. Such methods can be based on rational drug design. Structural analogs or mimetics of the cannabinoid can be produced based on rational drug design with the aim of improving drug potency, efficacy and stability, and reducing side effects. Methods known in the art for rational drug design can be used in the present invention. See, e.g., Hodgson et al., Bio/Technology, 9:19-21 (1991); U.S. Pat. Nos. 5,800,998 and 5,891,628, all of which are incorporated herein by reference.
[0104]Molecular modeling programs can be used to determine whether a small molecule can fit into a functionally relevant portion, for example, an active site, of the TRPV2 polypeptide. Basic information on molecular modeling is provided in, for example, M. Schlecht, Molecular Modeling on the PC, 1998, John Wiley & Sons; Gans et al., Fundamental Principals of Molecular Modeling, 1996, Plenum Pub. Corp.; N. C. Cohen (editor), Guidebook on Molecular Modeling in Drug Design, 1996, Academic Press; and W. B. Smith, Introduction to Theoretical Organic Chemistry and Molecular Modeling, 1996. U.S. patents that provide detailed information on molecular modeling include U.S. Pat. Nos. 6,093,573; 6,080,576; 5,612,894; and 5,583,973.
[0105]Programs that can be useful for molecular modeling studies include, for example, GRID (Goodford, P. J., "A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules" J. Med. Chem., 28, pp. 849-857, 1985), available from Oxford University, Oxford, UK; MCSS (Miranker, A. and M. Karplus, "Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method." Proteins: Structure, Function and Genetics, 11, pp. 29-34, 1991), available from Molecular Simulations, Burlington, Mass.; AUTODOCK (Goodsell, D. S. and A. J. Olsen, "Automated Docking of Substrates to Proteins by Simulated Annealing" Proteins: Structure. Function, and Genetics, 8, pp. 195-202, 1990); available from Scripps Research Institute, La Jolla, Calif.; and DOCK (Kuntz, I. D. et al., "A Geometric Approach to Macromolecule-Ligand Interactions" J. Mol. Biol., 161, pp. 269-288, 1982), available from University of California, San Francisco, Calif.
[0106]In this respect, structural information on the TRPV2-cannabinoid complex is obtained. Preferably, atomic coordinates defining a three-dimensional structure of the complex can be obtained. For example, the interacting TRPV2-cannabinoid complex can be studied using various biophysical techniques including, e.g., X-ray crystallography, NMR, computer modeling, mass spectrometry, and the like. Likewise, structural information can also be obtained from protein complexes formed by interacting proteins and a compound that initiates or stabilizes the interaction of the proteins. Methods for obtaining such atomic coordinates by X-ray crystallography, NMR, and the like are known in the art and the application thereof to the target protein or protein complex of the present invention should be apparent to skilled persons in the art of structural biology. See Smyth and Martin, Mol. Pathol., 53:8-14 (2000); Oakley and Wilce, Clin. Exp. Pharmacol. Physiol., 27(3):145-151 (2000); Ferentz and Wagner, Q. Rev. Biophys., 33:29-65 (2000); Hicks, Curr. Med. Chem., 8(6):627-650 (2001); and Roberts, Curr. Opin. Biotechnol., 10:42-47 (1999).
[0107]The domains, residues or moieties of a cannabinoid critical to TRPV2-cannabinoid interaction constitute the active region of the cannabinoid known as its "pharmacophore." Once the pharmacophore has been elucidated, a structural model can be established by a modeling process that may incorporate data from NMR analysis, X-ray diffraction data, alanine scanning, spectroscopic techniques and the like. Various techniques including computational analysis (e.g., molecular modeling and simulated annealing), similarity mapping and the like can all be used in this modeling process. See e.g., Perry et al., in OSAR: Quantitative Structure-Activity Relationships in Drug Design, pp. 189-193, Alan R. Liss, Inc., 1989; Rotivinen et al., Acta Pharmaceutical Fennica, 97:159-166 (1988); Lewis et al., Proc. R. Soc. Lond., 236:125-140 (1989); McKinaly et al., Annu. Rev. Pharmacol. Toxiciol., 29:111-122 (1989). Commercially available molecular modeling systems from Polygen Corporation, Waltham, Mass., include the CHARMm program, which performs energy minimization and molecular dynamics functions, and QUANTA program, which performs construction, graphic modeling and analysis of molecular structure. Such programs allow interactive construction, modification, and visualization of molecules. Other computer modeling programs are also available from BioDesign, Inc. (Pasadena, Calif.), Hypercube, Inc. (Cambridge, Ontario), and Allelix, Inc. (Mississauga, Ontario, Canada).
[0108]A template can be formed based on the established model. Various compounds can then be designed by linking various chemical groups or moieties to the template. Various moieties of the template can also be replaced. In addition, in the case of a peptide lead compound, the peptide or mimetics thereof can be cyclized, e.g., by linking the N-terminus and C-terminus together, to increase its stability. These rationally designed compounds are further tested. In this manner, pharmacologically acceptable and stable compounds with improved potency/efficacy and reduced side effects can be developed. The compounds identified in accordance with the present invention can be incorporated into a pharmaceutical formulation suitable for administration to an individual.
[0109]In addition, the structural models or atomic coordinates defining a three-dimensional structure of the target protein or protein complex can also be used in virtual screen to select compounds capable of activating TRPV2. Various methods of computer-based virtual screen using atomic coordinates are generally known in the art. For example, U.S. Pat. No. 5,798,247 (which is incorporated herein by reference) discloses a method of identifying a compound (specifically, an interleukin converting enzyme inhibitor) by determining binding interactions between an organic compound and binding sites of a binding cavity within the target protein. The binding sites are defined by atomic coordinates.
[0110]The compounds designed or selected based on rational drug design or virtual screen can be tested for their ability to modulate (interfere with or strengthen) the interaction between the interacting partners within the protein complexes of the present invention. In addition, the compounds can further be tested in TRPV2 channel conductivity assays or animal models as described supra.
[0111]Following the selection of desirable compounds according to the methods disclosed above, the methods of the present invention further provide for the manufacture of the selected compounds. The compounds can be manufactured for further experimental studies, or for therapeutic use. The compounds identified in the screening methods of the present invention can be made into therapeutically or prophylactically effective drugs for preventing or ameliorating diseases, disorders or symptoms caused by or associated with TRPV2, such as pain, or inflammation, etc.
Example 1
Expression of Rat and Human TRPV2 in HEK293 Cells
[0112]A cDNA fragment encoding the full-length rat TRPV2 was subcloned into pCI-neo (Promega, Madison, Wis.) mammalian expression vector. The expression construct was then transfected into HEK293 cells with FuGene6 transfection reagent (Roche, Indianapolis, Ind.) according to the vendor's protocol. Stable cell lines were selected by growth in the presence of 400 .mu.g/ml G418. Single G418 resistant clones were isolated and purified. Stable expression of rat TRPV2 in these cells was confirmed by Western blot analysis with an anti-rat TRPV2 specific antibody (Chemicon, Temecula, Calif.), Ca.sup.2+ imaging assay (FLIPR) and whole cell patch clamp analyses.
[0113]A cDNA fragment encoding the full-length human TRPV2 was subcloned into pCI-neo or pcDNA3 mammalian expression vectors. The expression constructs were then transfected into HEK293 cells with FuGene6 transfection reagent (Roche, Indianapolis, Ind.) according to vendor's protocol. TRPV2-expressing HEK293 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 .mu.g/ml streptomycin for 48-72 hr and either evaluated for transient expression and/or activity, or dosed with 400 .mu.g/ml G418 to select for stably-transfected TRPV2-expressing cell clones. Cells were maintained at 37.degree. C. and in 5% CO.sub.2.
Example 2
TRPV2 is activated by .DELTA..sup.9-tetrahydrocannabinol (.DELTA..sup.9-THC)
[0114]To search for pharmacological activators of TRPV2, .DELTA..sup.9-THC, a major psychoactive constituent of marijuana derived from Cannabis, was tested. The rat TRPV2-expressing HEK293 cells were seeded in a 384-well plate at a concentration of 5.times.10.sup.3 cells/well and incubated overnight at 37.degree. C. The following day, the cells were loaded with buffer and calcium dye 3 (Molecular Devices, Sunnyvale, Calif.) in a final volume of 50 .mu.l and incubated for 30 minutes at 37.degree. C./5% CO.sub.2 followed by 30 additional minutes at room temperature. The fluorescence intensity was measured by a fluorescent plate reader (FLIPR) before, during and after the addition of test compounds.
[0115]As shown in FIG. 4A, addition of 100 .mu.M .DELTA..sup.9-THC solid line but not buffer (dotted line), caused a robust elevation of intracellular Ca.sup.2+ in rat TRPV2-expressing HEK293 cells. In contrast, no significant intracellular Ca.sup.2+ elevation was observed in untransfected HEK293 cells at the same concentration of .DELTA..sup.9-THC (dashed line), suggesting that the elevation of intracellular Ca.sup.2+ was mediated by rat TRPV2. Activation of rat TRPV2 by .DELTA..sup.9-THC was dose-dependent with an EC.sub.50 value of 15.7 uM and Hill slope of 1.04 (FIG. 4B).
[0116]To further confirm the .DELTA..sup.9-THC effect on TRPV2, whole-cell patch clamp studies were performed. The extracellular solution contained (in mM): NaCl, 132; CaCl, 1.8; KCl, 5.4; MgCl.sub.2, 0.8; HEPES, 10; glucose, 10; pH=7.4. The intracellular solution used to fill recording pipettes contained (in mM): CsCl, 145; EGTA, 5; HEPES, 10; glucose, 5; pH=7.4. Recordings were performed using the conventional whole-cell patch clamp technique, 2-3 days after transient transfection of human TRPV2 into HEK293 cell or 1-2 days after plating HEK293 cells stably expressing rat TRPV2 onto glass coverslips. Currents were amplified by a patch clamp amplifier and filtered at 2 kHz (Axopatch 200B), sampled at 10 kHz using Digidata 1322A and acquired and analyzed with pClamp 9.0 (all instruments from Molecular Devices, CA). A 600 ms voltage ramp was given once every five seconds from -100 mV to +60 mV. The holding potential between voltage ramps was -100 mV. Extracellular solutions were applied to the cell at 0.5 ml/min via a gravity-fed perfusion system. All experiments were performed at 22.degree. C.
[0117]As shown in FIG. 5B, upon application of 100 .mu.M .DELTA..sup.9-THC, there was a significant increase of the whole-cell current amplitude (gray solid line) in HEK293 cells expressing rTRPV2 compared to control (black dotted line) at both hyperpolarized and depolarized membrane potentials. However, this effect was more pronounced at depolarized potentials. The same concentration of .DELTA..sup.9-THC elicited no current above control level in untransfected HEK293 cells (data not shown). The .DELTA..sup.9-THC-activated current had a reversal potential near 0 mV, indicating the relatively unselective (at least for the cations used in these experiments) nature of the channel. Similar effects induced by .DELTA..sup.9-THC were also observed in human TRPV2 (FIG. 5A). Furthermore, the .DELTA..sup.9-THC-activated currents were significantly inhibited by 10 .mu.M ruthenium red (RR), a non-selective TRP channel inhibitor (black line) in both rat and human TRPV2. Taken together, these results indicate that .DELTA..sup.9-THC activates currents mediated by TRPV2 in these cells.
Example 3
TRPV2 is Activated by Other Cannabinoids
[0118]To further explore activation of TRPV2 by cannabinoids, several different classes of cannabinoids were tested in a FLIPR calcium mobilization assay using 100 .mu.M compound concentrations (except for anandamide which was at 120 .mu.M) as evaluated using rat TRPV2-expressing HEK293 cells. All data was normalized to that observed for 100 .mu.M .DELTA..sup.9-THC. The tested compounds included: the non-psychoactive constituents of marijuana (cannabidiol and cannabinol); synthetic analogs of THC (nabilone, CP 55,940, HU210, HU211, HU-308, HU331, 11-hydroxy-.DELTA.9-THC, and O-1821); several endocannabinoids (anandamide, 2-arachidonoyl-glycerol (2-AG) and their analog palmitoylethanolamide (PEA)); a cannabinoid transport blocker (AM404); other synthetic cannabinoid receptor agonists (WIN55, 212-2, WIN55, 212-3, JWH015, JWH133, O-1918 and CAY10429); and the non-selective agonist, 2-APB The ability of these compounds to activate rat TRPV2 is shown in Table 1. The data suggest that TRPV2 could be activated by more than one class of cannabinoids.
TABLE-US-00001 TABLE 1 Activation of rat TRPV2 by Cannabinoids Compound Compound Structure % Response EC.sub.50 .DELTA..sup.9-THC ##STR00001## 100 15.5 uM Cannabinol ##STR00002## 68 77.7 uM Nabilone ##STR00003## 59 CP55,940 ##STR00004## 43 HU-210 ##STR00005## 39 WIN-55,212-2 ##STR00006## -2 JWH015 ##STR00007## 14 Anandamide ##STR00008## 0 2-AG ##STR00009## 30 PEA ##STR00010## 5 AM404 ##STR00011## 6 Cannabidiol ##STR00012## 163 3.7 uM 11-hydroxy-.DELTA..sup.9-THC ##STR00013## 58 O-1821 ##STR00014## 95 ~20 uM O-1918 ##STR00015## 6 CAY10429 ##STR00016## 0 WIN 55,212-3 ##STR00017## 7 HU-211 ##STR00018## 31 HU-308 ##STR00019## 10 HU-331 ##STR00020## 47 ~14 uM JWH-133 ##STR00021## 5 2-APB ##STR00022## 102 8.0 uM
Example 4
.DELTA..sup.9-THC Selectively Activates TRPV2
[0119]A selected number of cannabinoids were also tested against HEK293 cells expressing human TRPV1 and canine TRPM8 in the FLIRP assay. As summarized in Table 2, .DELTA..sup.9-THC (240 .mu.M) and cannabinol (600 .mu.M) showed no significant activation of TRPV1, whereas anandamide, 2-AG and AM404 activated TRPV1, consistent with previous reports. None of these cannabinoids showed agonist effect against TRPM8.
TABLE-US-00002 TABLE 2 Activation of TRP channels by cannabinoids TRPV1 TRPV2 TRPM8 .DELTA..sup.9-THC (240 .mu.M) - + - Cannabinol (600 .mu.M) - + - Anandamide (120 .mu.M) + - - 2-AG (120 .mu.M) + + - AM404 (120 .mu.M) + - -
Example 5
Deletion Mutants of TRPV2 were Activated by Cannabinoids
[0120]TRPV2 deletion mutants were constructed and tested for activation by .DELTA..sup.9-THC, 2-APB and high temperature stimulation. Methods of this Example can be used to construct and test any type of TRPV2 deletion mutants, including, but not limited to, mutants having one or more amino acid residues deleted at the N-terminal of the TRPV2, at the C-terminal of the TRPV2, and/or at any other location of the TRPV2.
[0121]DNA molecules encoding the deletion mutants were amplified by PCR using the rat TRPV2 cDNA as the template. For amino-terminal deletions, a series N-terminal forward primers encoding an initiating methionine in frame with sequences matching adjacent regions of the desired start at residues G21 (mutant N20, SEQ ID NO:11), P33 (N32, SEQ ID NO:12), A66 (N65, SEQ ID NO:13) and V84 (N83, SEQ ID NO:14) paired with a C-terminal reverse primer were used for PCR amplification. While for carboxyl-terminal deletions, a forward N-terminal primer paired with a series C-terminal reverse primers ending at residues R706 (C51, SEQ ID NO:7), P729 (C32, SEQ ID NO:8), P738 (C23, SEQ ID NO:9) and E750 (C11, SEQ ID NO: 10) with an in-frame stop codon at the terminal end of the open reading frame were used for amplification. After PCR amplification and purification, the DNA molecules encoding the deletion mutants were subcloned into the pCI-neo mammalian expression vector and the constructs were confirmed by DNA sequencing. The DNA molecules that encoded for the various TRPV2 deletion mutants comprised the nucleotide sequences of: SEQ ID NO:18 (N20), SEQ ID NO:19 (N32), SEQ ID NO:20 (N65), SEQ ID NO:21 (N83), SEQ ID NO:22 (C51), SEQ ID NO:23 (C32), SEQ ID NO:24 (C23), and SEQ ID NO:25 (C11).
[0122]The deletion mutant constructs were then transfected into HEK293 cells using Fugene 6 reagent (Roche) as per the manufacturer's instructions. At 24 hours post-transfection, the cells were harvested and replated in fresh DMEM medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 .mu.g/ml streptomycin and. The cells were distributed onto poly-D-Lysine coated 96- or 384-well plates at a density of approximately 40,000 and 10,000 cells per well, respectively. At approximately 48 hours post-transfection, the medium was removed from the assay plate and replaced with Calcium 3 Dye Buffer (Molecular Devices) using the protocol available from the manufacturer. Calcium mobilization was triggered using .DELTA..sup.9-THC or 2-APB or elevated temperature buffer and measured using either FLIPR or FLEX STATION instruments.
[0123]It was found that deletion mutants of rat TRPV2 lacking the N-terminal 20, 33, 66, or lacking the C-terminal 11, 23, or 32 amino acid residues were still active in their responses to .DELTA..sup.9-THC (FIG. 6), 2-APB and an elevated temperature of about 53.degree. C. (data not shown).
Example 6
Activation of TRPV2 Chimera
[0124]The domain-swapping chimeras between rat and human TRPV2s were also made and tested for activation by .DELTA..sup.9-THC, 2-APB and high temperature stimulation. Methods of this Example can be used to construct and test any type of TRPV2 chimeras, including, but not limited to, the domain-swapping chimeras between TRPV2s from different animals, and the chimeras between TRPV2 and other TRPV channels such as TRPV1 and TRPV3.
[0125]Based on the predicted topology and primary sequence features of TRPV2, TRPV2 are divided into 3 major domains: 1) the amino-terminal intracellular domain; 2) the transmembrane domain; and 3) the carboxyl-terminal intracellular domain. For a chimera, each domain can be of rat (R) or human (H) origin. Three rat and human TRPV2 chimera were constructed and tested herein. RRH (SEQ ID NO:15) was a chimera comprising rat 1-392 aa, rat 393-646 aa, and human 647-764 aa. RHR (SEQ ID NO:16) was a chimera comprising rat 1-392, human 391-646, and rat 647-761. HRR (SEQ ID NO:17) was a chimera comprising human 1-390, rat 393-646, and rat 647-761.
[0126]DNA molecules encoding the three rat and human TRPV2 chimeras were obtained by fusion PCR. First, DNA molecules encoding the desired TRPV2 domains were amplified by PCR using the rat or human TRPV2 cDNA as the template with synthetic primer DNA containing in-frame sequence that overlapped with the other species domain to be linked. After PCR amplification and purification, DNA molecules encoding the desired TRPV2 domains from human and rat were combined and used as templates for fusion PCR with primer DNA matching the 5' and 3' end sequences of the coding sequence for the full length TRPV2 chimera. The DNA molecules that encoded the various TRPV2 chimeras comprised the nucleotide sequences of: SEQ ID NO: 26 (RRH), SEQ ID NO:27 (RHR), and SEQ ID NO:28 (HRR).
[0127]The DNA molecules encoding the TRPV2 chimera were then subcloned into the mammalian expression vector, pCI-neo and the resulting constructs confirmed by DNA sequencing. The chimeras were transiently expressed in HEK293 cells and their responses to a variety of stimulators were also tested as described in Example 5.
[0128]Chimera RHR was fully responsive to the addition of .DELTA..sup.9-THC (FIG. 7A),). Although the HEK cells expressing chimera RRH or HRR separately were poorly, or not active, respectively, the cells co-expressing both chimeras (RRH+HHR) were fully responsive to .DELTA..sup.9-THC (FIG. 7B. Similar responses by the above-listed chimeras to 2-APB stimulation were observed (data not shown). The gain of function study by coexpression of two inactive mutants suggests that a functional TRPV2 channel is a complex with multiple subunits and some of the critical functional domains act in trans rather than in cis.
Sequence CWU
1
2812295DNAHomo sapiens 1atgacctcac cctccagctc tccagttttc aggttggaga
cattagatgg aggccaagaa 60gatggctctg aggcggacag aggaaagctg gattttggga
gcgggctgcc tcccatggag 120tcacagttcc agggcgagga ccggaaattc gcccctcaga
taagagtcaa cctcaactac 180cgaaagggaa caggtgccag tcagccggat ccaaaccgat
ttgaccgaga tcggctcttc 240aatgcggtct cccggggtgt ccccgaggat ctggctggac
ttccagagta cctgagcaag 300accagcaagt acctcaccga ctcggaatac acagagggct
ccacaggtaa gacgtgcctg 360atgaaggctg tgctgaacct taaggacgga gtcaatgcct
gcattctgcc actgctgcag 420atcgacaggg actctggcaa tcctcagccc ctggtaaatg
cccagtgcac agatgactat 480taccgaggcc acagcgctct gcacatcgcc attgagaaga
ggagtctgca gtgtgtgaag 540ctcctggtgg agaatggggc caatgtgcat gcccgggcct
gcggccgctt cttccagaag 600ggccaaggga cttgctttta tttcggtgag ctacccctct
ctttggccgc ttgcaccaag 660cagtgggatg tggtaagcta cctcctggag aacccacacc
agcccgccag cctgcaggcc 720actgactccc agggcaacac agtcctgcat gccctagtga
tgatctcgga caactcagct 780gagaacattg cactggtgac cagcatgtat gatgggctcc
tccaagctgg ggcccgcctc 840tgccctaccg tgcagcttga ggacatccgc aacctgcagg
atctcacgcc tctgaagctg 900gccgccaagg agggcaagat cgagattttc aggcacatcc
tgcagcggga gttttcagga 960ctgagccacc tttcccgaaa gttcaccgag tggtgctatg
ggcctgtccg ggtgtcgctg 1020tatgacctgg cttctgtgga cagctgtgag gagaactcag
tgctggagat cattgccttt 1080cattgcaaga gcccgcaccg acaccgaatg gtcgttttgg
agcccctgaa caaactgctg 1140caggcgaaat gggatctgct catccccaag ttcttcttaa
acttcctgtg taatctgatc 1200tacatgttca tcttcaccgc tgttgcctac catcagccta
ccctgaagaa gcaggccgcc 1260cctcacctga aagcggaggt tggaaactcc atgctgctga
cgggccacat ccttatcctg 1320ctagggggga tctacctcct cgtgggccag ctgtggtact
tctggcggcg ccacgtgttc 1380atctggatct cgttcataga cagctacttt gaaatcctct
tcctgttcca ggccctgctc 1440acagtggtgt cccaggtgct gtgtttcctg gccatcgagt
ggtacctgcc cctgcttgtg 1500tctgcgctgg tgctgggctg gctgaacctg ctttactata
cacgtggctt ccagcacaca 1560ggcatctaca gtgtcatgat ccagaaggtc atcctgcggg
acctgctgcg cttccttctg 1620atctacttag tcttcctttt cggcttcgct gtagccctgg
tgagcctgag ccaggaggct 1680tggcgccccg aagctcctac aggccccaat gccacagagt
cagtgcagcc catggaggga 1740caggaggacg agggcaacgg ggcccagtac aggggtatcc
tggaagcctc cttggagctc 1800ttcaaattca ccatcggcat gggcgagctg gccttccagg
agcagctgca cttccgcggc 1860atggtgctgc tgctgctgct ggcctacgtg ctgctcacct
acatcctgct gctcaacatg 1920ctcatcgccc tcatgagcga gaccgtcaac agtgtcgcca
ctgacagctg gagcatctgg 1980aagctgcaga aagccatctc tgtcctggag atggagaatg
gctattggtg gtgcaggaag 2040aagcagcggg caggtgtgat gctgaccgtt ggcactaagc
cagatggcag ccccgatgag 2100cgctggtgct tcagggtgga ggaggtgaac tgggcttcat
gggagcagac gctgcctacg 2160ctgtgtgagg acccgtcagg ggcaggtgtc cctcgaactc
tcgagaaccc tgtcctggct 2220tcccctccca aggaggatga ggatggtgcc tctgaggaaa
actatgtgcc cgtccagctc 2280ctccagtcca actga
22952764PRTHomo sapiens 2Met Thr Ser Pro Ser Ser
Ser Pro Val Phe Arg Leu Glu Thr Leu Asp1 5
10 15Gly Gly Gln Glu Asp Gly Ser Glu Ala Asp Arg Gly
Lys Leu Asp Phe 20 25 30Gly
Ser Gly Leu Pro Pro Met Glu Ser Gln Phe Gln Gly Glu Asp Arg 35
40 45Lys Phe Ala Pro Gln Ile Arg Val Asn
Leu Asn Tyr Arg Lys Gly Thr 50 55
60Gly Ala Ser Gln Pro Asp Pro Asn Arg Phe Asp Arg Asp Arg Leu Phe65
70 75 80Asn Ala Val Ser Arg
Gly Val Pro Glu Asp Leu Ala Gly Leu Pro Glu 85
90 95Tyr Leu Ser Lys Thr Ser Lys Tyr Leu Thr Asp
Ser Glu Tyr Thr Glu 100 105
110Gly Ser Thr Gly Lys Thr Cys Leu Met Lys Ala Val Leu Asn Leu Lys
115 120 125Asp Gly Val Asn Ala Cys Ile
Leu Pro Leu Leu Gln Ile Asp Arg Asp 130 135
140Ser Gly Asn Pro Gln Pro Leu Val Asn Ala Gln Cys Thr Asp Asp
Tyr145 150 155 160Tyr Arg
Gly His Ser Ala Leu His Ile Ala Ile Glu Lys Arg Ser Leu
165 170 175Gln Cys Val Lys Leu Leu Val
Glu Asn Gly Ala Asn Val His Ala Arg 180 185
190Ala Cys Gly Arg Phe Phe Gln Lys Gly Gln Gly Thr Cys Phe
Tyr Phe 195 200 205Gly Glu Leu Pro
Leu Ser Leu Ala Ala Cys Thr Lys Gln Trp Asp Val 210
215 220Val Ser Tyr Leu Leu Glu Asn Pro His Gln Pro Ala
Ser Leu Gln Ala225 230 235
240Thr Asp Ser Gln Gly Asn Thr Val Leu His Ala Leu Val Met Ile Ser
245 250 255Asp Asn Ser Ala Glu
Asn Ile Ala Leu Val Thr Ser Met Tyr Asp Gly 260
265 270Leu Leu Gln Ala Gly Ala Arg Leu Cys Pro Thr Val
Gln Leu Glu Asp 275 280 285Ile Arg
Asn Leu Gln Asp Leu Thr Pro Leu Lys Leu Ala Ala Lys Glu 290
295 300Gly Lys Ile Glu Ile Phe Arg His Ile Leu Gln
Arg Glu Phe Ser Gly305 310 315
320Leu Ser His Leu Ser Arg Lys Phe Thr Glu Trp Cys Tyr Gly Pro Val
325 330 335Arg Val Ser Leu
Tyr Asp Leu Ala Ser Val Asp Ser Cys Glu Glu Asn 340
345 350Ser Val Leu Glu Ile Ile Ala Phe His Cys Lys
Ser Pro His Arg His 355 360 365Arg
Met Val Val Leu Glu Pro Leu Asn Lys Leu Leu Gln Ala Lys Trp 370
375 380Asp Leu Leu Ile Pro Lys Phe Phe Leu Asn
Phe Leu Cys Asn Leu Ile385 390 395
400Tyr Met Phe Ile Phe Thr Ala Val Ala Tyr His Gln Pro Thr Leu
Lys 405 410 415Lys Gln Ala
Ala Pro His Leu Lys Ala Glu Val Gly Asn Ser Met Leu 420
425 430Leu Thr Gly His Ile Leu Ile Leu Leu Gly
Gly Ile Tyr Leu Leu Val 435 440
445Gly Gln Leu Trp Tyr Phe Trp Arg Arg His Val Phe Ile Trp Ile Ser 450
455 460Phe Ile Asp Ser Tyr Phe Glu Ile
Leu Phe Leu Phe Gln Ala Leu Leu465 470
475 480Thr Val Val Ser Gln Val Leu Cys Phe Leu Ala Ile
Glu Trp Tyr Leu 485 490
495Pro Leu Leu Val Ser Ala Leu Val Leu Gly Trp Leu Asn Leu Leu Tyr
500 505 510Tyr Thr Arg Gly Phe Gln
His Thr Gly Ile Tyr Ser Val Met Ile Gln 515 520
525Lys Val Ile Leu Arg Asp Leu Leu Arg Phe Leu Leu Ile Tyr
Leu Val 530 535 540Phe Leu Phe Gly Phe
Ala Val Ala Leu Val Ser Leu Ser Gln Glu Ala545 550
555 560Trp Arg Pro Glu Ala Pro Thr Gly Pro Asn
Ala Thr Glu Ser Val Gln 565 570
575Pro Met Glu Gly Gln Glu Asp Glu Gly Asn Gly Ala Gln Tyr Arg Gly
580 585 590Ile Leu Glu Ala Ser
Leu Glu Leu Phe Lys Phe Thr Ile Gly Met Gly 595
600 605Glu Leu Ala Phe Gln Glu Gln Leu His Phe Arg Gly
Met Val Leu Leu 610 615 620Leu Leu Leu
Ala Tyr Val Leu Leu Thr Tyr Ile Leu Leu Leu Asn Met625
630 635 640Leu Ile Ala Leu Met Ser Glu
Thr Val Asn Ser Val Ala Thr Asp Ser 645
650 655Trp Ser Ile Trp Lys Leu Gln Lys Ala Ile Ser Val
Leu Glu Met Glu 660 665 670Asn
Gly Tyr Trp Trp Cys Arg Lys Lys Gln Arg Ala Gly Val Met Leu 675
680 685Thr Val Gly Thr Lys Pro Asp Gly Ser
Pro Asp Glu Arg Trp Cys Phe 690 695
700Arg Val Glu Glu Val Asn Trp Ala Ser Trp Glu Gln Thr Leu Pro Thr705
710 715 720Leu Cys Glu Asp
Pro Ser Gly Ala Gly Val Pro Arg Thr Leu Glu Asn 725
730 735Pro Val Leu Ala Ser Pro Pro Lys Glu Asp
Glu Asp Gly Ala Ser Glu 740 745
750Glu Asn Tyr Val Pro Val Gln Leu Leu Gln Ser Asn 755
76032286DNARattus norvegicus 3atgacttcag cctccagccc cccagctttc
aggctggaga cttccgatgg agatgaagag 60ggcaatgctg aggtgaacaa ggggaagcag
gaaccgcccc ccatggagtc accattccag 120agggaggacc ggaattcctc ccctcagatc
aaagtgaacc tcaacttcat aaagagacct 180cctaaaaaca cttctgctcc cagccagcag
gagccagatc ggtttgaccg tgaccgactc 240ttcagtgtgg tctcccgggg tgtccccgag
gaactgactg gactgctaga atacctgcgc 300tggaacagca agtacctcac tgactctgca
tacacagaag gctccactgg aaagacgtgc 360ctgatgaagg ctgtgctgaa ccttcaggat
ggggtcaatg cctgcatcat gccgctgctg 420cagattgaca aggattccgg caatcccaag
cccctcgtca atgcccagtg catcgatgag 480ttctaccaag gccacagtgc gctgcacatc
gccatagaga agaggagcct gcagtgcgtg 540aagctgctgg tagagaatgg agcggatgtt
cacctccgag cctgtggccg cttcttccaa 600aagcaccaag gaacttgttt ctattttgga
gagctacctc tttctctggc tgcgtgcacc 660aagcagtggg atgtggtgac ctacctcctg
gagaacccac accagccggc cagcctggag 720gccaccgact ccctgggcaa cacagtcctg
catgctctgg taatgattgc agataactcg 780cctgagaaca gtgccctggt gatccacatg
tacgacgggc ttctacaaat gggggcgcgc 840ctctgcccca ctgtgcagct tgaggaaatc
tccaaccacc aaggcctcac acccctgaaa 900ctagccgcca aggaaggcaa aatcgagatt
ttcaggcaca ttctgcagcg ggaattctca 960ggaccgtacc agcccctttc ccgaaagttt
actgagtggt gttacggtcc tgtgcgggta 1020tcgctgtacg acctgtcctc tgtggacagc
tgggaaaaga actcggtgct ggagatcatc 1080gcttttcatt gcaagagccc gaaccggcac
cgcatggtgg ttttagaacc actgaacaag 1140cttctgcagg agaaatggga tcggctcgtc
tcaagattct tcttcaactt cgcctgctac 1200ttggtctaca tgttcatctt caccgtcgtt
gcctaccacc agccttccct ggatcagcca 1260gccatcccct catcaaaagc gacttttggg
gaatccatgc tgctgctggg ccacattctg 1320atcctgcttg ggggtattta cctcttactg
ggccagctgt ggtacttttg gcggcggcgc 1380ctgttcatct ggatctcatt catggacagc
tactttgaaa tcctctttct ccttcaggct 1440ctgctcacag tgctgtccca ggtgctgcgc
ttcatggaga ctgaatggta cctacccctg 1500ctagtgttat ccctagtgct gggctggctg
aacctgcttt actacacacg gggctttcag 1560cacacaggca tctacagtgt catgatccag
aaggtcatcc ttcgagacct gctccgtttc 1620ctgctggtct acctggtctt ccttttcggc
tttgctgtag ccctagtaag cttgagcaga 1680gaggcccgaa gtcccaaagc ccctgaagat
aacaactcca cagtgacgga acagcccacg 1740gtgggccagg aggaggagcc agctccatat
cggagcattc tggatgcctc cctagagctg 1800ttcaagttca ccattggtat gggggagctg
gctttccagg aacagctgcg ttttcgtggg 1860gtggtcctgc tgttgctgtt ggcctacgtc
cttctcacct acgtcctgct gctcaacatg 1920ctcattgctc tcatgagcga aactgtcaac
cacgttgctg acaacagctg gagcatctgg 1980aagttgcaga aagccatctc tgtcttggag
atggagaatg gttactggtg gtgccggagg 2040aagaaacatc gtgaagggag gctgctgaaa
gtcggcacca ggggggatgg tacccctgat 2100gagcgctggt gcttcagggt ggaggaagta
aattgggttg cttgggagaa gactcttccc 2160accttatctg aggatccatc agggccaggc
atcactggta ataaaaagaa cccaacctct 2220aaaccgggga agaacagtgc ctcagaggaa
gaccatctgc cccttcaggt cctccagtcc 2280ccctga
22864761PRTRattus norvegicus 4Met Thr
Ser Ala Ser Ser Pro Pro Ala Phe Arg Leu Glu Thr Ser Asp1 5
10 15Gly Asp Glu Glu Gly Asn Ala Glu
Val Asn Lys Gly Lys Gln Glu Pro 20 25
30Pro Pro Met Glu Ser Pro Phe Gln Arg Glu Asp Arg Asn Ser Ser
Pro 35 40 45Gln Ile Lys Val Asn
Leu Asn Phe Ile Lys Arg Pro Pro Lys Asn Thr 50 55
60Ser Ala Pro Ser Gln Gln Glu Pro Asp Arg Phe Asp Arg Asp
Arg Leu65 70 75 80Phe
Ser Val Val Ser Arg Gly Val Pro Glu Glu Leu Thr Gly Leu Leu
85 90 95Glu Tyr Leu Arg Trp Asn Ser
Lys Tyr Leu Thr Asp Ser Ala Tyr Thr 100 105
110Glu Gly Ser Thr Gly Lys Thr Cys Leu Met Lys Ala Val Leu
Asn Leu 115 120 125Gln Asp Gly Val
Asn Ala Cys Ile Met Pro Leu Leu Gln Ile Asp Lys 130
135 140Asp Ser Gly Asn Pro Lys Pro Leu Val Asn Ala Gln
Cys Ile Asp Glu145 150 155
160Phe Tyr Gln Gly His Ser Ala Leu His Ile Ala Ile Glu Lys Arg Ser
165 170 175Leu Gln Cys Val Lys
Leu Leu Val Glu Asn Gly Ala Asp Val His Leu 180
185 190Arg Ala Cys Gly Arg Phe Phe Gln Lys His Gln Gly
Thr Cys Phe Tyr 195 200 205Phe Gly
Glu Leu Pro Leu Ser Leu Ala Ala Cys Thr Lys Gln Trp Asp 210
215 220Val Val Thr Tyr Leu Leu Glu Asn Pro His Gln
Pro Ala Ser Leu Glu225 230 235
240Ala Thr Asp Ser Leu Gly Asn Thr Val Leu His Ala Leu Val Met Ile
245 250 255Ala Asp Asn Ser
Pro Glu Asn Ser Ala Leu Val Ile His Met Tyr Asp 260
265 270Gly Leu Leu Gln Met Gly Ala Arg Leu Cys Pro
Thr Val Gln Leu Glu 275 280 285Glu
Ile Ser Asn His Gln Gly Leu Thr Pro Leu Lys Leu Ala Ala Lys 290
295 300Glu Gly Lys Ile Glu Ile Phe Arg His Ile
Leu Gln Arg Glu Phe Ser305 310 315
320Gly Pro Tyr Gln Pro Leu Ser Arg Lys Phe Thr Glu Trp Cys Tyr
Gly 325 330 335Pro Val Arg
Val Ser Leu Tyr Asp Leu Ser Ser Val Asp Ser Trp Glu 340
345 350Lys Asn Ser Val Leu Glu Ile Ile Ala Phe
His Cys Lys Ser Pro Asn 355 360
365Arg His Arg Met Val Val Leu Glu Pro Leu Asn Lys Leu Leu Gln Glu 370
375 380Lys Trp Asp Arg Leu Val Ser Arg
Phe Phe Phe Asn Phe Ala Cys Tyr385 390
395 400Leu Val Tyr Met Phe Ile Phe Thr Val Val Ala Tyr
His Gln Pro Ser 405 410
415Leu Asp Gln Pro Ala Ile Pro Ser Ser Lys Ala Thr Phe Gly Glu Ser
420 425 430Met Leu Leu Leu Gly His
Ile Leu Ile Leu Leu Gly Gly Ile Tyr Leu 435 440
445Leu Leu Gly Gln Leu Trp Tyr Phe Trp Arg Arg Arg Leu Phe
Ile Trp 450 455 460Ile Ser Phe Met Asp
Ser Tyr Phe Glu Ile Leu Phe Leu Leu Gln Ala465 470
475 480Leu Leu Thr Val Leu Ser Gln Val Leu Arg
Phe Met Glu Thr Glu Trp 485 490
495Tyr Leu Pro Leu Leu Val Leu Ser Leu Val Leu Gly Trp Leu Asn Leu
500 505 510Leu Tyr Tyr Thr Arg
Gly Phe Gln His Thr Gly Ile Tyr Ser Val Met 515
520 525Ile Gln Lys Val Ile Leu Arg Asp Leu Leu Arg Phe
Leu Leu Val Tyr 530 535 540Leu Val Phe
Leu Phe Gly Phe Ala Val Ala Leu Val Ser Leu Ser Arg545
550 555 560Glu Ala Arg Ser Pro Lys Ala
Pro Glu Asp Asn Asn Ser Thr Val Thr 565
570 575Glu Gln Pro Thr Val Gly Gln Glu Glu Glu Pro Ala
Pro Tyr Arg Ser 580 585 590Ile
Leu Asp Ala Ser Leu Glu Leu Phe Lys Phe Thr Ile Gly Met Gly 595
600 605Glu Leu Ala Phe Gln Glu Gln Leu Arg
Phe Arg Gly Val Val Leu Leu 610 615
620Leu Leu Leu Ala Tyr Val Leu Leu Thr Tyr Val Leu Leu Leu Asn Met625
630 635 640Leu Ile Ala Leu
Met Ser Glu Thr Val Asn His Val Ala Asp Asn Ser 645
650 655Trp Ser Ile Trp Lys Leu Gln Lys Ala Ile
Ser Val Leu Glu Met Glu 660 665
670Asn Gly Tyr Trp Trp Cys Arg Arg Lys Lys His Arg Glu Gly Arg Leu
675 680 685Leu Lys Val Gly Thr Arg Gly
Asp Gly Thr Pro Asp Glu Arg Trp Cys 690 695
700Phe Arg Val Glu Glu Val Asn Trp Val Ala Trp Glu Lys Thr Leu
Pro705 710 715 720Thr Leu
Ser Glu Asp Pro Ser Gly Pro Gly Ile Thr Gly Asn Lys Lys
725 730 735Asn Pro Thr Ser Lys Pro Gly
Lys Asn Ser Ala Ser Glu Glu Asp His 740 745
750Leu Pro Leu Gln Val Leu Gln Ser Pro 755
76052271DNAMus musculus 5atgacttcag cctccaaccc cccagctttt aggctggaga
cgtccgatgg agatgaagaa 60ggcagtgctg aggtgaacaa aggaaagaat gagccacccc
ccatggagtc tcccttccag 120ggagaggacc ggaacttctc ccctcagatt aaagtgaatc
tcaactaccg aaagggactg 180ggtcccagcc aacaggaccc aaatcggttt gaccgtgacc
gactcttcag tgtggtctcc 240cggggtgtcc ccgaggagct gactggactg ctagagtacc
tgcgccggac cagcaagtac 300ctcactgact cggcatacac agaaggctcc actggaaaga
cgtgcctgat gaaggctgtg 360ctgaaccttc aggatggggt caatgcctgt atcctgccgc
tgctgcagat tgacagggat 420tccggcaatc ctcagcccct tgtcaatgcc cagtgcaccg
atgagttcta ccgaggccac 480agtgcgctgc acatcgccat agagaagagg agcctgtggt
gcgtgaagct gctggtagag 540aatggagcga atgttcacat ccgagcctgt ggccgcttct
tccaaaagca ccaaggaact 600tgtttctatt ttggagagct acctctttct ctggcagcgt
gcaccaagca gtgggatgtg 660gtgacctacc tcctggagaa cccacaccag cctgccagcc
tggaggccac cgactccctg 720ggcaacacag tcctgcatgc tctggtaatg attgcagaca
actcacctga gaacagtgcg 780ctggtgatcc acatgtatga cagccttctc caaatggggg
cccgcctctg ccccactgta 840cagcttgagg atatctgcaa ccatcaaggc ttaacacccc
tgaagttggc tgccaaggaa 900ggtaaaattg agatcttcag gcacatcctg cagcgggagt
tctcagggct gtaccagccc 960ctttcccgaa agttcaccga gtggtgctac ggtcctgtcc
gagtgtcact gtacgacctg 1020tcctctgtgg acagttggga aaagaactcg gtcctggaaa
tcatcgcttt ccattgcaag 1080agcccgcacc ggcaccgcat ggtggtttta gagccactga
acaagcttct gcaggagaaa 1140tgggatcggc tcatcccaag attcttcttc aacttcgcct
gttacttggt ctacatgatc 1200atcttcacca tagttgccta ccaccagcct tccctggagc
agccagccat tccctcatca 1260aaagcgactt ttggggactc catgctgctg ttgggccaca
ttctgatcct gcttgggggt 1320atttacctct tactgggcca gctgtggtac ttttggcggc
ggcgcctgtt catctggatc 1380tcgttcatgg acagttactt tgaaatcctc ttccttgtcc
aggcgctgct cacagtgctg 1440tcccaggtgc tgcgcttcgt ggagactgaa tggtacctcc
ccctgttagt gtcatcccta 1500gtgctgggct ggctgaacct gctttattat acacgtggct
ttcagcacac aggcatctac 1560agtgtcatga tccaaaaggt cattctgcga gacctgctcc
gcttcctgct ggtctaccta 1620gtcttccttt tcggctttgc tgtagcccta gtaagcttga
gccgggaggc ccgaagtccc 1680aaagcccctg aaaatagcaa caccactgtg acggaaaagc
ccacgctggg tcaggaggag 1740gagccagtcc catatggggg cattctggat gcctccctag
agctgttcaa gttcaccatt 1800ggtatgggtg agctggcttt ccaggaacag ctgcgctttc
gtggggtggt gctgctgttg 1860ctgttggcct acgtcctcct cacctacgtc ctactgctca
acatgctcat tgccctcatg 1920agtgagactg tcaacagcgt tgccactgac agctggagca
tctggaagtt gcagaaagcc 1980atctctgtct tggagatgga gaatggttac tggtggtgca
ggaggaaaag gcatcgcgca 2040gggaggctgc tgaaagttgg caccaaaggg gatggtatac
ctgatgagcg ctggtgcttc 2100agggtggagg aagtaaactg ggctgcatgg gagaagaccc
ttcccacctt atctgaggat 2160ccatcagggg caggcatcac tggttataaa aagaacccaa
cctctaaacc tgggaagaac 2220agtgcctcag aggaagacca tctgcctctt caggtcctcc
agtcccactg a 22716756PRTMus musculus 6Met Thr Ser Ala Ser Asn
Pro Pro Ala Phe Arg Leu Glu Thr Ser Asp1 5
10 15Gly Asp Glu Glu Gly Ser Ala Glu Val Asn Lys Gly
Lys Asn Glu Pro 20 25 30Pro
Pro Met Glu Ser Pro Phe Gln Gly Glu Asp Arg Asn Phe Ser Pro 35
40 45Gln Ile Lys Val Asn Leu Asn Tyr Arg
Lys Gly Leu Gly Pro Ser Gln 50 55
60Gln Asp Pro Asn Arg Phe Asp Arg Asp Arg Leu Phe Ser Val Val Ser65
70 75 80Arg Gly Val Pro Glu
Glu Leu Thr Gly Leu Leu Glu Tyr Leu Arg Arg 85
90 95Thr Ser Lys Tyr Leu Thr Asp Ser Ala Tyr Thr
Glu Gly Ser Thr Gly 100 105
110Lys Thr Cys Leu Met Lys Ala Val Leu Asn Leu Gln Asp Gly Val Asn
115 120 125Ala Cys Ile Leu Pro Leu Leu
Gln Ile Asp Arg Asp Ser Gly Asn Pro 130 135
140Gln Pro Leu Val Asn Ala Gln Cys Thr Asp Glu Phe Tyr Arg Gly
His145 150 155 160Ser Ala
Leu His Ile Ala Ile Glu Lys Arg Ser Leu Trp Cys Val Lys
165 170 175Leu Leu Val Glu Asn Gly Ala
Asn Val His Ile Arg Ala Cys Gly Arg 180 185
190Phe Phe Gln Lys His Gln Gly Thr Cys Phe Tyr Phe Gly Glu
Leu Pro 195 200 205Leu Ser Leu Ala
Ala Cys Thr Lys Gln Trp Asp Val Val Thr Tyr Leu 210
215 220Leu Glu Asn Pro His Gln Pro Ala Ser Leu Glu Ala
Thr Asp Ser Leu225 230 235
240Gly Asn Thr Val Leu His Ala Leu Val Met Ile Ala Asp Asn Ser Pro
245 250 255Glu Asn Ser Ala Leu
Val Ile His Met Tyr Asp Ser Leu Leu Gln Met 260
265 270Gly Ala Arg Leu Cys Pro Thr Val Gln Leu Glu Asp
Ile Cys Asn His 275 280 285Gln Gly
Leu Thr Pro Leu Lys Leu Ala Ala Lys Glu Gly Lys Ile Glu 290
295 300Ile Phe Arg His Ile Leu Gln Arg Glu Phe Ser
Gly Leu Tyr Gln Pro305 310 315
320Leu Ser Arg Lys Phe Thr Glu Trp Cys Tyr Gly Pro Val Arg Val Ser
325 330 335Leu Tyr Asp Leu
Ser Ser Val Asp Ser Trp Glu Lys Asn Ser Val Leu 340
345 350Glu Ile Ile Ala Phe His Cys Lys Ser Pro His
Arg His Arg Met Val 355 360 365Val
Leu Glu Pro Leu Asn Lys Leu Leu Gln Glu Lys Trp Asp Arg Leu 370
375 380Ile Pro Arg Phe Phe Phe Asn Phe Ala Cys
Tyr Leu Val Tyr Met Ile385 390 395
400Ile Phe Thr Ile Val Ala Tyr His Gln Pro Ser Leu Glu Gln Pro
Ala 405 410 415Ile Pro Ser
Ser Lys Ala Thr Phe Gly Asp Ser Met Leu Leu Leu Gly 420
425 430His Ile Leu Ile Leu Leu Gly Gly Ile Tyr
Leu Leu Leu Gly Gln Leu 435 440
445Trp Tyr Phe Trp Arg Arg Arg Leu Phe Ile Trp Ile Ser Phe Met Asp 450
455 460Ser Tyr Phe Glu Ile Leu Phe Leu
Val Gln Ala Leu Leu Thr Val Leu465 470
475 480Ser Gln Val Leu Arg Phe Val Glu Thr Glu Trp Tyr
Leu Pro Leu Leu 485 490
495Val Ser Ser Leu Val Leu Gly Trp Leu Asn Leu Leu Tyr Tyr Thr Arg
500 505 510Gly Phe Gln His Thr Gly
Ile Tyr Ser Val Met Ile Gln Lys Val Ile 515 520
525Leu Arg Asp Leu Leu Arg Phe Leu Leu Val Tyr Leu Val Phe
Leu Phe 530 535 540Gly Phe Ala Val Ala
Leu Val Ser Leu Ser Arg Glu Ala Arg Ser Pro545 550
555 560Lys Ala Pro Glu Asn Ser Asn Thr Thr Val
Thr Glu Lys Pro Thr Leu 565 570
575Gly Gln Glu Glu Glu Pro Val Pro Tyr Gly Gly Ile Leu Asp Ala Ser
580 585 590Leu Glu Leu Phe Lys
Phe Thr Ile Gly Met Gly Glu Leu Ala Phe Gln 595
600 605Glu Gln Leu Arg Phe Arg Gly Val Val Leu Leu Leu
Leu Leu Ala Tyr 610 615 620Val Leu Leu
Thr Tyr Val Leu Leu Leu Asn Met Leu Ile Ala Leu Met625
630 635 640Ser Glu Thr Val Asn Ser Val
Ala Thr Asp Ser Trp Ser Ile Trp Lys 645
650 655Leu Gln Lys Ala Ile Ser Val Leu Glu Met Glu Asn
Gly Tyr Trp Trp 660 665 670Cys
Arg Arg Lys Arg His Arg Ala Gly Arg Leu Leu Lys Val Gly Thr 675
680 685Lys Gly Asp Gly Ile Pro Asp Glu Arg
Trp Cys Phe Arg Val Glu Glu 690 695
700Val Asn Trp Ala Ala Trp Glu Lys Thr Leu Pro Thr Leu Ser Glu Asp705
710 715 720Pro Ser Gly Ala
Gly Ile Thr Gly Tyr Lys Lys Asn Pro Thr Ser Lys 725
730 735Pro Gly Lys Asn Ser Ala Ser Glu Glu Asp
His Leu Pro Leu Gln Val 740 745
750Leu Gln Ser His 7557706PRTRattus norvegicus 7Met Thr Ser Ala
Ser Ser Pro Pro Ala Phe Arg Leu Glu Thr Ser Asp1 5
10 15Gly Asp Glu Glu Gly Asn Ala Glu Val Asn
Lys Gly Lys Gln Glu Pro 20 25
30Pro Pro Met Glu Ser Pro Phe Gln Arg Glu Asp Arg Asn Ser Ser Pro
35 40 45Gln Ile Lys Val Asn Leu Asn Phe
Ile Lys Arg Pro Pro Lys Asn Thr 50 55
60Ser Ala Pro Ser Gln Gln Glu Pro Asp Arg Phe Asp Arg Asp Arg Leu65
70 75 80Phe Ser Val Val Ser
Arg Gly Val Pro Glu Glu Leu Thr Gly Leu Leu 85
90 95Glu Tyr Leu Arg Trp Asn Ser Lys Tyr Leu Thr
Asp Ser Ala Tyr Thr 100 105
110Glu Gly Ser Thr Gly Lys Thr Cys Leu Met Lys Ala Val Leu Asn Leu
115 120 125Gln Asp Gly Val Asn Ala Cys
Ile Met Pro Leu Leu Gln Ile Asp Lys 130 135
140Asp Ser Gly Asn Pro Lys Pro Leu Val Asn Ala Gln Cys Ile Asp
Glu145 150 155 160Phe Tyr
Gln Gly His Ser Ala Leu His Ile Ala Ile Glu Lys Arg Ser
165 170 175Leu Gln Cys Val Lys Leu Leu
Val Glu Asn Gly Ala Asp Val His Leu 180 185
190Arg Ala Cys Gly Arg Phe Phe Gln Lys His Gln Gly Thr Cys
Phe Tyr 195 200 205Phe Gly Glu Leu
Pro Leu Ser Leu Ala Ala Cys Thr Lys Gln Trp Asp 210
215 220Val Val Thr Tyr Leu Leu Glu Asn Pro His Gln Pro
Ala Ser Leu Glu225 230 235
240Ala Thr Asp Ser Leu Gly Asn Thr Val Leu His Ala Leu Val Met Ile
245 250 255Ala Asp Asn Ser Pro
Glu Asn Ser Ala Leu Val Ile His Met Tyr Asp 260
265 270Gly Leu Leu Gln Met Gly Ala Arg Leu Cys Pro Thr
Val Gln Leu Glu 275 280 285Glu Ile
Ser Asn His Gln Gly Leu Thr Pro Leu Lys Leu Ala Ala Lys 290
295 300Glu Gly Lys Ile Glu Ile Phe Arg His Ile Leu
Gln Arg Glu Phe Ser305 310 315
320Gly Pro Tyr Gln Pro Leu Ser Arg Lys Phe Thr Glu Trp Cys Tyr Gly
325 330 335Pro Val Arg Val
Ser Leu Tyr Asp Leu Ser Ser Val Asp Ser Trp Glu 340
345 350Lys Asn Ser Val Leu Glu Ile Ile Ala Phe His
Cys Lys Ser Pro Asn 355 360 365Arg
His Arg Met Val Val Leu Glu Pro Leu Asn Lys Leu Leu Gln Glu 370
375 380Lys Trp Asp Arg Leu Val Ser Arg Phe Phe
Phe Asn Phe Ala Cys Tyr385 390 395
400Leu Val Tyr Met Phe Ile Phe Thr Val Val Ala Tyr His Gln Pro
Ser 405 410 415Leu Asp Gln
Pro Ala Ile Pro Ser Ser Lys Ala Thr Phe Gly Glu Ser 420
425 430Met Leu Leu Leu Gly His Ile Leu Ile Leu
Leu Gly Gly Ile Tyr Leu 435 440
445Leu Leu Gly Gln Leu Trp Tyr Phe Trp Arg Arg Arg Leu Phe Ile Trp 450
455 460Ile Ser Phe Met Asp Ser Tyr Phe
Glu Ile Leu Phe Leu Leu Gln Ala465 470
475 480Leu Leu Thr Val Leu Ser Gln Val Leu Arg Phe Met
Glu Thr Glu Trp 485 490
495Tyr Leu Pro Leu Leu Val Leu Ser Leu Val Leu Gly Trp Leu Asn Leu
500 505 510Leu Tyr Tyr Thr Arg Gly
Phe Gln His Thr Gly Ile Tyr Ser Val Met 515 520
525Ile Gln Lys Val Ile Leu Arg Asp Leu Leu Arg Phe Leu Leu
Val Tyr 530 535 540Leu Val Phe Leu Phe
Gly Phe Ala Val Ala Leu Val Ser Leu Ser Arg545 550
555 560Glu Ala Arg Ser Pro Lys Ala Pro Glu Asp
Asn Asn Ser Thr Val Thr 565 570
575Glu Gln Pro Thr Val Gly Gln Glu Glu Glu Pro Ala Pro Tyr Arg Ser
580 585 590Ile Leu Asp Ala Ser
Leu Glu Leu Phe Lys Phe Thr Ile Gly Met Gly 595
600 605Glu Leu Ala Phe Gln Glu Gln Leu Arg Phe Arg Gly
Val Val Leu Leu 610 615 620Leu Leu Leu
Ala Tyr Val Leu Leu Thr Tyr Val Leu Leu Leu Asn Met625
630 635 640Leu Ile Ala Leu Met Ser Glu
Thr Val Asn His Val Ala Asp Asn Ser 645
650 655Trp Ser Ile Trp Lys Leu Gln Lys Ala Ile Ser Val
Leu Glu Met Glu 660 665 670Asn
Gly Tyr Trp Trp Cys Arg Arg Lys Lys His Arg Glu Gly Arg Leu 675
680 685Leu Lys Val Gly Thr Arg Gly Asp Gly
Thr Pro Asp Glu Arg Trp Cys 690 695
700Phe Arg7058729PRTRattus norvegicus 8Met Thr Ser Ala Ser Ser Pro Pro
Ala Phe Arg Leu Glu Thr Ser Asp1 5 10
15Gly Asp Glu Glu Gly Asn Ala Glu Val Asn Lys Gly Lys Gln
Glu Pro 20 25 30Pro Pro Met
Glu Ser Pro Phe Gln Arg Glu Asp Arg Asn Ser Ser Pro 35
40 45Gln Ile Lys Val Asn Leu Asn Phe Ile Lys Arg
Pro Pro Lys Asn Thr 50 55 60Ser Ala
Pro Ser Gln Gln Glu Pro Asp Arg Phe Asp Arg Asp Arg Leu65
70 75 80Phe Ser Val Val Ser Arg Gly
Val Pro Glu Glu Leu Thr Gly Leu Leu 85 90
95Glu Tyr Leu Arg Trp Asn Ser Lys Tyr Leu Thr Asp Ser
Ala Tyr Thr 100 105 110Glu Gly
Ser Thr Gly Lys Thr Cys Leu Met Lys Ala Val Leu Asn Leu 115
120 125Gln Asp Gly Val Asn Ala Cys Ile Met Pro
Leu Leu Gln Ile Asp Lys 130 135 140Asp
Ser Gly Asn Pro Lys Pro Leu Val Asn Ala Gln Cys Ile Asp Glu145
150 155 160Phe Tyr Gln Gly His Ser
Ala Leu His Ile Ala Ile Glu Lys Arg Ser 165
170 175Leu Gln Cys Val Lys Leu Leu Val Glu Asn Gly Ala
Asp Val His Leu 180 185 190Arg
Ala Cys Gly Arg Phe Phe Gln Lys His Gln Gly Thr Cys Phe Tyr 195
200 205Phe Gly Glu Leu Pro Leu Ser Leu Ala
Ala Cys Thr Lys Gln Trp Asp 210 215
220Val Val Thr Tyr Leu Leu Glu Asn Pro His Gln Pro Ala Ser Leu Glu225
230 235 240Ala Thr Asp Ser
Leu Gly Asn Thr Val Leu His Ala Leu Val Met Ile 245
250 255Ala Asp Asn Ser Pro Glu Asn Ser Ala Leu
Val Ile His Met Tyr Asp 260 265
270Gly Leu Leu Gln Met Gly Ala Arg Leu Cys Pro Thr Val Gln Leu Glu
275 280 285Glu Ile Ser Asn His Gln Gly
Leu Thr Pro Leu Lys Leu Ala Ala Lys 290 295
300Glu Gly Lys Ile Glu Ile Phe Arg His Ile Leu Gln Arg Glu Phe
Ser305 310 315 320Gly Pro
Tyr Gln Pro Leu Ser Arg Lys Phe Thr Glu Trp Cys Tyr Gly
325 330 335Pro Val Arg Val Ser Leu Tyr
Asp Leu Ser Ser Val Asp Ser Trp Glu 340 345
350Lys Asn Ser Val Leu Glu Ile Ile Ala Phe His Cys Lys Ser
Pro Asn 355 360 365Arg His Arg Met
Val Val Leu Glu Pro Leu Asn Lys Leu Leu Gln Glu 370
375 380Lys Trp Asp Arg Leu Val Ser Arg Phe Phe Phe Asn
Phe Ala Cys Tyr385 390 395
400Leu Val Tyr Met Phe Ile Phe Thr Val Val Ala Tyr His Gln Pro Ser
405 410 415Leu Asp Gln Pro Ala
Ile Pro Ser Ser Lys Ala Thr Phe Gly Glu Ser 420
425 430Met Leu Leu Leu Gly His Ile Leu Ile Leu Leu Gly
Gly Ile Tyr Leu 435 440 445Leu Leu
Gly Gln Leu Trp Tyr Phe Trp Arg Arg Arg Leu Phe Ile Trp 450
455 460Ile Ser Phe Met Asp Ser Tyr Phe Glu Ile Leu
Phe Leu Leu Gln Ala465 470 475
480Leu Leu Thr Val Leu Ser Gln Val Leu Arg Phe Met Glu Thr Glu Trp
485 490 495Tyr Leu Pro Leu
Leu Val Leu Ser Leu Val Leu Gly Trp Leu Asn Leu 500
505 510Leu Tyr Tyr Thr Arg Gly Phe Gln His Thr Gly
Ile Tyr Ser Val Met 515 520 525Ile
Gln Lys Val Ile Leu Arg Asp Leu Leu Arg Phe Leu Leu Val Tyr 530
535 540Leu Val Phe Leu Phe Gly Phe Ala Val Ala
Leu Val Ser Leu Ser Arg545 550 555
560Glu Ala Arg Ser Pro Lys Ala Pro Glu Asp Asn Asn Ser Thr Val
Thr 565 570 575Glu Gln Pro
Thr Val Gly Gln Glu Glu Glu Pro Ala Pro Tyr Arg Ser 580
585 590Ile Leu Asp Ala Ser Leu Glu Leu Phe Lys
Phe Thr Ile Gly Met Gly 595 600
605Glu Leu Ala Phe Gln Glu Gln Leu Arg Phe Arg Gly Val Val Leu Leu 610
615 620Leu Leu Leu Ala Tyr Val Leu Leu
Thr Tyr Val Leu Leu Leu Asn Met625 630
635 640Leu Ile Ala Leu Met Ser Glu Thr Val Asn His Val
Ala Asp Asn Ser 645 650
655Trp Ser Ile Trp Lys Leu Gln Lys Ala Ile Ser Val Leu Glu Met Glu
660 665 670Asn Gly Tyr Trp Trp Cys
Arg Arg Lys Lys His Arg Glu Gly Arg Leu 675 680
685Leu Lys Val Gly Thr Arg Gly Asp Gly Thr Pro Asp Glu Arg
Trp Cys 690 695 700Phe Arg Val Glu Glu
Val Asn Trp Val Ala Trp Glu Lys Thr Leu Pro705 710
715 720Thr Leu Ser Glu Asp Pro Ser Gly Pro
7259738PRTRattus norvegicus 9Met Thr Ser Ala Ser Ser Pro Pro Ala
Phe Arg Leu Glu Thr Ser Asp1 5 10
15Gly Asp Glu Glu Gly Asn Ala Glu Val Asn Lys Gly Lys Gln Glu
Pro 20 25 30Pro Pro Met Glu
Ser Pro Phe Gln Arg Glu Asp Arg Asn Ser Ser Pro 35
40 45Gln Ile Lys Val Asn Leu Asn Phe Ile Lys Arg Pro
Pro Lys Asn Thr 50 55 60Ser Ala Pro
Ser Gln Gln Glu Pro Asp Arg Phe Asp Arg Asp Arg Leu65 70
75 80Phe Ser Val Val Ser Arg Gly Val
Pro Glu Glu Leu Thr Gly Leu Leu 85 90
95Glu Tyr Leu Arg Trp Asn Ser Lys Tyr Leu Thr Asp Ser Ala
Tyr Thr 100 105 110Glu Gly Ser
Thr Gly Lys Thr Cys Leu Met Lys Ala Val Leu Asn Leu 115
120 125Gln Asp Gly Val Asn Ala Cys Ile Met Pro Leu
Leu Gln Ile Asp Lys 130 135 140Asp Ser
Gly Asn Pro Lys Pro Leu Val Asn Ala Gln Cys Ile Asp Glu145
150 155 160Phe Tyr Gln Gly His Ser Ala
Leu His Ile Ala Ile Glu Lys Arg Ser 165
170 175Leu Gln Cys Val Lys Leu Leu Val Glu Asn Gly Ala
Asp Val His Leu 180 185 190Arg
Ala Cys Gly Arg Phe Phe Gln Lys His Gln Gly Thr Cys Phe Tyr 195
200 205Phe Gly Glu Leu Pro Leu Ser Leu Ala
Ala Cys Thr Lys Gln Trp Asp 210 215
220Val Val Thr Tyr Leu Leu Glu Asn Pro His Gln Pro Ala Ser Leu Glu225
230 235 240Ala Thr Asp Ser
Leu Gly Asn Thr Val Leu His Ala Leu Val Met Ile 245
250 255Ala Asp Asn Ser Pro Glu Asn Ser Ala Leu
Val Ile His Met Tyr Asp 260 265
270Gly Leu Leu Gln Met Gly Ala Arg Leu Cys Pro Thr Val Gln Leu Glu
275 280 285Glu Ile Ser Asn His Gln Gly
Leu Thr Pro Leu Lys Leu Ala Ala Lys 290 295
300Glu Gly Lys Ile Glu Ile Phe Arg His Ile Leu Gln Arg Glu Phe
Ser305 310 315 320Gly Pro
Tyr Gln Pro Leu Ser Arg Lys Phe Thr Glu Trp Cys Tyr Gly
325 330 335Pro Val Arg Val Ser Leu Tyr
Asp Leu Ser Ser Val Asp Ser Trp Glu 340 345
350Lys Asn Ser Val Leu Glu Ile Ile Ala Phe His Cys Lys Ser
Pro Asn 355 360 365Arg His Arg Met
Val Val Leu Glu Pro Leu Asn Lys Leu Leu Gln Glu 370
375 380Lys Trp Asp Arg Leu Val Ser Arg Phe Phe Phe Asn
Phe Ala Cys Tyr385 390 395
400Leu Val Tyr Met Phe Ile Phe Thr Val Val Ala Tyr His Gln Pro Ser
405 410 415Leu Asp Gln Pro Ala
Ile Pro Ser Ser Lys Ala Thr Phe Gly Glu Ser 420
425 430Met Leu Leu Leu Gly His Ile Leu Ile Leu Leu Gly
Gly Ile Tyr Leu 435 440 445Leu Leu
Gly Gln Leu Trp Tyr Phe Trp Arg Arg Arg Leu Phe Ile Trp 450
455 460Ile Ser Phe Met Asp Ser Tyr Phe Glu Ile Leu
Phe Leu Leu Gln Ala465 470 475
480Leu Leu Thr Val Leu Ser Gln Val Leu Arg Phe Met Glu Thr Glu Trp
485 490 495Tyr Leu Pro Leu
Leu Val Leu Ser Leu Val Leu Gly Trp Leu Asn Leu 500
505 510Leu Tyr Tyr Thr Arg Gly Phe Gln His Thr Gly
Ile Tyr Ser Val Met 515 520 525Ile
Gln Lys Val Ile Leu Arg Asp Leu Leu Arg Phe Leu Leu Val Tyr 530
535 540Leu Val Phe Leu Phe Gly Phe Ala Val Ala
Leu Val Ser Leu Ser Arg545 550 555
560Glu Ala Arg Ser Pro Lys Ala Pro Glu Asp Asn Asn Ser Thr Val
Thr 565 570 575Glu Gln Pro
Thr Val Gly Gln Glu Glu Glu Pro Ala Pro Tyr Arg Ser 580
585 590Ile Leu Asp Ala Ser Leu Glu Leu Phe Lys
Phe Thr Ile Gly Met Gly 595 600
605Glu Leu Ala Phe Gln Glu Gln Leu Arg Phe Arg Gly Val Val Leu Leu 610
615 620Leu Leu Leu Ala Tyr Val Leu Leu
Thr Tyr Val Leu Leu Leu Asn Met625 630
635 640Leu Ile Ala Leu Met Ser Glu Thr Val Asn His Val
Ala Asp Asn Ser 645 650
655Trp Ser Ile Trp Lys Leu Gln Lys Ala Ile Ser Val Leu Glu Met Glu
660 665 670Asn Gly Tyr Trp Trp Cys
Arg Arg Lys Lys His Arg Glu Gly Arg Leu 675 680
685Leu Lys Val Gly Thr Arg Gly Asp Gly Thr Pro Asp Glu Arg
Trp Cys 690 695 700Phe Arg Val Glu Glu
Val Asn Trp Val Ala Trp Glu Lys Thr Leu Pro705 710
715 720Thr Leu Ser Glu Asp Pro Ser Gly Pro Gly
Ile Thr Gly Asn Lys Lys 725 730
735Asn Pro10750PRTRattus norvegicus 10Met Thr Ser Ala Ser Ser Pro
Pro Ala Phe Arg Leu Glu Thr Ser Asp1 5 10
15Gly Asp Glu Glu Gly Asn Ala Glu Val Asn Lys Gly Lys
Gln Glu Pro 20 25 30Pro Pro
Met Glu Ser Pro Phe Gln Arg Glu Asp Arg Asn Ser Ser Pro 35
40 45Gln Ile Lys Val Asn Leu Asn Phe Ile Lys
Arg Pro Pro Lys Asn Thr 50 55 60Ser
Ala Pro Ser Gln Gln Glu Pro Asp Arg Phe Asp Arg Asp Arg Leu65
70 75 80Phe Ser Val Val Ser Arg
Gly Val Pro Glu Glu Leu Thr Gly Leu Leu 85
90 95Glu Tyr Leu Arg Trp Asn Ser Lys Tyr Leu Thr Asp
Ser Ala Tyr Thr 100 105 110Glu
Gly Ser Thr Gly Lys Thr Cys Leu Met Lys Ala Val Leu Asn Leu 115
120 125Gln Asp Gly Val Asn Ala Cys Ile Met
Pro Leu Leu Gln Ile Asp Lys 130 135
140Asp Ser Gly Asn Pro Lys Pro Leu Val Asn Ala Gln Cys Ile Asp Glu145
150 155 160Phe Tyr Gln Gly
His Ser Ala Leu His Ile Ala Ile Glu Lys Arg Ser 165
170 175Leu Gln Cys Val Lys Leu Leu Val Glu Asn
Gly Ala Asp Val His Leu 180 185
190Arg Ala Cys Gly Arg Phe Phe Gln Lys His Gln Gly Thr Cys Phe Tyr
195 200 205Phe Gly Glu Leu Pro Leu Ser
Leu Ala Ala Cys Thr Lys Gln Trp Asp 210 215
220Val Val Thr Tyr Leu Leu Glu Asn Pro His Gln Pro Ala Ser Leu
Glu225 230 235 240Ala Thr
Asp Ser Leu Gly Asn Thr Val Leu His Ala Leu Val Met Ile
245 250 255Ala Asp Asn Ser Pro Glu Asn
Ser Ala Leu Val Ile His Met Tyr Asp 260 265
270Gly Leu Leu Gln Met Gly Ala Arg Leu Cys Pro Thr Val Gln
Leu Glu 275 280 285Glu Ile Ser Asn
His Gln Gly Leu Thr Pro Leu Lys Leu Ala Ala Lys 290
295 300Glu Gly Lys Ile Glu Ile Phe Arg His Ile Leu Gln
Arg Glu Phe Ser305 310 315
320Gly Pro Tyr Gln Pro Leu Ser Arg Lys Phe Thr Glu Trp Cys Tyr Gly
325 330 335Pro Val Arg Val Ser
Leu Tyr Asp Leu Ser Ser Val Asp Ser Trp Glu 340
345 350Lys Asn Ser Val Leu Glu Ile Ile Ala Phe His Cys
Lys Ser Pro Asn 355 360 365Arg His
Arg Met Val Val Leu Glu Pro Leu Asn Lys Leu Leu Gln Glu 370
375 380Lys Trp Asp Arg Leu Val Ser Arg Phe Phe Phe
Asn Phe Ala Cys Tyr385 390 395
400Leu Val Tyr Met Phe Ile Phe Thr Val Val Ala Tyr His Gln Pro Ser
405 410 415Leu Asp Gln Pro
Ala Ile Pro Ser Ser Lys Ala Thr Phe Gly Glu Ser 420
425 430Met Leu Leu Leu Gly His Ile Leu Ile Leu Leu
Gly Gly Ile Tyr Leu 435 440 445Leu
Leu Gly Gln Leu Trp Tyr Phe Trp Arg Arg Arg Leu Phe Ile Trp 450
455 460Ile Ser Phe Met Asp Ser Tyr Phe Glu Ile
Leu Phe Leu Leu Gln Ala465 470 475
480Leu Leu Thr Val Leu Ser Gln Val Leu Arg Phe Met Glu Thr Glu
Trp 485 490 495Tyr Leu Pro
Leu Leu Val Leu Ser Leu Val Leu Gly Trp Leu Asn Leu 500
505 510Leu Tyr Tyr Thr Arg Gly Phe Gln His Thr
Gly Ile Tyr Ser Val Met 515 520
525Ile Gln Lys Val Ile Leu Arg Asp Leu Leu Arg Phe Leu Leu Val Tyr 530
535 540Leu Val Phe Leu Phe Gly Phe Ala
Val Ala Leu Val Ser Leu Ser Arg545 550
555 560Glu Ala Arg Ser Pro Lys Ala Pro Glu Asp Asn Asn
Ser Thr Val Thr 565 570
575Glu Gln Pro Thr Val Gly Gln Glu Glu Glu Pro Ala Pro Tyr Arg Ser
580 585 590Ile Leu Asp Ala Ser Leu
Glu Leu Phe Lys Phe Thr Ile Gly Met Gly 595 600
605Glu Leu Ala Phe Gln Glu Gln Leu Arg Phe Arg Gly Val Val
Leu Leu 610 615 620Leu Leu Leu Ala Tyr
Val Leu Leu Thr Tyr Val Leu Leu Leu Asn Met625 630
635 640Leu Ile Ala Leu Met Ser Glu Thr Val Asn
His Val Ala Asp Asn Ser 645 650
655Trp Ser Ile Trp Lys Leu Gln Lys Ala Ile Ser Val Leu Glu Met Glu
660 665 670Asn Gly Tyr Trp Trp
Cys Arg Arg Lys Lys His Arg Glu Gly Arg Leu 675
680 685Leu Lys Val Gly Thr Arg Gly Asp Gly Thr Pro Asp
Glu Arg Trp Cys 690 695 700Phe Arg Val
Glu Glu Val Asn Trp Val Ala Trp Glu Lys Thr Leu Pro705
710 715 720Thr Leu Ser Glu Asp Pro Ser
Gly Pro Gly Ile Thr Gly Asn Lys Lys 725
730 735Asn Pro Thr Ser Lys Pro Gly Lys Asn Ser Ala Ser
Glu Glu 740 745
75011741PRTRattus norvegicus 11Gly Asn Ala Glu Val Asn Lys Gly Lys Gln
Glu Pro Pro Pro Met Glu1 5 10
15Ser Pro Phe Gln Arg Glu Asp Arg Asn Ser Ser Pro Gln Ile Lys Val
20 25 30Asn Leu Asn Phe Ile Lys
Arg Pro Pro Lys Asn Thr Ser Ala Pro Ser 35 40
45Gln Gln Glu Pro Asp Arg Phe Asp Arg Asp Arg Leu Phe Ser
Val Val 50 55 60Ser Arg Gly Val Pro
Glu Glu Leu Thr Gly Leu Leu Glu Tyr Leu Arg65 70
75 80Trp Asn Ser Lys Tyr Leu Thr Asp Ser Ala
Tyr Thr Glu Gly Ser Thr 85 90
95Gly Lys Thr Cys Leu Met Lys Ala Val Leu Asn Leu Gln Asp Gly Val
100 105 110Asn Ala Cys Ile Met
Pro Leu Leu Gln Ile Asp Lys Asp Ser Gly Asn 115
120 125Pro Lys Pro Leu Val Asn Ala Gln Cys Ile Asp Glu
Phe Tyr Gln Gly 130 135 140His Ser Ala
Leu His Ile Ala Ile Glu Lys Arg Ser Leu Gln Cys Val145
150 155 160Lys Leu Leu Val Glu Asn Gly
Ala Asp Val His Leu Arg Ala Cys Gly 165
170 175Arg Phe Phe Gln Lys His Gln Gly Thr Cys Phe Tyr
Phe Gly Glu Leu 180 185 190Pro
Leu Ser Leu Ala Ala Cys Thr Lys Gln Trp Asp Val Val Thr Tyr 195
200 205Leu Leu Glu Asn Pro His Gln Pro Ala
Ser Leu Glu Ala Thr Asp Ser 210 215
220Leu Gly Asn Thr Val Leu His Ala Leu Val Met Ile Ala Asp Asn Ser225
230 235 240Pro Glu Asn Ser
Ala Leu Val Ile His Met Tyr Asp Gly Leu Leu Gln 245
250 255Met Gly Ala Arg Leu Cys Pro Thr Val Gln
Leu Glu Glu Ile Ser Asn 260 265
270His Gln Gly Leu Thr Pro Leu Lys Leu Ala Ala Lys Glu Gly Lys Ile
275 280 285Glu Ile Phe Arg His Ile Leu
Gln Arg Glu Phe Ser Gly Pro Tyr Gln 290 295
300Pro Leu Ser Arg Lys Phe Thr Glu Trp Cys Tyr Gly Pro Val Arg
Val305 310 315 320Ser Leu
Tyr Asp Leu Ser Ser Val Asp Ser Trp Glu Lys Asn Ser Val
325 330 335Leu Glu Ile Ile Ala Phe His
Cys Lys Ser Pro Asn Arg His Arg Met 340 345
350Val Val Leu Glu Pro Leu Asn Lys Leu Leu Gln Glu Lys Trp
Asp Arg 355 360 365Leu Val Ser Arg
Phe Phe Phe Asn Phe Ala Cys Tyr Leu Val Tyr Met 370
375 380Phe Ile Phe Thr Val Val Ala Tyr His Gln Pro Ser
Leu Asp Gln Pro385 390 395
400Ala Ile Pro Ser Ser Lys Ala Thr Phe Gly Glu Ser Met Leu Leu Leu
405 410 415Gly His Ile Leu Ile
Leu Leu Gly Gly Ile Tyr Leu Leu Leu Gly Gln 420
425 430Leu Trp Tyr Phe Trp Arg Arg Arg Leu Phe Ile Trp
Ile Ser Phe Met 435 440 445Asp Ser
Tyr Phe Glu Ile Leu Phe Leu Leu Gln Ala Leu Leu Thr Val 450
455 460Leu Ser Gln Val Leu Arg Phe Met Glu Thr Glu
Trp Tyr Leu Pro Leu465 470 475
480Leu Val Leu Ser Leu Val Leu Gly Trp Leu Asn Leu Leu Tyr Tyr Thr
485 490 495Arg Gly Phe Gln
His Thr Gly Ile Tyr Ser Val Met Ile Gln Lys Val 500
505 510Ile Leu Arg Asp Leu Leu Arg Phe Leu Leu Val
Tyr Leu Val Phe Leu 515 520 525Phe
Gly Phe Ala Val Ala Leu Val Ser Leu Ser Arg Glu Ala Arg Ser 530
535 540Pro Lys Ala Pro Glu Asp Asn Asn Ser Thr
Val Thr Glu Gln Pro Thr545 550 555
560Val Gly Gln Glu Glu Glu Pro Ala Pro Tyr Arg Ser Ile Leu Asp
Ala 565 570 575Ser Leu Glu
Leu Phe Lys Phe Thr Ile Gly Met Gly Glu Leu Ala Phe 580
585 590Gln Glu Gln Leu Arg Phe Arg Gly Val Val
Leu Leu Leu Leu Leu Ala 595 600
605Tyr Val Leu Leu Thr Tyr Val Leu Leu Leu Asn Met Leu Ile Ala Leu 610
615 620Met Ser Glu Thr Val Asn His Val
Ala Asp Asn Ser Trp Ser Ile Trp625 630
635 640Lys Leu Gln Lys Ala Ile Ser Val Leu Glu Met Glu
Asn Gly Tyr Trp 645 650
655Trp Cys Arg Arg Lys Lys His Arg Glu Gly Arg Leu Leu Lys Val Gly
660 665 670Thr Arg Gly Asp Gly Thr
Pro Asp Glu Arg Trp Cys Phe Arg Val Glu 675 680
685Glu Val Asn Trp Val Ala Trp Glu Lys Thr Leu Pro Thr Leu
Ser Glu 690 695 700Asp Pro Ser Gly Pro
Gly Ile Thr Gly Asn Lys Lys Asn Pro Thr Ser705 710
715 720Lys Pro Gly Lys Asn Ser Ala Ser Glu Glu
Asp His Leu Pro Leu Gln 725 730
735Val Leu Gln Ser Pro 74012728PRTRattus norvegicus 12Pro
Pro Met Glu Ser Pro Phe Gln Arg Glu Asp Arg Asn Ser Ser Pro1
5 10 15Gln Ile Lys Val Asn Leu Asn
Phe Ile Lys Arg Pro Pro Lys Asn Thr 20 25
30Ser Ala Pro Ser Gln Gln Glu Pro Asp Arg Phe Asp Arg Asp
Arg Leu 35 40 45Phe Ser Val Val
Ser Arg Gly Val Pro Glu Glu Leu Thr Gly Leu Leu 50 55
60Glu Tyr Leu Arg Trp Asn Ser Lys Tyr Leu Thr Asp Ser
Ala Tyr Thr65 70 75
80Glu Gly Ser Thr Gly Lys Thr Cys Leu Met Lys Ala Val Leu Asn Leu
85 90 95Gln Asp Gly Val Asn Ala
Cys Ile Met Pro Leu Leu Gln Ile Asp Lys 100
105 110Asp Ser Gly Asn Pro Lys Pro Leu Val Asn Ala Gln
Cys Ile Asp Glu 115 120 125Phe Tyr
Gln Gly His Ser Ala Leu His Ile Ala Ile Glu Lys Arg Ser 130
135 140Leu Gln Cys Val Lys Leu Leu Val Glu Asn Gly
Ala Asp Val His Leu145 150 155
160Arg Ala Cys Gly Arg Phe Phe Gln Lys His Gln Gly Thr Cys Phe Tyr
165 170 175Phe Gly Glu Leu
Pro Leu Ser Leu Ala Ala Cys Thr Lys Gln Trp Asp 180
185 190Val Val Thr Tyr Leu Leu Glu Asn Pro His Gln
Pro Ala Ser Leu Glu 195 200 205Ala
Thr Asp Ser Leu Gly Asn Thr Val Leu His Ala Leu Val Met Ile 210
215 220Ala Asp Asn Ser Pro Glu Asn Ser Ala Leu
Val Ile His Met Tyr Asp225 230 235
240Gly Leu Leu Gln Met Gly Ala Arg Leu Cys Pro Thr Val Gln Leu
Glu 245 250 255Glu Ile Ser
Asn His Gln Gly Leu Thr Pro Leu Lys Leu Ala Ala Lys 260
265 270Glu Gly Lys Ile Glu Ile Phe Arg His Ile
Leu Gln Arg Glu Phe Ser 275 280
285Gly Pro Tyr Gln Pro Leu Ser Arg Lys Phe Thr Glu Trp Cys Tyr Gly 290
295 300Pro Val Arg Val Ser Leu Tyr Asp
Leu Ser Ser Val Asp Ser Trp Glu305 310
315 320Lys Asn Ser Val Leu Glu Ile Ile Ala Phe His Cys
Lys Ser Pro Asn 325 330
335Arg His Arg Met Val Val Leu Glu Pro Leu Asn Lys Leu Leu Gln Glu
340 345 350Lys Trp Asp Arg Leu Val
Ser Arg Phe Phe Phe Asn Phe Ala Cys Tyr 355 360
365Leu Val Tyr Met Phe Ile Phe Thr Val Val Ala Tyr His Gln
Pro Ser 370 375 380Leu Asp Gln Pro Ala
Ile Pro Ser Ser Lys Ala Thr Phe Gly Glu Ser385 390
395 400Met Leu Leu Leu Gly His Ile Leu Ile Leu
Leu Gly Gly Ile Tyr Leu 405 410
415Leu Leu Gly Gln Leu Trp Tyr Phe Trp Arg Arg Arg Leu Phe Ile Trp
420 425 430Ile Ser Phe Met Asp
Ser Tyr Phe Glu Ile Leu Phe Leu Leu Gln Ala 435
440 445Leu Leu Thr Val Leu Ser Gln Val Leu Arg Phe Met
Glu Thr Glu Trp 450 455 460Tyr Leu Pro
Leu Leu Val Leu Ser Leu Val Leu Gly Trp Leu Asn Leu465
470 475 480Leu Tyr Tyr Thr Arg Gly Phe
Gln His Thr Gly Ile Tyr Ser Val Met 485
490 495Ile Gln Lys Val Ile Leu Arg Asp Leu Leu Arg Phe
Leu Leu Val Tyr 500 505 510Leu
Val Phe Leu Phe Gly Phe Ala Val Ala Leu Val Ser Leu Ser Arg 515
520 525Glu Ala Arg Ser Pro Lys Ala Pro Glu
Asp Asn Asn Ser Thr Val Thr 530 535
540Glu Gln Pro Thr Val Gly Gln Glu Glu Glu Pro Ala Pro Tyr Arg Ser545
550 555 560Ile Leu Asp Ala
Ser Leu Glu Leu Phe Lys Phe Thr Ile Gly Met Gly 565
570 575Glu Leu Ala Phe Gln Glu Gln Leu Arg Phe
Arg Gly Val Val Leu Leu 580 585
590Leu Leu Leu Ala Tyr Val Leu Leu Thr Tyr Val Leu Leu Leu Asn Met
595 600 605Leu Ile Ala Leu Met Ser Glu
Thr Val Asn His Val Ala Asp Asn Ser 610 615
620Trp Ser Ile Trp Lys Leu Gln Lys Ala Ile Ser Val Leu Glu Met
Glu625 630 635 640Asn Gly
Tyr Trp Trp Cys Arg Arg Lys Lys His Arg Glu Gly Arg Leu
645 650 655Leu Lys Val Gly Thr Arg Gly
Asp Gly Thr Pro Asp Glu Arg Trp Cys 660 665
670Phe Arg Val Glu Glu Val Asn Trp Val Ala Trp Glu Lys Thr
Leu Pro 675 680 685Thr Leu Ser Glu
Asp Pro Ser Gly Pro Gly Ile Thr Gly Asn Lys Lys 690
695 700Asn Pro Thr Ser Lys Pro Gly Lys Asn Ser Ala Ser
Glu Glu Asp His705 710 715
720Leu Pro Leu Gln Val Leu Gln Ser 72513696PRTRattus
norvegicus 13Ala Pro Ser Gln Gln Glu Pro Asp Arg Phe Asp Arg Asp Arg Leu
Phe1 5 10 15Ser Val Val
Ser Arg Gly Val Pro Glu Glu Leu Thr Gly Leu Leu Glu 20
25 30Tyr Leu Arg Trp Asn Ser Lys Tyr Leu Thr
Asp Ser Ala Tyr Thr Glu 35 40
45Gly Ser Thr Gly Lys Thr Cys Leu Met Lys Ala Val Leu Asn Leu Gln 50
55 60Asp Gly Val Asn Ala Cys Ile Met Pro
Leu Leu Gln Ile Asp Lys Asp65 70 75
80Ser Gly Asn Pro Lys Pro Leu Val Asn Ala Gln Cys Ile Asp
Glu Phe 85 90 95Tyr Gln
Gly His Ser Ala Leu His Ile Ala Ile Glu Lys Arg Ser Leu 100
105 110Gln Cys Val Lys Leu Leu Val Glu Asn
Gly Ala Asp Val His Leu Arg 115 120
125Ala Cys Gly Arg Phe Phe Gln Lys His Gln Gly Thr Cys Phe Tyr Phe
130 135 140Gly Glu Leu Pro Leu Ser Leu
Ala Ala Cys Thr Lys Gln Trp Asp Val145 150
155 160Val Thr Tyr Leu Leu Glu Asn Pro His Gln Pro Ala
Ser Leu Glu Ala 165 170
175Thr Asp Ser Leu Gly Asn Thr Val Leu His Ala Leu Val Met Ile Ala
180 185 190Asp Asn Ser Pro Glu Asn
Ser Ala Leu Val Ile His Met Tyr Asp Gly 195 200
205Leu Leu Gln Met Gly Ala Arg Leu Cys Pro Thr Val Gln Leu
Glu Glu 210 215 220Ile Ser Asn His Gln
Gly Leu Thr Pro Leu Lys Leu Ala Ala Lys Glu225 230
235 240Gly Lys Ile Glu Ile Phe Arg His Ile Leu
Gln Arg Glu Phe Ser Gly 245 250
255Pro Tyr Gln Pro Leu Ser Arg Lys Phe Thr Glu Trp Cys Tyr Gly Pro
260 265 270Val Arg Val Ser Leu
Tyr Asp Leu Ser Ser Val Asp Ser Trp Glu Lys 275
280 285Asn Ser Val Leu Glu Ile Ile Ala Phe His Cys Lys
Ser Pro Asn Arg 290 295 300His Arg Met
Val Val Leu Glu Pro Leu Asn Lys Leu Leu Gln Glu Lys305
310 315 320Trp Asp Arg Leu Val Ser Arg
Phe Phe Phe Asn Phe Ala Cys Tyr Leu 325
330 335Val Tyr Met Phe Ile Phe Thr Val Val Ala Tyr His
Gln Pro Ser Leu 340 345 350Asp
Gln Pro Ala Ile Pro Ser Ser Lys Ala Thr Phe Gly Glu Ser Met 355
360 365Leu Leu Leu Gly His Ile Leu Ile Leu
Leu Gly Gly Ile Tyr Leu Leu 370 375
380Leu Gly Gln Leu Trp Tyr Phe Trp Arg Arg Arg Leu Phe Ile Trp Ile385
390 395 400Ser Phe Met Asp
Ser Tyr Phe Glu Ile Leu Phe Leu Leu Gln Ala Leu 405
410 415Leu Thr Val Leu Ser Gln Val Leu Arg Phe
Met Glu Thr Glu Trp Tyr 420 425
430Leu Pro Leu Leu Val Leu Ser Leu Val Leu Gly Trp Leu Asn Leu Leu
435 440 445Tyr Tyr Thr Arg Gly Phe Gln
His Thr Gly Ile Tyr Ser Val Met Ile 450 455
460Gln Lys Val Ile Leu Arg Asp Leu Leu Arg Phe Leu Leu Val Tyr
Leu465 470 475 480Val Phe
Leu Phe Gly Phe Ala Val Ala Leu Val Ser Leu Ser Arg Glu
485 490 495Ala Arg Ser Pro Lys Ala Pro
Glu Asp Asn Asn Ser Thr Val Thr Glu 500 505
510Gln Pro Thr Val Gly Gln Glu Glu Glu Pro Ala Pro Tyr Arg
Ser Ile 515 520 525Leu Asp Ala Ser
Leu Glu Leu Phe Lys Phe Thr Ile Gly Met Gly Glu 530
535 540Leu Ala Phe Gln Glu Gln Leu Arg Phe Arg Gly Val
Val Leu Leu Leu545 550 555
560Leu Leu Ala Tyr Val Leu Leu Thr Tyr Val Leu Leu Leu Asn Met Leu
565 570 575Ile Ala Leu Met Ser
Glu Thr Val Asn His Val Ala Asp Asn Ser Trp 580
585 590Ser Ile Trp Lys Leu Gln Lys Ala Ile Ser Val Leu
Glu Met Glu Asn 595 600 605Gly Tyr
Trp Trp Cys Arg Arg Lys Lys His Arg Glu Gly Arg Leu Leu 610
615 620Lys Val Gly Thr Arg Gly Asp Gly Thr Pro Asp
Glu Arg Trp Cys Phe625 630 635
640Arg Val Glu Glu Val Asn Trp Val Ala Trp Glu Lys Thr Leu Pro Thr
645 650 655Leu Ser Glu Asp
Pro Ser Gly Pro Gly Ile Thr Gly Asn Lys Lys Asn 660
665 670Pro Thr Ser Lys Pro Gly Lys Asn Ser Ala Ser
Glu Glu Asp His Leu 675 680 685Pro
Leu Gln Val Leu Gln Ser Pro 690 69514678PRTRattus
norvegicus 14Val Ser Arg Gly Val Pro Glu Glu Leu Thr Gly Leu Leu Glu Tyr
Leu1 5 10 15Arg Trp Asn
Ser Lys Tyr Leu Thr Asp Ser Ala Tyr Thr Glu Gly Ser 20
25 30Thr Gly Lys Thr Cys Leu Met Lys Ala Val
Leu Asn Leu Gln Asp Gly 35 40
45Val Asn Ala Cys Ile Met Pro Leu Leu Gln Ile Asp Lys Asp Ser Gly 50
55 60Asn Pro Lys Pro Leu Val Asn Ala Gln
Cys Ile Asp Glu Phe Tyr Gln65 70 75
80Gly His Ser Ala Leu His Ile Ala Ile Glu Lys Arg Ser Leu
Gln Cys 85 90 95Val Lys
Leu Leu Val Glu Asn Gly Ala Asp Val His Leu Arg Ala Cys 100
105 110Gly Arg Phe Phe Gln Lys His Gln Gly
Thr Cys Phe Tyr Phe Gly Glu 115 120
125Leu Pro Leu Ser Leu Ala Ala Cys Thr Lys Gln Trp Asp Val Val Thr
130 135 140Tyr Leu Leu Glu Asn Pro His
Gln Pro Ala Ser Leu Glu Ala Thr Asp145 150
155 160Ser Leu Gly Asn Thr Val Leu His Ala Leu Val Met
Ile Ala Asp Asn 165 170
175Ser Pro Glu Asn Ser Ala Leu Val Ile His Met Tyr Asp Gly Leu Leu
180 185 190Gln Met Gly Ala Arg Leu
Cys Pro Thr Val Gln Leu Glu Glu Ile Ser 195 200
205Asn His Gln Gly Leu Thr Pro Leu Lys Leu Ala Ala Lys Glu
Gly Lys 210 215 220Ile Glu Ile Phe Arg
His Ile Leu Gln Arg Glu Phe Ser Gly Pro Tyr225 230
235 240Gln Pro Leu Ser Arg Lys Phe Thr Glu Trp
Cys Tyr Gly Pro Val Arg 245 250
255Val Ser Leu Tyr Asp Leu Ser Ser Val Asp Ser Trp Glu Lys Asn Ser
260 265 270Val Leu Glu Ile Ile
Ala Phe His Cys Lys Ser Pro Asn Arg His Arg 275
280 285Met Val Val Leu Glu Pro Leu Asn Lys Leu Leu Gln
Glu Lys Trp Asp 290 295 300Arg Leu Val
Ser Arg Phe Phe Phe Asn Phe Ala Cys Tyr Leu Val Tyr305
310 315 320Met Phe Ile Phe Thr Val Val
Ala Tyr His Gln Pro Ser Leu Asp Gln 325
330 335Pro Ala Ile Pro Ser Ser Lys Ala Thr Phe Gly Glu
Ser Met Leu Leu 340 345 350Leu
Gly His Ile Leu Ile Leu Leu Gly Gly Ile Tyr Leu Leu Leu Gly 355
360 365Gln Leu Trp Tyr Phe Trp Arg Arg Arg
Leu Phe Ile Trp Ile Ser Phe 370 375
380Met Asp Ser Tyr Phe Glu Ile Leu Phe Leu Leu Gln Ala Leu Leu Thr385
390 395 400Val Leu Ser Gln
Val Leu Arg Phe Met Glu Thr Glu Trp Tyr Leu Pro 405
410 415Leu Leu Val Leu Ser Leu Val Leu Gly Trp
Leu Asn Leu Leu Tyr Tyr 420 425
430Thr Arg Gly Phe Gln His Thr Gly Ile Tyr Ser Val Met Ile Gln Lys
435 440 445Val Ile Leu Arg Asp Leu Leu
Arg Phe Leu Leu Val Tyr Leu Val Phe 450 455
460Leu Phe Gly Phe Ala Val Ala Leu Val Ser Leu Ser Arg Glu Ala
Arg465 470 475 480Ser Pro
Lys Ala Pro Glu Asp Asn Asn Ser Thr Val Thr Glu Gln Pro
485 490 495Thr Val Gly Gln Glu Glu Glu
Pro Ala Pro Tyr Arg Ser Ile Leu Asp 500 505
510Ala Ser Leu Glu Leu Phe Lys Phe Thr Ile Gly Met Gly Glu
Leu Ala 515 520 525Phe Gln Glu Gln
Leu Arg Phe Arg Gly Val Val Leu Leu Leu Leu Leu 530
535 540Ala Tyr Val Leu Leu Thr Tyr Val Leu Leu Leu Asn
Met Leu Ile Ala545 550 555
560Leu Met Ser Glu Thr Val Asn His Val Ala Asp Asn Ser Trp Ser Ile
565 570 575Trp Lys Leu Gln Lys
Ala Ile Ser Val Leu Glu Met Glu Asn Gly Tyr 580
585 590Trp Trp Cys Arg Arg Lys Lys His Arg Glu Gly Arg
Leu Leu Lys Val 595 600 605Gly Thr
Arg Gly Asp Gly Thr Pro Asp Glu Arg Trp Cys Phe Arg Val 610
615 620Glu Glu Val Asn Trp Val Ala Trp Glu Lys Thr
Leu Pro Thr Leu Ser625 630 635
640Glu Asp Pro Ser Gly Pro Gly Ile Thr Gly Asn Lys Lys Asn Pro Thr
645 650 655Ser Lys Pro Gly
Lys Asn Ser Ala Ser Glu Glu Asp His Leu Pro Leu 660
665 670Gln Val Leu Gln Ser Pro
67515764PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 15Met Thr Ser Ala Ser Ser Pro Pro Ala Phe Arg
Leu Glu Thr Ser Asp1 5 10
15Gly Asp Glu Glu Gly Asn Ala Glu Val Asn Lys Gly Lys Gln Glu Pro
20 25 30Pro Pro Met Glu Ser Pro Phe
Gln Arg Glu Asp Arg Asn Ser Ser Pro 35 40
45Gln Ile Lys Val Asn Leu Asn Phe Ile Lys Arg Pro Pro Lys Asn
Thr 50 55 60Ser Ala Pro Ser Gln Gln
Glu Pro Asp Arg Phe Asp Arg Asp Arg Leu65 70
75 80Phe Ser Val Val Ser Arg Gly Val Pro Glu Glu
Leu Thr Gly Leu Leu 85 90
95Glu Tyr Leu Arg Trp Asn Ser Lys Tyr Leu Thr Asp Ser Ala Tyr Thr
100 105 110Glu Gly Ser Thr Gly Lys
Thr Cys Leu Met Lys Ala Val Leu Asn Leu 115 120
125Gln Asp Gly Val Asn Ala Cys Ile Met Pro Leu Leu Gln Ile
Asp Lys 130 135 140Asp Ser Gly Asn Pro
Lys Pro Leu Val Asn Ala Gln Cys Ile Asp Glu145 150
155 160Phe Tyr Gln Gly His Ser Ala Leu His Ile
Ala Ile Glu Lys Arg Ser 165 170
175Leu Gln Cys Val Lys Leu Leu Val Glu Asn Gly Ala Asp Val His Leu
180 185 190Arg Ala Cys Gly Arg
Phe Phe Gln Lys His Gln Gly Thr Cys Phe Tyr 195
200 205Phe Gly Glu Leu Pro Leu Ser Leu Ala Ala Cys Thr
Lys Gln Trp Asp 210 215 220Val Val Thr
Tyr Leu Leu Glu Asn Pro His Gln Pro Ala Ser Leu Glu225
230 235 240Ala Thr Asp Ser Leu Gly Asn
Thr Val Leu His Ala Leu Val Met Ile 245
250 255Ala Asp Asn Ser Pro Glu Asn Ser Ala Leu Val Ile
His Met Tyr Asp 260 265 270Gly
Leu Leu Gln Met Gly Ala Arg Leu Cys Pro Thr Val Gln Leu Glu 275
280 285Glu Ile Ser Asn His Gln Gly Leu Thr
Pro Leu Lys Leu Ala Ala Lys 290 295
300Glu Gly Lys Ile Glu Ile Phe Arg His Ile Leu Gln Arg Glu Phe Ser305
310 315 320Gly Pro Tyr Gln
Pro Leu Ser Arg Lys Phe Thr Glu Trp Cys Tyr Gly 325
330 335Pro Val Arg Val Ser Leu Tyr Asp Leu Ser
Ser Val Asp Ser Trp Glu 340 345
350Lys Asn Ser Val Leu Glu Ile Ile Ala Phe His Cys Lys Ser Pro Asn
355 360 365Arg His Arg Met Val Val Leu
Glu Pro Leu Asn Lys Leu Leu Gln Glu 370 375
380Lys Trp Asp Arg Leu Val Ser Arg Phe Phe Phe Asn Phe Ala Cys
Tyr385 390 395 400Leu Val
Tyr Met Phe Ile Phe Thr Val Val Ala Tyr His Gln Pro Ser
405 410 415Leu Asp Gln Pro Ala Ile Pro
Ser Ser Lys Ala Thr Phe Gly Glu Ser 420 425
430Met Leu Leu Leu Gly His Ile Leu Ile Leu Leu Gly Gly Ile
Tyr Leu 435 440 445Leu Leu Gly Gln
Leu Trp Tyr Phe Trp Arg Arg Arg Leu Phe Ile Trp 450
455 460Ile Ser Phe Met Asp Ser Tyr Phe Glu Ile Leu Phe
Leu Leu Gln Ala465 470 475
480Leu Leu Thr Val Leu Ser Gln Val Leu Arg Phe Met Glu Thr Glu Trp
485 490 495Tyr Leu Pro Leu Leu
Val Leu Ser Leu Val Leu Gly Trp Leu Asn Leu 500
505 510Leu Tyr Tyr Thr Arg Gly Phe Gln His Thr Gly Ile
Tyr Ser Val Met 515 520 525Ile Gln
Lys Val Ile Leu Arg Asp Leu Leu Arg Phe Leu Leu Val Tyr 530
535 540Leu Val Phe Leu Phe Gly Phe Ala Val Ala Leu
Val Ser Leu Ser Arg545 550 555
560Glu Ala Arg Ser Pro Lys Ala Pro Glu Asp Asn Asn Ser Thr Val Thr
565 570 575Glu Gln Pro Thr
Val Gly Gln Glu Glu Glu Pro Ala Pro Tyr Arg Ser 580
585 590Ile Leu Asp Ala Ser Leu Glu Leu Phe Lys Phe
Thr Ile Gly Met Gly 595 600 605Glu
Leu Ala Phe Gln Glu Gln Leu Arg Phe Arg Gly Val Val Leu Leu 610
615 620Leu Leu Leu Ala Tyr Val Leu Leu Thr Tyr
Val Leu Leu Leu Asn Met625 630 635
640Leu Ile Ala Leu Met Ser Glu Thr Val Asn Ser Val Ala Thr Asp
Ser 645 650 655Trp Ser Ile
Trp Lys Leu Gln Lys Ala Ile Ser Val Leu Glu Met Glu 660
665 670Asn Gly Tyr Trp Trp Cys Arg Lys Lys Gln
Arg Ala Gly Val Met Leu 675 680
685Thr Val Gly Thr Lys Pro Asp Gly Ser Pro Asp Glu Arg Trp Cys Phe 690
695 700Arg Val Glu Glu Val Asn Trp Ala
Ser Trp Glu Gln Thr Leu Pro Thr705 710
715 720Leu Cys Glu Asp Pro Ser Gly Ala Gly Val Pro Arg
Thr Leu Glu Asn 725 730
735Pro Val Leu Ala Ser Pro Pro Lys Glu Asp Glu Asp Gly Ala Ser Glu
740 745 750Glu Asn Tyr Val Pro Val
Gln Leu Leu Gln Ser Asn 755 76016763PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
16Met Thr Ser Ala Ser Ser Pro Pro Ala Phe Arg Leu Glu Thr Ser Asp1
5 10 15Gly Asp Glu Glu Gly Asn
Ala Glu Val Asn Lys Gly Lys Gln Glu Pro 20 25
30Pro Pro Met Glu Ser Pro Phe Gln Arg Glu Asp Arg Asn
Ser Ser Pro 35 40 45Gln Ile Lys
Val Asn Leu Asn Phe Ile Lys Arg Pro Pro Lys Asn Thr 50
55 60Ser Ala Pro Ser Gln Gln Glu Pro Asp Arg Phe Asp
Arg Asp Arg Leu65 70 75
80Phe Ser Val Val Ser Arg Gly Val Pro Glu Glu Leu Thr Gly Leu Leu
85 90 95Glu Tyr Leu Arg Trp Asn
Ser Lys Tyr Leu Thr Asp Ser Ala Tyr Thr 100
105 110Glu Gly Ser Thr Gly Lys Thr Cys Leu Met Lys Ala
Val Leu Asn Leu 115 120 125Gln Asp
Gly Val Asn Ala Cys Ile Met Pro Leu Leu Gln Ile Asp Lys 130
135 140Asp Ser Gly Asn Pro Lys Pro Leu Val Asn Ala
Gln Cys Ile Asp Glu145 150 155
160Phe Tyr Gln Gly His Ser Ala Leu His Ile Ala Ile Glu Lys Arg Ser
165 170 175Leu Gln Cys Val
Lys Leu Leu Val Glu Asn Gly Ala Asp Val His Leu 180
185 190Arg Ala Cys Gly Arg Phe Phe Gln Lys His Gln
Gly Thr Cys Phe Tyr 195 200 205Phe
Gly Glu Leu Pro Leu Ser Leu Ala Ala Cys Thr Lys Gln Trp Asp 210
215 220Val Val Thr Tyr Leu Leu Glu Asn Pro His
Gln Pro Ala Ser Leu Glu225 230 235
240Ala Thr Asp Ser Leu Gly Asn Thr Val Leu His Ala Leu Val Met
Ile 245 250 255Ala Asp Asn
Ser Pro Glu Asn Ser Ala Leu Val Ile His Met Tyr Asp 260
265 270Gly Leu Leu Gln Met Gly Ala Arg Leu Cys
Pro Thr Val Gln Leu Glu 275 280
285Glu Ile Ser Asn His Gln Gly Leu Thr Pro Leu Lys Leu Ala Ala Lys 290
295 300Glu Gly Lys Ile Glu Ile Phe Arg
His Ile Leu Gln Arg Glu Phe Ser305 310
315 320Gly Pro Tyr Gln Pro Leu Ser Arg Lys Phe Thr Glu
Trp Cys Tyr Gly 325 330
335Pro Val Arg Val Ser Leu Tyr Asp Leu Ser Ser Val Asp Ser Trp Glu
340 345 350Lys Asn Ser Val Leu Glu
Ile Ile Ala Phe His Cys Lys Ser Pro Asn 355 360
365Arg His Arg Met Val Val Leu Glu Pro Leu Asn Lys Leu Leu
Gln Glu 370 375 380Lys Trp Asp Arg Leu
Val Ser Arg Phe Phe Leu Asn Phe Leu Cys Asn385 390
395 400Leu Ile Tyr Met Phe Ile Phe Thr Ala Val
Ala Tyr His Gln Pro Thr 405 410
415Leu Lys Lys Gln Ala Ala Pro His Leu Lys Ala Glu Val Gly Asn Ser
420 425 430Met Leu Leu Thr Gly
His Ile Leu Ile Leu Leu Gly Gly Ile Tyr Leu 435
440 445Leu Val Gly Gln Leu Trp Tyr Phe Trp Arg Arg His
Val Phe Ile Trp 450 455 460Ile Ser Phe
Ile Asp Ser Tyr Phe Glu Ile Leu Phe Leu Phe Gln Ala465
470 475 480Leu Leu Thr Val Val Ser Gln
Val Leu Cys Phe Leu Ala Ile Glu Trp 485
490 495Tyr Leu Pro Leu Leu Val Ser Ala Leu Val Leu Gly
Trp Leu Asn Leu 500 505 510Leu
Tyr Tyr Thr Arg Gly Phe Gln His Thr Gly Ile Tyr Ser Val Met 515
520 525Ile Gln Lys Val Ile Leu Arg Asp Leu
Leu Arg Phe Leu Leu Ile Tyr 530 535
540Leu Val Phe Leu Phe Gly Phe Ala Val Ala Leu Val Ser Leu Ser Gln545
550 555 560Glu Ala Trp Arg
Pro Glu Ala Pro Thr Gly Pro Asn Ala Thr Glu Ser 565
570 575Val Gln Pro Met Glu Gly Gln Glu Asp Glu
Gly Asn Gly Ala Gln Tyr 580 585
590Arg Gly Ile Leu Glu Ala Ser Leu Glu Leu Phe Lys Phe Thr Ile Gly
595 600 605Met Gly Glu Leu Ala Phe Gln
Glu Gln Leu His Phe Arg Gly Met Val 610 615
620Leu Leu Leu Leu Leu Ala Tyr Val Leu Leu Thr Tyr Ile Leu Leu
Leu625 630 635 640Asn Met
Leu Ile Ala Leu Met Ser Glu Thr Val Asn His Val Ala Asp
645 650 655Asn Ser Trp Ser Ile Trp Lys
Leu Gln Lys Ala Ile Ser Val Leu Glu 660 665
670Met Glu Asn Gly Tyr Trp Trp Cys Arg Arg Lys Lys His Arg
Glu Gly 675 680 685Arg Leu Leu Lys
Val Gly Thr Arg Gly Asp Gly Thr Pro Asp Glu Arg 690
695 700Trp Cys Phe Arg Val Glu Glu Val Asn Trp Val Ala
Trp Glu Lys Thr705 710 715
720Leu Pro Thr Leu Ser Glu Asp Pro Ser Gly Pro Gly Ile Thr Gly Asn
725 730 735Lys Lys Asn Pro Thr
Ser Lys Pro Gly Lys Asn Ser Ala Ser Glu Glu 740
745 750Asp His Leu Pro Leu Gln Val Leu Gln Ser Pro
755 76017759PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 17Met Thr Ser Pro Ser Ser
Ser Pro Val Phe Arg Leu Glu Thr Leu Asp1 5
10 15Gly Gly Gln Glu Asp Gly Ser Glu Ala Asp Arg Gly
Lys Leu Asp Phe 20 25 30Gly
Ser Gly Leu Pro Pro Met Glu Ser Gln Phe Gln Gly Glu Asp Arg 35
40 45Lys Phe Ala Pro Gln Ile Arg Val Asn
Leu Asn Tyr Arg Lys Gly Thr 50 55
60Gly Ala Ser Gln Pro Asp Pro Asn Arg Phe Asp Arg Asp Arg Leu Phe65
70 75 80Asn Ala Val Ser Arg
Gly Val Pro Glu Asp Leu Ala Gly Leu Pro Glu 85
90 95Tyr Leu Ser Lys Thr Ser Lys Tyr Leu Thr Asp
Ser Glu Tyr Thr Glu 100 105
110Gly Ser Thr Gly Lys Thr Cys Leu Met Lys Ala Val Leu Asn Leu Lys
115 120 125Asp Gly Val Asn Ala Cys Ile
Leu Pro Leu Leu Gln Ile Asp Arg Asp 130 135
140Ser Gly Asn Pro Gln Pro Leu Val Asn Ala Gln Cys Thr Asp Asp
Tyr145 150 155 160Tyr Arg
Gly His Ser Ala Leu His Ile Ala Ile Glu Lys Arg Ser Leu
165 170 175Gln Cys Val Lys Leu Leu Val
Glu Asn Gly Ala Asn Val His Ala Arg 180 185
190Ala Cys Gly Arg Phe Phe Gln Lys Gly Gln Gly Thr Cys Phe
Tyr Phe 195 200 205Gly Glu Leu Pro
Leu Ser Leu Ala Ala Cys Thr Lys Gln Trp Asp Val 210
215 220Val Ser Tyr Leu Leu Glu Asn Pro His Gln Pro Ala
Ser Leu Gln Ala225 230 235
240Thr Asp Ser Gln Gly Asn Thr Val Leu His Ala Leu Val Met Ile Ser
245 250 255Asp Asn Ser Ala Glu
Asn Ile Ala Leu Val Thr Ser Met Tyr Asp Gly 260
265 270Leu Leu Gln Ala Gly Ala Arg Leu Cys Pro Thr Val
Gln Leu Glu Asp 275 280 285Ile Arg
Asn Leu Gln Asp Leu Thr Pro Leu Lys Leu Ala Ala Lys Glu 290
295 300Gly Lys Ile Glu Ile Phe Arg His Ile Leu Gln
Arg Glu Phe Ser Gly305 310 315
320Leu Ser His Leu Ser Arg Lys Phe Thr Glu Trp Cys Tyr Gly Pro Val
325 330 335Arg Val Ser Leu
Tyr Asp Leu Ala Ser Val Asp Ser Cys Glu Glu Asn 340
345 350Ser Val Leu Glu Ile Ile Ala Phe His Cys Lys
Ser Pro His Arg His 355 360 365Arg
Met Val Val Leu Glu Pro Leu Asn Lys Leu Leu Gln Ala Lys Trp 370
375 380Asp Leu Leu Ile Pro Lys Phe Phe Phe Asn
Phe Ala Cys Tyr Leu Val385 390 395
400Tyr Met Phe Ile Phe Thr Val Val Ala Tyr His Gln Pro Ser Leu
Asp 405 410 415Gln Pro Ala
Ile Pro Ser Ser Lys Ala Thr Phe Gly Glu Ser Met Leu 420
425 430Leu Leu Gly His Ile Leu Ile Leu Leu Gly
Gly Ile Tyr Leu Leu Leu 435 440
445Gly Gln Leu Trp Tyr Phe Trp Arg Arg Arg Leu Phe Ile Trp Ile Ser 450
455 460Phe Met Asp Ser Tyr Phe Glu Ile
Leu Phe Leu Leu Gln Ala Leu Leu465 470
475 480Thr Val Leu Ser Gln Val Leu Arg Phe Met Glu Thr
Glu Trp Tyr Leu 485 490
495Pro Leu Leu Val Leu Ser Leu Val Leu Gly Trp Leu Asn Leu Leu Tyr
500 505 510Tyr Thr Arg Gly Phe Gln
His Thr Gly Ile Tyr Ser Val Met Ile Gln 515 520
525Lys Val Ile Leu Arg Asp Leu Leu Arg Phe Leu Leu Val Tyr
Leu Val 530 535 540Phe Leu Phe Gly Phe
Ala Val Ala Leu Val Ser Leu Ser Arg Glu Ala545 550
555 560Arg Ser Pro Lys Ala Pro Glu Asp Asn Asn
Ser Thr Val Thr Glu Gln 565 570
575Pro Thr Val Gly Gln Glu Glu Glu Pro Ala Pro Tyr Arg Ser Ile Leu
580 585 590Asp Ala Ser Leu Glu
Leu Phe Lys Phe Thr Ile Gly Met Gly Glu Leu 595
600 605Ala Phe Gln Glu Gln Leu Arg Phe Arg Gly Val Val
Leu Leu Leu Leu 610 615 620Leu Ala Tyr
Val Leu Leu Thr Tyr Val Leu Leu Leu Asn Met Leu Ile625
630 635 640Ala Leu Met Ser Glu Thr Val
Asn His Val Ala Asp Asn Ser Trp Ser 645
650 655Ile Trp Lys Leu Gln Lys Ala Ile Ser Val Leu Glu
Met Glu Asn Gly 660 665 670Tyr
Trp Trp Cys Arg Arg Lys Lys His Arg Glu Gly Arg Leu Leu Lys 675
680 685Val Gly Thr Arg Gly Asp Gly Thr Pro
Asp Glu Arg Trp Cys Phe Arg 690 695
700Val Glu Glu Val Asn Trp Val Ala Trp Glu Lys Thr Leu Pro Thr Leu705
710 715 720Ser Glu Asp Pro
Ser Gly Pro Gly Ile Thr Gly Asn Lys Lys Asn Pro 725
730 735Thr Ser Lys Pro Gly Lys Asn Ser Ala Ser
Glu Glu Asp His Leu Pro 740 745
750Leu Gln Val Leu Gln Ser Pro 755182226DNARattus norvegicus
18ggcaatgctg aggtgaacaa ggggaagcag gaaccgcccc ccatggagtc accattccag
60agggaggacc ggaattcctc ccctcagatc aaagtgaacc tcaacttcat aaagagacct
120cctaaaaaca cttctgctcc cagccagcag gagccagatc ggtttgaccg tgaccgactc
180ttcagtgtgg tctcccgggg tgtccccgag gaactgactg gactgctaga atacctgcgc
240tggaacagca agtacctcac tgactctgca tacacagaag gctccactgg aaagacgtgc
300ctgatgaagg ctgtgctgaa ccttcaggat ggggtcaatg cctgcatcat gccgctgctg
360cagattgaca aggattccgg caatcccaag cccctcgtca atgcccagtg catcgatgag
420ttctaccaag gccacagtgc gctgcacatc gccatagaga agaggagcct gcagtgcgtg
480aagctgctgg tagagaatgg agcggatgtt cacctccgag cctgtggccg cttcttccaa
540aagcaccaag gaacttgttt ctattttgga gagctacctc tttctctggc tgcgtgcacc
600aagcagtggg atgtggtgac ctacctcctg gagaacccac accagccggc cagcctggag
660gccaccgact ccctgggcaa cacagtcctg catgctctgg taatgattgc agataactcg
720cctgagaaca gtgccctggt gatccacatg tacgacgggc ttctacaaat gggggcgcgc
780ctctgcccca ctgtgcagct tgaggaaatc tccaaccacc aaggcctcac acccctgaaa
840ctagccgcca aggaaggcaa aatcgagatt ttcaggcaca ttctgcagcg ggaattctca
900ggaccgtacc agcccctttc ccgaaagttt actgagtggt gttacggtcc tgtgcgggta
960tcgctgtacg acctgtcctc tgtggacagc tgggaaaaga actcggtgct ggagatcatc
1020gcttttcatt gcaagagccc gaaccggcac cgcatggtgg ttttagaacc actgaacaag
1080cttctgcagg agaaatggga tcggctcgtc tcaagattct tcttcaactt cgcctgctac
1140ttggtctaca tgttcatctt caccgtcgtt gcctaccacc agccttccct ggatcagcca
1200gccatcccct catcaaaagc gacttttggg gaatccatgc tgctgctggg ccacattctg
1260atcctgcttg ggggtattta cctcttactg ggccagctgt ggtacttttg gcggcggcgc
1320ctgttcatct ggatctcatt catggacagc tactttgaaa tcctctttct ccttcaggct
1380ctgctcacag tgctgtccca ggtgctgcgc ttcatggaga ctgaatggta cctacccctg
1440ctagtgttat ccctagtgct gggctggctg aacctgcttt actacacacg gggctttcag
1500cacacaggca tctacagtgt catgatccag aaggtcatcc ttcgagacct gctccgtttc
1560ctgctggtct acctggtctt ccttttcggc tttgctgtag ccctagtaag cttgagcaga
1620gaggcccgaa gtcccaaagc ccctgaagat aacaactcca cagtgacgga acagcccacg
1680gtgggccagg aggaggagcc agctccatat cggagcattc tggatgcctc cctagagctg
1740ttcaagttca ccattggtat gggggagctg gctttccagg aacagctgcg ttttcgtggg
1800gtggtcctgc tgttgctgtt ggcctacgtc cttctcacct acgtcctgct gctcaacatg
1860ctcattgctc tcatgagcga aactgtcaac cacgttgctg acaacagctg gagcatctgg
1920aagttgcaga aagccatctc tgtcttggag atggagaatg gttactggtg gtgccggagg
1980aagaaacatc gtgaagggag gctgctgaaa gtcggcacca ggggggatgg tacccctgat
2040gagcgctggt gcttcagggt ggaggaagta aattgggttg cttgggagaa gactcttccc
2100accttatctg aggatccatc agggccaggc atcactggta ataaaaagaa cccaacctct
2160aaaccgggga agaacagtgc ctcagaggaa gaccatctgc cccttcaggt cctccagtcc
2220ccctga
2226192190DNARattus norvegicus 19ccccccatgg agtcaccatt ccagagggag
gaccggaatt cctcccctca gatcaaagtg 60aacctcaact tcataaagag acctcctaaa
aacacttctg ctcccagcca gcaggagcca 120gatcggtttg accgtgaccg actcttcagt
gtggtctccc ggggtgtccc cgaggaactg 180actggactgc tagaatacct gcgctggaac
agcaagtacc tcactgactc tgcatacaca 240gaaggctcca ctggaaagac gtgcctgatg
aaggctgtgc tgaaccttca ggatggggtc 300aatgcctgca tcatgccgct gctgcagatt
gacaaggatt ccggcaatcc caagcccctc 360gtcaatgccc agtgcatcga tgagttctac
caaggccaca gtgcgctgca catcgccata 420gagaagagga gcctgcagtg cgtgaagctg
ctggtagaga atggagcgga tgttcacctc 480cgagcctgtg gccgcttctt ccaaaagcac
caaggaactt gtttctattt tggagagcta 540cctctttctc tggctgcgtg caccaagcag
tgggatgtgg tgacctacct cctggagaac 600ccacaccagc cggccagcct ggaggccacc
gactccctgg gcaacacagt cctgcatgct 660ctggtaatga ttgcagataa ctcgcctgag
aacagtgccc tggtgatcca catgtacgac 720gggcttctac aaatgggggc gcgcctctgc
cccactgtgc agcttgagga aatctccaac 780caccaaggcc tcacacccct gaaactagcc
gccaaggaag gcaaaatcga gattttcagg 840cacattctgc agcgggaatt ctcaggaccg
taccagcccc tttcccgaaa gtttactgag 900tggtgttacg gtcctgtgcg ggtatcgctg
tacgacctgt cctctgtgga cagctgggaa 960aagaactcgg tgctggagat catcgctttt
cattgcaaga gcccgaaccg gcaccgcatg 1020gtggttttag aaccactgaa caagcttctg
caggagaaat gggatcggct cgtctcaaga 1080ttcttcttca acttcgcctg ctacttggtc
tacatgttca tcttcaccgt cgttgcctac 1140caccagcctt ccctggatca gccagccatc
ccctcatcaa aagcgacttt tggggaatcc 1200atgctgctgc tgggccacat tctgatcctg
cttgggggta tttacctctt actgggccag 1260ctgtggtact tttggcggcg gcgcctgttc
atctggatct cattcatgga cagctacttt 1320gaaatcctct ttctccttca ggctctgctc
acagtgctgt cccaggtgct gcgcttcatg 1380gagactgaat ggtacctacc cctgctagtg
ttatccctag tgctgggctg gctgaacctg 1440ctttactaca cacggggctt tcagcacaca
ggcatctaca gtgtcatgat ccagaaggtc 1500atccttcgag acctgctccg tttcctgctg
gtctacctgg tcttcctttt cggctttgct 1560gtagccctag taagcttgag cagagaggcc
cgaagtccca aagcccctga agataacaac 1620tccacagtga cggaacagcc cacggtgggc
caggaggagg agccagctcc atatcggagc 1680attctggatg cctccctaga gctgttcaag
ttcaccattg gtatggggga gctggctttc 1740caggaacagc tgcgttttcg tggggtggtc
ctgctgttgc tgttggccta cgtccttctc 1800acctacgtcc tgctgctcaa catgctcatt
gctctcatga gcgaaactgt caaccacgtt 1860gctgacaaca gctggagcat ctggaagttg
cagaaagcca tctctgtctt ggagatggag 1920aatggttact ggtggtgccg gaggaagaaa
catcgtgaag ggaggctgct gaaagtcggc 1980accagggggg atggtacccc tgatgagcgc
tggtgcttca gggtggagga agtaaattgg 2040gttgcttggg agaagactct tcccacctta
tctgaggatc catcagggcc aggcatcact 2100ggtaataaaa agaacccaac ctctaaaccg
gggaagaaca gtgcctcaga ggaagaccat 2160ctgccccttc aggtcctcca gtccccctga
2190202091DNARattus norvegicus
20ttcagtgtgg tctcccgggg tgtccccgag gaactgactg gactgctaga atacctgcgc
60tggaacagca agtacctcac tgactctgca tacacagaag gctccactgg aaagacgtgc
120ctgatgaagg ctgtgctgaa ccttcaggat ggggtcaatg cctgcatcat gccgctgctg
180cagattgaca aggattccgg caatcccaag cccctcgtca atgcccagtg catcgatgag
240ttctaccaag gccacagtgc gctgcacatc gccatagaga agaggagcct gcagtgcgtg
300aagctgctgg tagagaatgg agcggatgtt cacctccgag cctgtggccg cttcttccaa
360aagcaccaag gaacttgttt ctattttgga gagctacctc tttctctggc tgcgtgcacc
420aagcagtggg atgtggtgac ctacctcctg gagaacccac accagccggc cagcctggag
480gccaccgact ccctgggcaa cacagtcctg catgctctgg taatgattgc agataactcg
540cctgagaaca gtgccctggt gatccacatg tacgacgggc ttctacaaat gggggcgcgc
600ctctgcccca ctgtgcagct tgaggaaatc tccaaccacc aaggcctcac acccctgaaa
660ctagccgcca aggaaggcaa aatcgagatt ttcaggcaca ttctgcagcg ggaattctca
720ggaccgtacc agcccctttc ccgaaagttt actgagtggt gttacggtcc tgtgcgggta
780tcgctgtacg acctgtcctc tgtggacagc tgggaaaaga actcggtgct ggagatcatc
840gcttttcatt gcaagagccc gaaccggcac cgcatggtgg ttttagaacc actgaacaag
900cttctgcagg agaaatggga tcggctcgtc tcaagattct tcttcaactt cgcctgctac
960ttggtctaca tgttcatctt caccgtcgtt gcctaccacc agccttccct ggatcagcca
1020gccatcccct catcaaaagc gacttttggg gaatccatgc tgctgctggg ccacattctg
1080atcctgcttg ggggtattta cctcttactg ggccagctgt ggtacttttg gcggcggcgc
1140ctgttcatct ggatctcatt catggacagc tactttgaaa tcctctttct ccttcaggct
1200ctgctcacag tgctgtccca ggtgctgcgc ttcatggaga ctgaatggta cctacccctg
1260ctagtgttat ccctagtgct gggctggctg aacctgcttt actacacacg gggctttcag
1320cacacaggca tctacagtgt catgatccag aaggtcatcc ttcgagacct gctccgtttc
1380ctgctggtct acctggtctt ccttttcggc tttgctgtag ccctagtaag cttgagcaga
1440gaggcccgaa gtcccaaagc ccctgaagat aacaactcca cagtgacgga acagcccacg
1500gtgggccagg aggaggagcc agctccatat cggagcattc tggatgcctc cctagagctg
1560ttcaagttca ccattggtat gggggagctg gctttccagg aacagctgcg ttttcgtggg
1620gtggtcctgc tgttgctgtt ggcctacgtc cttctcacct acgtcctgct gctcaacatg
1680ctcattgctc tcatgagcga aactgtcaac cacgttgctg acaacagctg gagcatctgg
1740aagttgcaga aagccatctc tgtcttggag atggagaatg gttactggtg gtgccggagg
1800aagaaacatc gtgaagggag gctgctgaaa gtcggcacca ggggggatgg tacccctgat
1860gagcgctggt gcttcagggt ggaggaagta aattgggttg cttgggagaa gactcttccc
1920accttatctg aggatccatc agggccaggc atcactggta ataaaaagaa cccaacctct
1980aaaccgggga agaacagtgc ctcagaggaa gaccatctgc cccttcaggt cctccagtcc
2040ccctgagctc ccagccagca ggagccagat cggtttgacc gtgaccgact c
2091212037DNARattus norvegicus 21gaatacctgc gctggaacag caagtacctc
actgactctg catacacaga aggctccact 60ggaaagacgt gcctgatgaa ggctgtgctg
aaccttcagg atggggtcaa tgcctgcatc 120atgccgctgc tgcagattga caaggattcc
ggcaatccca agcccctcgt caatgcccag 180tgcatcgatg agttctacca aggccacagt
gcgctgcaca tcgccataga gaagaggagc 240ctgcagtgcg tgaagctgct ggtagagaat
ggagcggatg ttcacctccg agcctgtggc 300cgcttcttcc aaaagcacca aggaacttgt
ttctattttg gagagctacc tctttctctg 360gctgcgtgca ccaagcagtg ggatgtggtg
acctacctcc tggagaaccc acaccagccg 420gccagcctgg aggccaccga ctccctgggc
aacacagtcc tgcatgctct ggtaatgatt 480gcagataact cgcctgagaa cagtgccctg
gtgatccaca tgtacgacgg gcttctacaa 540atgggggcgc gcctctgccc cactgtgcag
cttgaggaaa tctccaacca ccaaggcctc 600acacccctga aactagccgc caaggaaggc
aaaatcgaga ttttcaggca cattctgcag 660cgggaattct caggaccgta ccagcccctt
tcccgaaagt ttactgagtg gtgttacggt 720cctgtgcggg tatcgctgta cgacctgtcc
tctgtggaca gctgggaaaa gaactcggtg 780ctggagatca tcgcttttca ttgcaagagc
ccgaaccggc accgcatggt ggttttagaa 840ccactgaaca agcttctgca ggagaaatgg
gatcggctcg tctcaagatt cttcttcaac 900ttcgcctgct acttggtcta catgttcatc
ttcaccgtcg ttgcctacca ccagccttcc 960ctggatcagc cagccatccc ctcatcaaaa
gcgacttttg gggaatccat gctgctgctg 1020ggccacattc tgatcctgct tgggggtatt
tacctcttac tgggccagct gtggtacttt 1080tggcggcggc gcctgttcat ctggatctca
ttcatggaca gctactttga aatcctcttt 1140ctccttcagg ctctgctcac agtgctgtcc
caggtgctgc gcttcatgga gactgaatgg 1200tacctacccc tgctagtgtt atccctagtg
ctgggctggc tgaacctgct ttactacaca 1260cggggctttc agcacacagg catctacagt
gtcatgatcc agaaggtcat ccttcgagac 1320ctgctccgtt tcctgctggt ctacctggtc
ttccttttcg gctttgctgt agccctagta 1380agcttgagca gagaggcccg aagtcccaaa
gcccctgaag ataacaactc cacagtgacg 1440gaacagccca cggtgggcca ggaggaggag
ccagctccat atcggagcat tctggatgcc 1500tccctagagc tgttcaagtt caccattggt
atgggggagc tggctttcca ggaacagctg 1560cgttttcgtg gggtggtcct gctgttgctg
ttggcctacg tccttctcac ctacgtcctg 1620ctgctcaaca tgctcattgc tctcatgagc
gaaactgtca accacgttgc tgacaacagc 1680tggagcatct ggaagttgca gaaagccatc
tctgtcttgg agatggagaa tggttactgg 1740tggtgccgga ggaagaaaca tcgtgaaggg
aggctgctga aagtcggcac caggggggat 1800ggtacccctg atgagcgctg gtgcttcagg
gtggaggaag taaattgggt tgcttgggag 1860aagactcttc ccaccttatc tgaggatcca
tcagggccag gcatcactgg taataaaaag 1920aacccaacct ctaaaccggg gaagaacagt
gcctcagagg aagaccatct gccccttcag 1980gtcctccagt ccccctgagt ctcccggggt
gtccccgagg aactgactgg actgcta 2037222118DNARattus norvegicus
22gaatacctgc gctggaacag caagtacctc actgactctg catacacaga aggctccact
60ggaaagacgt gcctgatgaa ggctgtgctg aaccttcagg atggggtcaa tgcctgcatc
120atgccgctgc tgcagattga caaggattcc ggcaatccca agcccctcgt caatgcccag
180tgcatcgatg agttctacca aggccacagt gcgctgcaca tcgccataga gaagaggagc
240ctgcagtgcg tgaagctgct ggtagagaat ggagcggatg ttcacctccg agcctgtggc
300cgcttcttcc aaaagcacca aggaacttgt ttctattttg gagagctacc tctttctctg
360gctgcgtgca ccaagcagtg ggatgtggtg acctacctcc tggagaaccc acaccagccg
420gccagcctgg aggccaccga ctccctgggc aacacagtcc tgcatgctct ggtaatgatt
480gcagataact cgcctgagaa cagtgccctg gtgatccaca tgtacgacgg gcttctacaa
540atgggggcgc gcctctgccc cactgtgcag cttgaggaaa tctccaacca ccaaggcctc
600acacccctga aactagccgc caaggaaggc aaaatcgaga ttttcaggca cattctgcag
660cgggaattct caggaccgta ccagcccctt tcccgaaagt ttactgagtg gtgttacggt
720cctgtgcggg tatcgctgta cgacctgtcc tctgtggaca gctgggaaaa gaactcggtg
780ctggagatca tcgcttttca ttgcaagagc ccgaaccggc accgcatggt ggttttagaa
840ccactgaaca agcttctgca ggagaaatgg gatcggctcg tctcaagatt cttcttcaac
900ttcgcctgct acttggtcta catgttcatc ttcaccgtcg ttgcctacca ccagccttcc
960ctggatcagc cagccatccc ctcatcaaaa gcgacttttg gggaatccat gctgctgctg
1020ggccacattc tgatcctgct tgggggtatt tacctcttac tgggccagct gtggtacttt
1080tggcggcggc gcctgttcat ctggatctca ttcatggaca gctactttga aatcctcttt
1140ctccttcagg ctctgctcac agtgctgtcc caggtgctgc gcttcatgga gactgaatgg
1200tacctacccc tgctagtgtt atccctagtg ctgggctggc tgaacctgct ttactacaca
1260cggggctttc agcacacagg catctacagt gtcatgatcc agaaggtcat ccttcgagac
1320ctgctccgtt tcctgctggt ctacctggtc ttccttttcg gctttgctgt agccctagta
1380agcttgagca gagaggcccg aagtcccaaa gcccctgaag ataacaactc cacagtgacg
1440gaacagccca cggtgggcca ggaggaggag ccagctccat atcggagcat tctggatgcc
1500tccctagagc tgttcaagtt caccattggt atgggggagc tggctttcca ggaacagctg
1560cgttttcgtg gggtggtcct gctgttgctg ttggcctacg tccttctcac ctacgtcctg
1620ctgctcaaca tgatgacttc agcctccagc cccccagctt tcaggctgga gacttccgat
1680ggagatgaag agggcaatgc tgaggtgaac aaggggaagc aggaaccgcc ccccatggag
1740tcaccattcc agagggagga ccggaattcc tcccctcaga tcaaagtgaa cctcaacttc
1800ataaagagac ctcctaaaaa cacttctgct cccagccagc aggagccaga tcggtttgac
1860cgtgaccgac tcttcagtgt ggtctcccgg ggtgtccccg aggaactgac tggactgcta
1920ctcattgctc tcatgagcga aactgtcaac cacgttgctg acaacagctg gagcatctgg
1980aagttgcaga aagccatctc tgtcttggag atggagaatg gttactggtg gtgccggagg
2040aagaaacatc gtgaagggag gctgctgaaa gtcggcacca ggggggatgg tacccctgat
2100gagcgctggt gcttcagg
2118232187DNARattus norvegicus 23gaatacctgc gctggaacag caagtacctc
actgactctg catacacaga aggctccact 60ggaaagacgt gcctgatgaa ggctgtgctg
aaccttcagg atggggtcaa tgcctgcatc 120atgccgctgc tgcagattga caaggattcc
ggcaatccca agcccctcgt caatgcccag 180tgcatcgatg agttctacca aggccacagt
gcgctgcaca tcgccataga gaagaggagc 240ctgcagtgcg tgaagctgct ggtagagaat
ggagcggatg ttcacctccg agcctgtggc 300cgcttcttcc aaaagcacca aggaacttgt
ttctattttg gagagctacc tctttctctg 360gctgcgtgca ccaagcagtg ggatgtggtg
acctacctcc tggagaaccc acaccagccg 420gccagcctgg aggccaccga ctccctgggc
aacacagtcc tgcatgctct ggtaatgatt 480gcagataact cgcctgagaa cagtgccctg
gtgatccaca tgtacgacgg gcttctacaa 540atgggggcgc gcctctgccc cactgtgcag
cttgaggaaa tctccaacca ccaaggcctc 600acacccctga aactagccgc caaggaaggc
aaaatcgaga ttttcaggca cattctgcag 660cgggaattct caggaccgta ccagcccctt
tcccgaaagt ttactgagtg gtgttacggt 720cctgtgcggg tatcgctgta cgacctgtcc
tctgtggaca gctgggaaaa gaactcggtg 780ctggagatca tcgcttttca ttgcaagagc
ccgaaccggc accgcatggt ggttttagaa 840ccactgaaca agcttctgca ggagaaatgg
gatcggctcg tctcaagatt cttcttcaac 900ttcgcctgct acttggtcta catgttcatc
ttcaccgtcg ttgcctacca ccagccttcc 960ctggatcagc cagccatccc ctcatcaaaa
gcgacttttg gggaatccat gctgctgctg 1020ggccacattc tgatcctgct tgggggtatt
tacctcttac tgggccagct gtggtacttt 1080tggcggcggc gcctgttcat ctggatctca
ttcatggaca gctactttga aatcctcttt 1140ctccttcagg ctctgctcac agtgctgtcc
caggtgctgc gcttcatgga gactgaatgg 1200tacctacccc tgctagtgtt atccctagtg
ctgggctggc tgaacctgct ttactacaca 1260cggggctttc agcacacagg catctacagt
gtcatgatcc agaaggtcat ccttcgagac 1320ctgctccgtt tcctgctggt ctacctggtc
ttccttttcg gctttgctgt agccctagta 1380agcttgagca gagaggcccg aagtcccaaa
gcccctgaag ataacaactc cacagtgacg 1440gaacagccca cggtgggcca ggaggaggag
ccagctccat atcggagcat tctggatgcc 1500tccctagagc tgttcaagtt caccattggt
atgggggagc tggctttcca ggaacagctg 1560cgttttcgtg gggtggtcct gctgttgctg
ttggcctacg tccttctcac ctacgtcctg 1620ctgctcaaca tgatgacttc agcctccagc
cccccagctt tcaggctgga gacttccgat 1680ggagatgaag agggcaatgc tgaggtgaac
aaggggaagc aggaaccgcc ccccatggag 1740tcaccattcc agagggagga ccggaattcc
tcccctcaga tcaaagtgaa cctcaacttc 1800ataaagagac ctcctaaaaa cacttctgct
cccagccagc aggagccaga tcggtttgac 1860cgtgaccgac tcttcagtgt ggtctcccgg
ggtgtccccg aggaactgac tggactgcta 1920ctcattgctc tcatgagcga aactgtcaac
cacgttgctg acaacagctg gagcatctgg 1980aagttgcaga aagccatctc tgtcttggag
atggagaatg gttactggtg gtgccggagg 2040aagaaacatc gtgaagggag gctgctgaaa
gtcggcacca ggggggatgg tacccctgat 2100gagcgctggt gcttcagggt ggaggaagta
aattgggttg cttgggagaa gactcttccc 2160accttatctg aggatccatc agggcca
2187242214DNARattus norvegicus
24gaatacctgc gctggaacag caagtacctc actgactctg catacacaga aggctccact
60ggaaagacgt gcctgatgaa ggctgtgctg aaccttcagg atggggtcaa tgcctgcatc
120atgccgctgc tgcagattga caaggattcc ggcaatccca agcccctcgt caatgcccag
180tgcatcgatg agttctacca aggccacagt gcgctgcaca tcgccataga gaagaggagc
240ctgcagtgcg tgaagctgct ggtagagaat ggagcggatg ttcacctccg agcctgtggc
300cgcttcttcc aaaagcacca aggaacttgt ttctattttg gagagctacc tctttctctg
360gctgcgtgca ccaagcagtg ggatgtggtg acctacctcc tggagaaccc acaccagccg
420gccagcctgg aggccaccga ctccctgggc aacacagtcc tgcatgctct ggtaatgatt
480gcagataact cgcctgagaa cagtgccctg gtgatccaca tgtacgacgg gcttctacaa
540atgggggcgc gcctctgccc cactgtgcag cttgaggaaa tctccaacca ccaaggcctc
600acacccctga aactagccgc caaggaaggc aaaatcgaga ttttcaggca cattctgcag
660cgggaattct caggaccgta ccagcccctt tcccgaaagt ttactgagtg gtgttacggt
720cctgtgcggg tatcgctgta cgacctgtcc tctgtggaca gctgggaaaa gaactcggtg
780ctggagatca tcgcttttca ttgcaagagc ccgaaccggc accgcatggt ggttttagaa
840ccactgaaca agcttctgca ggagaaatgg gatcggctcg tctcaagatt cttcttcaac
900ttcgcctgct acttggtcta catgttcatc ttcaccgtcg ttgcctacca ccagccttcc
960ctggatcagc cagccatccc ctcatcaaaa gcgacttttg gggaatccat gctgctgctg
1020ggccacattc tgatcctgct tgggggtatt tacctcttac tgggccagct gtggtacttt
1080tggcggcggc gcctgttcat ctggatctca ttcatggaca gctactttga aatcctcttt
1140ctccttcagg ctctgctcac agtgctgtcc caggtgctgc gcttcatgga gactgaatgg
1200tacctacccc tgctagtgtt atccctagtg ctgggctggc tgaacctgct ttactacaca
1260cggggctttc agcacacagg catctacagt gtcatgatcc agaaggtcat ccttcgagac
1320ctgctccgtt tcctgctggt ctacctggtc ttccttttcg gctttgctgt agccctagta
1380agcttgagca gagaggcccg aagtcccaaa gcccctgaag ataacaactc cacagtgacg
1440gaacagccca cggtgggcca ggaggaggag ccagctccat atcggagcat tctggatgcc
1500tccctagagc tgttcaagtt caccattggt atgggggagc tggctttcca ggaacagctg
1560cgttttcgtg gggtggtcct gctgttgctg ttggcctacg tccttctcac ctacgtcctg
1620ctgctcaaca tgatgacttc agcctccagc cccccagctt tcaggctgga gacttccgat
1680ggagatgaag agggcaatgc tgaggtgaac aaggggaagc aggaaccgcc ccccatggag
1740tcaccattcc agagggagga ccggaattcc tcccctcaga tcaaagtgaa cctcaacttc
1800ataaagagac ctcctaaaaa cacttctgct cccagccagc aggagccaga tcggtttgac
1860cgtgaccgac tcttcagtgt ggtctcccgg ggtgtccccg aggaactgac tggactgcta
1920ctcattgctc tcatgagcga aactgtcaac cacgttgctg acaacagctg gagcatctgg
1980aagttgcaga aagccatctc tgtcttggag atggagaatg gttactggtg gtgccggagg
2040aagaaacatc gtgaagggag gctgctgaaa gtcggcacca ggggggatgg tacccctgat
2100gagcgctggt gcttcagggt ggaggaagta aattgggttg cttgggagaa gactcttccc
2160accttatctg aggatccatc agggccaggc atcactggta ataaaaagaa ccca
2214252250DNARattus norvegicus 25gaatacctgc gctggaacag caagtacctc
actgactctg catacacaga aggctccact 60ggaaagacgt gcctgatgaa ggctgtgctg
aaccttcagg atggggtcaa tgcctgcatc 120atgccgctgc tgcagattga caaggattcc
ggcaatccca agcccctcgt caatgcccag 180tgcatcgatg agttctacca aggccacagt
gcgctgcaca tcgccataga gaagaggagc 240ctgcagtgcg tgaagctgct ggtagagaat
ggagcggatg ttcacctccg agcctgtggc 300cgcttcttcc aaaagcacca aggaacttgt
ttctattttg gagagctacc tctttctctg 360gctgcgtgca ccaagcagtg ggatgtggtg
acctacctcc tggagaaccc acaccagccg 420gccagcctgg aggccaccga ctccctgggc
aacacagtcc tgcatgctct ggtaatgatt 480gcagataact cgcctgagaa cagtgccctg
gtgatccaca tgtacgacgg gcttctacaa 540atgggggcgc gcctctgccc cactgtgcag
cttgaggaaa tctccaacca ccaaggcctc 600acacccctga aactagccgc caaggaaggc
aaaatcgaga ttttcaggca cattctgcag 660cgggaattct caggaccgta ccagcccctt
tcccgaaagt ttactgagtg gtgttacggt 720cctgtgcggg tatcgctgta cgacctgtcc
tctgtggaca gctgggaaaa gaactcggtg 780ctggagatca tcgcttttca ttgcaagagc
ccgaaccggc accgcatggt ggttttagaa 840ccactgaaca agcttctgca ggagaaatgg
gatcggctcg tctcaagatt cttcttcaac 900ttcgcctgct acttggtcta catgttcatc
ttcaccgtcg ttgcctacca ccagccttcc 960ctggatcagc cagccatccc ctcatcaaaa
gcgacttttg gggaatccat gctgctgctg 1020ggccacattc tgatcctgct tgggggtatt
tacctcttac tgggccagct gtggtacttt 1080tggcggcggc gcctgttcat ctggatctca
ttcatggaca gctactttga aatcctcttt 1140ctccttcagg ctctgctcac agtgctgtcc
caggtgctgc gcttcatgga gactgaatgg 1200tacctacccc tgctagtgtt atccctagtg
ctgggctggc tgaacctgct ttactacaca 1260cggggctttc agcacacagg catctacagt
gtcatgatcc agaaggtcat ccttcgagac 1320ctgctccgtt tcctgctggt ctacctggtc
ttccttttcg gctttgctgt agccctagta 1380agcttgagca gagaggcccg aagtcccaaa
gcccctgaag ataacaactc cacagtgacg 1440gaacagccca cggtgggcca ggaggaggag
ccagctccat atcggagcat tctggatgcc 1500tccctagagc tgttcaagtt caccattggt
atgggggagc tggctttcca ggaacagctg 1560cgttttcgtg gggtggtcct gctgttgctg
ttggcctacg tccttctcac ctacgtcctg 1620ctgctcaaca tgatgacttc agcctccagc
cccccagctt tcaggctgga gacttccgat 1680ggagatgaag agggcaatgc tgaggtgaac
aaggggaagc aggaaccgcc ccccatggag 1740tcaccattcc agagggagga ccggaattcc
tcccctcaga tcaaagtgaa cctcaacttc 1800ataaagagac ctcctaaaaa cacttctgct
cccagccagc aggagccaga tcggtttgac 1860cgtgaccgac tcttcagtgt ggtctcccgg
ggtgtccccg aggaactgac tggactgcta 1920ctcattgctc tcatgagcga aactgtcaac
cacgttgctg acaacagctg gagcatctgg 1980aagttgcaga aagccatctc tgtcttggag
atggagaatg gttactggtg gtgccggagg 2040aagaaacatc gtgaagggag gctgctgaaa
gtcggcacca ggggggatgg tacccctgat 2100gagcgctggt gcttcagggt ggaggaagta
aattgggttg cttgggagaa gactcttccc 2160accttatctg aggatccatc agggccaggc
atcactggta ataaaaagaa cccaacctct 2220aaaccgggga agaacagtgc ctcagaggaa
2250262295DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
26atgacttcag cctccagccc cccagctttc aggctggaga cttccgatgg agatgaagag
60ggcaatgctg aggtgaacaa ggggaagcag gaaccgcccc ccatggagtc accattccag
120agggaggacc ggaattcctc ccctcagatc aaagtgaacc tcaacttcat aaagagacct
180cctaaaaaca cttctgctcc cagccagcag gagccagatc ggtttgaccg tgaccgactc
240ttcagtgtgg tctcccgggg tgtccccgag gaactgactg gactgctaga atacctgcgc
300tggaacagca agtacctcac tgactctgca tacacagaag gctccactgg aaagacgtgc
360ctgatgaagg ctgtgctgaa ccttcaggat ggggtcaatg cctgcatcat gccgctgctg
420cagattgaca aggattccgg caatcccaag cccctcgtca atgcccagtg catcgatgag
480ttctaccaag gccacagtgc gctgcacatc gccatagaga agaggagcct gcagtgcgtg
540aagctgctgg tagagaatgg agcggatgtt cacctccgag cctgtggccg cttcttccaa
600aagcaccaag gaacttgttt ctattttgga gagctacctc tttctctggc tgcgtgcacc
660aagcagtggg atgtggtgac ctacctcctg gagaacccac accagccggc cagcctggag
720gccaccgact ccctgggcaa cacagtcctg catgctctgg taatgattgc agataactcg
780cctgagaaca gtgccctggt gatccacatg tacgacgggc ttctacaaat gggggcgcgc
840ctctgcccca ctgtgcagct tgaggaaatc tccaaccacc aaggcctcac acccctgaaa
900ctagccgcca aggaaggcaa aatcgagatt ttcaggcaca ttctgcagcg ggaattctca
960ggaccgtacc agcccctttc ccgaaagttt actgagtggt gttacggtcc tgtgcgggta
1020tcgctgtacg acctgtcctc tgtggacagc tgggaaaaga actcggtgct ggagatcatc
1080gcttttcatt gcaagagccc gaaccggcac cgcatggtgg ttttagaacc actgaacaag
1140cttctgcagg agaaatggga tcggctcgtc tcaagattct tcttcaactt cgcctgctac
1200ttggtctaca tgttcatctt caccgtcgtt gcctaccacc agccttccct ggatcagcca
1260gccatcccct catcaaaagc gacttttggg gaatccatgc tgctgctggg ccacattctg
1320atcctgcttg ggggtattta cctcttactg ggccagctgt ggtacttttg gcggcggcgc
1380ctgttcatct ggatctcatt catggacagc tactttgaaa tcctctttct ccttcaggct
1440ctgctcacag tgctgtccca ggtgctgcgc ttcatggaga ctgaatggta cctacccctg
1500ctagtgttat ccctagtgct gggctggctg aacctgcttt actacacacg gggctttcag
1560cacacaggca tctacagtgt catgatccag aaggtcatcc ttcgagacct gctccgtttc
1620ctgctggtct acctggtctt ccttttcggc tttgctgtag ccctagtaag cttgagcaga
1680gaggcccgaa gtcccaaagc ccctgaagat aacaactcca cagtgacgga acagcccacg
1740gtgggccagg aggaggagcc agctccatat cggagcattc tggatgcctc cctagagctg
1800ttcaagttca ccattggtat gggggagctg gctttccagg aacagctgcg ttttcgtggg
1860gtggtcctgc tgttgctgtt ggcctacgtc cttctcacct acgtcctgct gctcaacatg
1920ctcattgctc tcatgagcga gaccgtcaac agtgtcgcca ctgacagctg gagcatctgg
1980aagctgcaga aagccatctc tgtcctggag atggagaatg gctattggtg gtgcaggaag
2040aagcagcggg caggtgtgat gctgaccgtt ggcactaagc cagatggcag ccccgatgag
2100cgctggtgct tcagggtgga ggaggtgaac tgggcttcat gggagcagac gctgcctacg
2160ctgtgtgagg acccgtcagg ggcaggtgtc cctcgaactc tcgagaaccc tgtcctggct
2220tcccctccca aggaggatga ggatggtgcc tctgaggaaa actatgtgcc cgtccagctc
2280ctccagtcca actga
2295272292DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 27atgacttcag cctccagccc cccagctttc
aggctggaga cttccgatgg agatgaagag 60ggcaatgctg aggtgaacaa ggggaagcag
gaaccgcccc ccatggagtc accattccag 120agggaggacc ggaattcctc ccctcagatc
aaagtgaacc tcaacttcat aaagagacct 180cctaaaaaca cttctgctcc cagccagcag
gagccagatc ggtttgaccg tgaccgactc 240ttcagtgtgg tctcccgggg tgtccccgag
gaactgactg gactgctaga atacctgcgc 300tggaacagca agtacctcac tgactctgca
tacacagaag gctccactgg aaagacgtgc 360ctgatgaagg ctgtgctgaa ccttcaggat
ggggtcaatg cctgcatcat gccgctgctg 420cagattgaca aggattccgg caatcccaag
cccctcgtca atgcccagtg catcgatgag 480ttctaccaag gccacagtgc gctgcacatc
gccatagaga agaggagcct gcagtgcgtg 540aagctgctgg tagagaatgg agcggatgtt
cacctccgag cctgtggccg cttcttccaa 600aagcaccaag gaacttgttt ctattttgga
gagctacctc tttctctggc tgcgtgcacc 660aagcagtggg atgtggtgac ctacctcctg
gagaacccac accagccggc cagcctggag 720gccaccgact ccctgggcaa cacagtcctg
catgctctgg taatgattgc agataactcg 780cctgagaaca gtgccctggt gatccacatg
tacgacgggc ttctacaaat gggggcgcgc 840ctctgcccca ctgtgcagct tgaggaaatc
tccaaccacc aaggcctcac acccctgaaa 900ctagccgcca aggaaggcaa aatcgagatt
ttcaggcaca ttctgcagcg ggaattctca 960ggaccgtacc agcccctttc ccgaaagttt
actgagtggt gttacggtcc tgtgcgggta 1020tcgctgtacg acctgtcctc tgtggacagc
tgggaaaaga actcggtgct ggagatcatc 1080gcttttcatt gcaagagccc gaaccggcac
cgcatggtgg ttttagaacc actgaacaag 1140cttctgcagg agaaatggga tcggctcgtc
tcaagattct tcttaaactt cctgtgtaat 1200ctgatctaca tgttcatctt caccgctgtt
gcctaccatc agcctaccct gaagaagcag 1260gccgcccctc acctgaaagc ggaggttgga
aactccatgc tgctgacggg ccacatcctt 1320atcctgctag gggggatcta cctcctcgtg
ggccagctgt ggtacttctg gcggcgccac 1380gtgttcatct ggatctcgtt catagacagc
tactttgaaa tcctcttcct gttccaggcc 1440ctgctcacag tggtgtccca ggtgctgtgt
ttcctggcca tcgagtggta cctgcccctg 1500cttgtgtctg cgctggtgct gggctggctg
aacctgcttt actatacacg tggcttccag 1560cacacaggca tctacagtgt catgatccag
aaggtcatcc tgcgggacct gctgcgcttc 1620cttctgatct acttagtctt ccttttcggc
ttcgctgtag ccctggtgag cctgagccag 1680gaggcttggc gccccgaagc tcctacaggc
cccaatgcca cagagtcagt gcagcccatg 1740gagggacagg aggacgaggg caacggggcc
cagtacaggg gtatcctgga agcctccttg 1800gagctcttca aattcaccat cggcatgggc
gagctggcct tccaggagca gctgcacttc 1860cgcggcatgg tgctgctgct gctgctggcc
tacgtgctgc tcacctacat cctgctgctc 1920aacatgctca tcgccctcat gagcgaaact
gtcaaccacg ttgctgacaa cagctggagc 1980atctggaagt tgcagaaagc catctctgtc
ttggagatgg agaatggtta ctggtggtgc 2040cggaggaaga aacatcgtga agggaggctg
ctgaaagtcg gcaccagggg ggatggtacc 2100cctgatgagc gctggtgctt cagggtggag
gaagtaaatt gggttgcttg ggagaagact 2160cttcccacct tatctgagga tccatcaggg
ccaggcatca ctggtaataa aaagaaccca 2220acctctaaac cggggaagaa cagtgcctca
gaggaagacc atctgcccct tcaggtcctc 2280cagtccccct ga
2292282280DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
28atgacctcac cctccagctc tccagttttc aggttggaga cattagatgg aggccaagaa
60gatggctctg aggcggacag aggaaagctg gattttggga gcgggctgcc tcccatggag
120tcacagttcc agggcgagga ccggaaattc gcccctcaga taagagtcaa cctcaactac
180cgaaagggaa caggtgccag tcagccggat ccaaaccgat ttgaccgaga tcggctcttc
240aatgcggtct cccggggtgt ccccgaggat ctggctggac ttccagagta cctgagcaag
300accagcaagt acctcaccga ctcggaatac acagagggct ccacaggtaa gacgtgcctg
360atgaaggctg tgctgaacct taaggacgga gtcaatgcct gcattctgcc actgctgcag
420atcgacaggg actctggcaa tcctcagccc ctggtaaatg cccagtgcac agatgactat
480taccgaggcc acagcgctct gcacatcgcc attgagaaga ggagtctgca gtgtgtgaag
540ctcctggtgg agaatggggc caatgtgcat gcccgggcct gcggccgctt cttccagaag
600ggccaaggga cttgctttta tttcggtgag ctacccctct ctttggccgc ttgcaccaag
660cagtgggatg tggtaagcta cctcctggag aacccacacc agcccgccag cctgcaggcc
720actgactccc agggcaacac agtcctgcat gccctagtga tgatctcgga caactcagct
780gagaacattg cactggtgac cagcatgtat gatgggctcc tccaagctgg ggcccgcctc
840tgccctaccg tgcagcttga ggacatccgc aacctgcagg atctcacgcc tctgaagctg
900gccgccaagg agggcaagat cgagattttc aggcacatcc tgcagcggga gttttcagga
960ctgagccacc tttcccgaaa gttcaccgag tggtgctatg ggcctgtccg ggtgtcgctg
1020tatgacctgg cttctgtgga cagctgtgag gagaactcag tgctggagat cattgccttt
1080cattgcaaga gcccgcaccg acaccgaatg gtcgttttgg agcccctgaa caaactgctg
1140caggcgaaat gggatctgct catccccaag ttcttcttca acttcgcctg ctacttggtc
1200tacatgttca tcttcaccgt cgttgcctac caccagcctt ccctggatca gccagccatc
1260ccctcatcaa aagcgacttt tggggaatcc atgctgctgc tgggccacat tctgatcctg
1320cttgggggta tttacctctt actgggccag ctgtggtact tttggcggcg gcgcctgttc
1380atctggatct cattcatgga cagctacttt gaaatcctct ttctccttca ggctctgctc
1440acagtgctgt cccaggtgct gcgcttcatg gagactgaat ggtacctacc cctgctagtg
1500ttatccctag tgctgggctg gctgaacctg ctttactaca cacggggctt tcagcacaca
1560ggcatctaca gtgtcatgat ccagaaggtc atccttcgag acctgctccg tttcctgctg
1620gtctacctgg tcttcctttt cggctttgct gtagccctag taagcttgag cagagaggcc
1680cgaagtccca aagcccctga agataacaac tccacagtga cggaacagcc cacggtgggc
1740caggaggagg agccagctcc atatcggagc attctggatg cctccctaga gctgttcaag
1800ttcaccattg gtatggggga gctggctttc caggaacagc tgcgttttcg tggggtggtc
1860ctgctgttgc tgttggccta cgtccttctc acctacgtcc tgctgctcaa catgctcatt
1920gctctcatga gcgaaactgt caaccacgtt gctgacaaca gctggagcat ctggaagttg
1980cagaaagcca tctctgtctt ggagatggag aatggttact ggtggtgccg gaggaagaaa
2040catcgtgaag ggaggctgct gaaagtcggc accagggggg atggtacccc tgatgagcgc
2100tggtgcttca gggtggagga agtaaattgg gttgcttggg agaagactct tcccacctta
2160tctgaggatc catcagggcc aggcatcact ggtaataaaa agaacccaac ctctaaaccg
2220gggaagaaca gtgcctcaga ggaagaccat ctgccccttc aggtcctcca gtccccctga
2280
