Patent Document

[0001]    This application is a divisional of application Ser, No. 09/617,099, filed Jul. 14, 2000, which claims priority of Japanese Application No. 11-288372, filed Oct. 8, 1999. The entire disclosure of application No. 9/617,099 is expressly incorporated by reference herein in its entirety. 
     
    
     
       FIELD OF THE INVENTION  
         [0002]    The present invention relates to protein Rim2, which is a novel isoform of Rim, i.e., a protein that interacts with a low molecular G protein Rab3 and is proposed to serve as a regulator of Rab3-dependent synaptic vesicle fusion, and which specifically interacts with the GDP/GTP exchange factor (GEFII; a cAMP sensor). More specifically, the present invention relates to elucidation of the mechanisms of intracellular vesicle transport and secretion, and to the novel protein Rim2 which is useful in diagnosis of endocrine-related diseases or neuropathy and in development of agents for prevention and treatment thereof, the gene encoding Rim2 and an antibody addressed to Rim2 protein.  
           [0003]    Rim2 is considered to be a regulatory factor of vesicle fusion. It was found in the course of the present invention that the protein is expressed predominantly in endocrine tissues and endocrine- and neuroendocrine-derived cell lines. GTP-Rab3/GEFII/Rim complex is thought to participate in the regulation of exocytosis of neurons and endocrine cells, in a cAMP-dependent and protein kinase A (PKA) independent manner.  
         BACKGROUND OF THE INVENTION  
         [0004]    Transport of substances between cell organelles, which are unit membrane-enclosed structures such as endoplasmic reticulum, is conducted by intracellular vesicle transport. In endocrine cells including pancreatic β-cells and pituitary cells, peptides/proteins synthesized at ribosomes are received by the endoplasmic reticulum, from which they are transported in vesicles, which are transformed into secretory vesicles through the Golgi body and transported to the cell membrane, where they are released out of the cell via a step which includes fusion of the membranes. In neurons, neurotransmitter-containing precursors of synaptic vesicles are formed in Golgi bodies and transported by microtubules along the axon and stored at the synapse. Depolarization of the pre-synaptic membrane causes the vesicles to fuse with the pre-synaptic membrane and thus the neurotransmitters are released. This type of secretion based on the fusion of the vesicles and the cell membrane is called exocytosis.  
           [0005]    In contrast, when extracellular substances such as hormones including cell growth factors are bound to the cell membrane, the complexes thus formed are invaginated into the cell to form endosomes. This type of uptake of environmental substances is called endocytosis.  
           [0006]    Formation of vesicles, such as by budding, commonly observed both in exocytosis and endocytosis, and docking and fusion, the phenomena observed in process of their transportation and binding to other membrane systems, are regulated by a GTP-binding, low-molecular protein, called G protein. More than 30 types of this protein are known. The group of the proteins, which are also classified in Rab family, regulate the intracellular vesicle transport system.  
           [0007]    With regard to the intracellular vesicle transport system, it is understood today that a cell is in a resting state when Rab protein occurs in a bound form to guanine nucleotide diphosphate (GDP), and that budding, docking and fusion are triggered as a result of a process in which a protein having GEF activity act on Rab protein and converts it to GTP-binding Rab protein, to which GTP binds to form a GTP-Rab complex, which in turn binds to a corresponding target protein on the membrane.  
           [0008]    Stimulus-secretion coupling plays an important role in exocytosis observed in many cell types including neurons and endocrine cells [J. E. Rothman, Nature 372:55(1994); T. C. Sudhof, Nature 375:645 (1995)]. While a rise in intracellular Ca 2+  concentration is important in the regulation of exocytosis, other signals are also known to play important roles. cAMP (cyclic adenosine-3′,5′-monophosphate)/PKA (cAMP-dependent protein kinase A) signaling pathway is known to regulate exocytosis in many of neurons, neuroendocrine cells and endocrine cells. In particular, cAMP has been thought to mediate long-term potentiation by increasing neurotransmitter release in the brain [R. D. Hawkins et al. Ann. Rev. Neurosci. 16:625(1993); G. Lonart et al., Neuron 21:1141(1998)]. cAMP also regulates exocytosis responsible for insulin release from pancreatic β-cells and amylase release from parotid acinar cells [P. M. Jones and S. J., Persaud, Endocrine. Rev. 19:429(1998); E. Renstrom, et al., J. Physiol. 502:105(1997); K. Yoshimura, Biochim. Biophys. Acta 1402:171(1998)].  
           [0009]    In addition to its role in PKA-dependent phosphorylation of regulatory proteins associated with the process of exocytosis, it is known that cAMP also acts directly on the exocytotic machinery in neurons and non-neuronal cells [G. Lonart et al., Neuron 21:1141 (1998); E. Renstrom et al., J. Physiol. 502:105 (1997); K. Yoshimura, Biochim. Biophys. Acta, 1402:171(1998)].  
           [0010]    During the search by the yeast two-hybrid screen (i.e., a method for detection of the interaction between two proteins in yeast cells) for an intracellular signaling molecule directly coupling to a sulphonylurea receptor, a component of pancreatic β-cell ATP-sensitive K +  (K ATP ) channels [N. Inagaki et al. Proc. Natl. Acad. Sci. U.S.A. 91,2679 (1994)], a CAMP sensor protein (called “CAMPS”) was identified and it was found that the protein has two putative cAMP binding domains, a Pleckstrin homology domain (PH domain), and a guanine nucleotide exchange factor (GEF) homology domain.  
           [0011]    In the course of this study, two study groups independently reported CAMP binding proteins that activate Rap1, a member of the small G binding proteins [J. de Rooiji et al. Nature 396:474 (1998); H. Kawasaki et al. Science 282:2275 (1998)], and CAMPS was incidentally revealed to be a mouse homologue of cAMP-GEFII [H. Kawasaki et al. Science 282:2275 (1998)].  
           [0012]    Though the mechanisms of intracellular vesicle transport system have thus gradually been clarified, substantial part of them remains still unknown. Further progress is needed for the understanding of the mechanisms so as to provide diagnostic agents or therapeutics for a variety of diseases which involve neurons or endocrine cells.  
           [0013]    Unlike the former suggestion that only a single cAMP binding domain was present in CAMP-GEFII, the study by the present inventors suggested the presence of two putative CAMP binding domains (cAMP-A and cAMP-B), based on a sequence alignment of cAMP-GEFII sequence and regulatory subunits of PKA. FIG. 1 shows the sequence alignment of the CAMP binding domains. The CAMP binding domains A and B (cAMP-A and CAMP-B, respectively) of cAMP-GEFII and the cAMP binding domains A and B of the PKA regulatory subunit I α (RI α-A and RI α-B, respectively) are shown. The invariant residues in the different cAMP-binding domains are indicated by black boxes.  
           [0014]    As shown in FIG. 2, a glutathione-S-transferase (GST)-cAMP-A fusion protein bound to [ 3 H]cAMP with a dissociation constant (Kd) of ˜10 μM, while the binding of [ 3 H]CAMP to a GST-cAMP-B fusion protein was not evident under the same conditions.  
           [0015]    [0015]FIG. 2 shows the binding of cAMP to cAMP-A. GST-cAMP-A (filled circles) or GST-PKA RI α (open circles) was incubated with different concentrations of [ 3 H]CAMP (0-50 μM). The data for cAMP-A or PKA RI α are normalized relative to maximal cAMP binding activities. Kd values are 10.0±2.3 μM and 23.7±0.6 nM for cAMP and PKA RI α, respectively.  
           [0016]    In the cAMP-B domain, the amino acid residue 423, which originally is glutamic acid (Glu), is substituted with lysine (Lys). This glutamic acid residue is important for CAMP binding. Considering that a more rapid dissociation than the wild-type was observed with a PKA regulatory subunit having an equivalent mutation (E-200-K), CAMP-B may also dissociate CAMP rapidly. Thus, a possibility remains that CAMP binds to the CAMP-B domain.  
         SUMMARY OF THE INVENTION  
         [0017]    As identification of a target molecule of CAMPUS, CAMP-GEFII, would serve to show its physiological role, the present inventors attempted to find a molecule that interacts with CAMP-GEFII by means of a yeast two-hybrid screen (YTH) method on the MIN6 cDNA library (See “Identification of Interacting molecules by YTH Method”).  
           [0018]    Surprisingly, the present inventors found that cAMP-GEFII interacts with a novel isoform (named “Rim2” by the present inventors) of Rim (a molecule which specifically interacts with Rab3: Rab3-interacting molecule: Hereinafter referred to as “Rim1”). Rim1 protein is a putative effector of the small G protein Rab3 and is proposed to serve as a Rab3-dependent regulator of synaptic vesicle fusion [Y. Wang et al. Nature 388:593(1997)].  
           [0019]    The full-length novel protein Rim2 sequenced by the present inventors, which consists of 1590 amino acid residues, was found to have 61.6% identity with rat Rim1. As FIG. 3 shows, a zinc finger, PDZ and two C2 domains were found highly conserved between Rim-1 and Rim2.  
           [0020]    Based on the above findings, the present invention provides a protein having the amino acid sequence set forth under SEQ ID NO: 1 in the Sequence Listing.  
           [0021]    The present invention further provides a protein having an amino acid sequence with one or more amino acids deleted, substituted, inserted or added relative to the amino acid sequence set forth under SEQ ID NO: 1 in the Sequence Listing and which has a property to interact with GDP/GTP exchange factor II.  
           [0022]    The present invention further provides a mouse gene which encodes the following proteins (1) or (2):  
           [0023]    (1) a protein having the amino acid sequence set forth under SEQ ID NO: 1 in the Sequence Listing,  
           [0024]    (2) a protein having an amino acid sequence with one or more amino acids deleted, substituted, inserted or added relative to the above-identified amino acid sequence and which has a property to interact with GDP/GTP exchange factor II.  
           [0025]    In the present specification, “one or more” amino acid residues are generally several (e.g., 3 or 4) to 10 residues.  
           [0026]    The present invention further provides a DNA having a nucleotide sequence set forth under SEQ ID NO: 2 in the Sequence Listing, the DNA being a cDNA corresponding to the above protein having the amino acid sequence set forth under SEQ ID NO: 1 in the Sequence Listing.  
           [0027]    The present invention further provides a DNA having a nucleotide sequence with one or more nucleotides deleted, substituted, inserted or added relative to the nucleotide sequence set forth under SEQ ID NO: 2 in the Sequence Listing and encoding any one of the above proteins. Herein, “one or more” nucleotides are generally several (e.g., 3 or 4) to 10 nucleotides. A variety of such nucleotide sequences with one or more nucleotides deleted, substituted, inserted or added can be readily prepared by those skilled in the art by making use of the familiar knowledge on degeneracy of the genetic code.  
           [0028]    The present invention further provides a DNA having the nucleotide sequence of the coding region of the any one of the above DNA&#39;s or of a DNA having the nucleotide sequence set forth under SEQ ID NO: 2 in the Sequence Listing.  
           [0029]    The present invention further provides a DNA fragment consisting of a part of any one of the above DNA&#39;s.  
           [0030]    The present invention further provides a probe comprising a DNA which hybridizes with the DNA consisting of any one of the above nucleotide sequences.  
           [0031]    The present invention further provides a primer DNA fragment consisting of a partial sequence of any one of the above nucleotide sequences.  
           [0032]    The present invention further provides a recombinant vector having any one of the above DNA&#39;s.  
           [0033]    The present invention further provides a monoclonal or polyclonal antibody directed to any one of the above proteins.  
           [0034]    The present invention further provides a diagnostic agent for human use comprising any one of the above probes or antibodies. The diagnostic agent is useful in the test for such diseases as secretion disorders in secretory systems including pituitary, hypothalamus, pancreatic β-cells and parotid gland, or the test for brain-nervous system diseases.  
           [0035]    The present invention further provides a therapeutic agent for any one of the above diseases. 
       
    
    
     BRIEF DESCRIPTION OF THE FIGURES  
       [0036]    [0036]FIG. 1 illustrates a sequence alignment of the cAMP binding domains.  
         [0037]    [0037]FIG. 2 is a graph showing the binding of cAMP to cAMP-A.  
         [0038]    [0038]FIG. 3 illustrates a comparison of amino acid identity between Rim1 and Rim2, in zinc finger, PDZ and C2 domains.  
         [0039]    [0039]FIG. 4 shows the results of immunoblotting showing the interaction between cAMP-GEFII and Rim1 or Rim2.  
         [0040]    [0040]FIG. 5 shows the results of Northern blot analysis of cAMP-GEFII, Rim1 and Rim2 in various rat tissues and endocrine- and neuroendocrine-derived cell lines.  
         [0041]    [0041]FIG. 6 is the result of In situ hybridization showing the localization of Rim1 and Rim2 in mouse brain and pituitary.  
         [0042]    [0042]FIG. 7 is a graph showing the result of yeast two-hybrid assays.  
         [0043]    [0043]FIG. 8 illustrates the result of immunoblotting showing the interaction between Rab3A and Rim1 or Rim2 in vitro.  
         [0044]    [0044]FIG. 9 is a graph showing the time course for high K + -induced GH secretion from PC12 cells cotransfected with GH and CAMP-GEFII.  
         [0045]    [0045]FIG. 10 is a graph showing the effect of forskolin on GH secretion from transfected PC cells.  
         [0046]    [0046]FIG. 11 is a graph showing forskolin-induced GH secretion from PC12 cells transfected with various mutant CAMP-GEFII.  
         [0047]    [0047]FIG. 12 is a graph showing the effect of H-89 on forskolin-induced GH secretion from PC12 cells transfected with CAMP-GEFII.  
         [0048]    [0048]FIG. 13 is a schematic illustration showing a model for cAMP-dependent exocytosis. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0049]    A variety of mutants can be provided by means of recombinant DNA technology. First, mutations can be introduced into a DNA clone fragment through different chemical and/or enzymatic processes, and the mutant DNA&#39;s thus obtained are then sequenced to select particular mutants with intended merits. This method allows a systematic preparation of different mutants regardless of their phenotypes. General methods of preparing a mutant clone DNA are as follows.  
         [0050]    1. With the help of an oligonucleotide, substitution, deletion, insertion or addition can be directly effected in a given DNA sequence. This method enables to introduce a number of mutations in a small region of a given DNA.  
         [0051]    2. By using longer oligonucleotides, it is possible to synthesize a desired gene.  
         [0052]    3. By means of region-specific mutagenesis, a desired mutation can be introduced into a large (1-3 kb) DNA region.  
         [0053]    4. Linker-scanning mutagenesis of DNA is a method suited for introducing a cluster point mutation into a relatively small (4-10 bp) DNA region.  
         [0054]    5. PCR is also utilized as a method for direct introduction of a mutation. [References: Current Protocols in Molecular Biology., 3 Vols., Edited by Ausubel F. M. et al., John Wiley &amp; Sons, Inc., Current Protocols., Vol. 1, Chapter 8: Mutagenesis of Cloned DNA, pages 8.0.1-8.5.10] 
         [0055]    Also well known to those skilled in the art are methods of preparing plasmids or vectors which can express a desired gene including different mutations obtained by the above methods. That is, by inserting a DNA carrying a desired gene into a expression vector DNA using a combination of restriction enzymes and a ligase, a recombinant plasmid is readily constructed which carries the desired gene. The recombinant plasmid thus obtained is then introduced into different cells to transfect them, thereby producing transformed cells. Cells which may be utilized range from prokaryotes, e.g.  E. coli,  to yeast, insect, plant and animal cells. [References: Vectors Essential Data. Gacesa P. and Ramji D. P., 166 pages. BIOS Scientific Publishers Limited 1994., John Wiley &amp; Sons in association with BIOS Scientific Publishers Ltd. Expression vectors, pages 9-12.] 
         [0056]    Introduction of a recombinant plasmid into host cells is effected by calcium chloride method or electroporation. Calcium chloride method provides efficient transformation without requiring any special apparatus. For higher efficiency, electroporation is recommended.  
         [0057]    [References: Current Protocols in Molecular Biology, 3 Vols. Edited by Ausbel F. M. et al., John Wiley &amp; Sons, Inc., Current Protocols, Vol. 1, unit 1.8: Introduction of Plasmid DNA into Cells, pages 1.8.1-1.8.10] 
         [0058]    Two types are known of transfection generally carried out on animal cell lines, i.e., transient and permanent types. In transient transfection, transformed cells are cultured for 1-4 days to effect transcription and replication of the transfected gene, and then the cells are harvested and their DNA analyzed. Alternatively, in many studies, a stable transformant cell line is produced, in which the transfected gene is incorporated into the chromosomes. Examples of the method for transfection include calcium phosphate method, electroporation, and liposome fusion method.  
         [0059]    [Reference: Current protocols in molecular biology. 3 vols. Edited by Ausubel F. M. et al., John Wiley &amp; Son, Inc., Current Protocols. Vol. 1, chapter 9: Introduction of DNA into mammalian cells, pages 9.0.1-9.17.3.] 
         [0060]    Polyclonal and monoclonal antibodies directed to the proteins (polypeptides) coded by Rim2 gene of the present invention or their fragments and analogues as well, are readily prepared using techniques well known in the art. Antibodies obtained may be used as laboratory reagents and diagnostic agents for diseases associated with Rim2 gene. The antibodies obtained are also used for preparation of antibody columns, for immunoprecipitation as well as for identification of the antigen by Western blotting.  
         [0061]    A general method for preparing a monoclonal antibody in mg-scale directed to the proteins coded for by Rim2 gene of the present invention is as follows: Mice are inoculated with the antigen protein to immunize. The spleen is removed from the mice exhibiting a sufficient antibody titer. The spleen cells are dissociated, and selected B cells are fused with myeloma cells of B cell origin to form hybridoma cells which secrete the antibody. The monoclonal antibody secreted from the hybridoma cells is purified from the culture medium using an affinity column, ion-exchange, or gel filtration, etc. The polyclonal antibody of the present invention may be prepared by a conventional method: Using rabbits, horses, mice or guinea pigs as immunized animals, the antigen protein is inoculated along one of the schedules known in the art to immunize the animals, and then IgG, etc. are isolated from the collected serum.  
         [0062]    [Reference: Current protocols in molecular biology, 3 vols. Edited by Ausubel F. M. et al., John Wiley &amp; Sons, Inc., Current Protocols, Vol. 2, chapter 11: Immunology, pages 11.0.1-11.16.13.] 
         [0063]    In order to assess the specificity of interaction between cAMP-GEFII and Rim2, the present inventors evaluated the binding of a FLAG-tagged cAMP-GEFII protein to a GST-Rim2 fusion protein immobilized on glutathione beads (See “Study on Interaction between Rim2 and cAMP-GEFII: I”).  
         [0064]    Briefly, lysates from COS-1 cells transfected with FLAG-tagged cAMP-GEFII, from MIN6 cells or from mouse brain homogenate were evaluated for binding to GST-Rim1, GST-Rim2 or GST alone. cAMP was detected by immunoblotting with an anti-FLAG antibody (FIG. 4, left) or an anti-cAMP-GEFII antibody (FIG. 4, center and right), respectively. These results demonstrates that cAMP-GEFII protein interacts with GST-Rim2 protein. Likewise, GST-Rim1 protein also bound to cAMP-GEFII in the mouse brain homogenate (See “Study on Interaction between Rim1 and cAMP-GEFII”) (FIG. 4, right). These results confirms that cAMP-GEFII interacts with Rim 1 and Rim2.  
         [0065]    [0065]FIG. 5 shows the results of the northern blot analyses of cAMP-GEFII, Rim1 and Rim2 in various rat tissues and endocrine- and neuroendocrine-derived cell lines (See “Northern Blotting in Rat Tissues”). Ten μL g samples of total RNA from various tissues and cell lines (except 5 μg for pancreatic islets) were used. Hybridization and washing were performed under standard conditions. The faint signals seen in Rim2 mRNA blot analysis of cerebrum and cerebellum are due to cross-hybridization with the Rim1 cDNA probe used. FIG. 5 shows that Rim2 mRNA is expressed predominantly in endocrine tissues and endocrine- and neuroendocrine-derived cell lines, including pituitary, pancreatic Langerhans&#39; islet cells, MIN6 cells, and PC12 cells. Rim2 mRNA was detected in the brain by reverse transcriptase-PCR (data not shown). Rim1 mRNA, in contrast, was found to be expressed in cerebrum, cerebellum, and pituitary by a similar analysis.  
         [0066]    The major transcripts for Rim1 and Rim2 have 6.4 kb for Rim1, and 7.2 kb and 5.4 kb for Rim2. There are also found several minor transcripts, which occur due probably to alternative splicing.  
         [0067]    cAMP-GEFII mRNA is generally coexpressed with Rim1 or Rim2 mRNA in tissues and cell lines in which regulated exocytosis is known to occur. FIG. 6 illustrates the results of in situ hybridization showing the localization of Rim1 and Rim2 in mouse brain and pituitary. In the figure: (a) cAMP-GEFII; (b) Rim1; (c) Rim2; (d) pituitary. The scale bar corresponds to 1 mm. Abbreviations: Cb=cerebellum, Cp=caudoputamen, Cx=cortex, Hi=hippocampus, Ob=olfactory bulb, Po=pons, Th=thalamus  
         [0068]    Rim2 mRNA is found expressed only in the cerebellar cortex, while Rim1 mRNA is expressed in cerebral cortex, hippocampus (especially CA3 and dentate gyrus), olfactory bulb, and cerebellar cortex (See “In situ Hybridization in Mouse Brain”). The distribution of CAMP-GEFII mRNA overlaps largely with that of Rim1 mRNA in the brain. It is confirmed that Rim2 mRNA and cAMP-GEFII mRNA are coexpressed in anterior pituitary.  
         [0069]    Rim1 is proposed to be a Rab3 effector, a low molecular weight G protein [Y. Wang, et al., Nature 388,593 (1997)]. Using yeast two-hybrid assays (See “Study on Interaction between Rim2 and Rab3A”.), the present inventors found that Rim2, like Rim1, interacts with active Rab3A (Q81L) (FIG. 7). FIG. 7 shows the results of the yeast two-hybrid assays. Rim1, Rim2 or rabphilin3 and wild-type Rab3A or constitutively active Rab3A (Q81L) in various combinations were determined by transactivation of liquid β-galactosidase activity.  
         [0070]    In addition, the immobilized GST-Rim2 bound only to the GTPγS-bound form of Rab3A (FIG. 8). FIG. 8 shows the interaction between Rab3A and Rim1 or Rim2 in vitro, which is the result obtained by incubating GTPγS- or GDP γS-bound form of Rab3A with GST-Rim1 (residues 1-201) and GST-Rim2 (residues 1-345) immobilized on glutathione beads, respectively. Rab3A was detected by immunoblotting with anti-Rab3A antibody. These results indicate that Rim2, like Rim1, binds to the GTP-activated form of Rab3A.  
         [0071]    The interaction of CAMP-GEFII and Rim2 protein strongly suggests that cAMP-GEFII is involved in regulated-exocytosis. To determine its functional role, the present inventors examined the effect of cAMP on Ca2+-dependent secretion in PC12 cells cotransfected with growth hormone (GH) and CAMP-GEFII (See “Study on GH secretion from Transfected PC12 Cells”).  
         [0072]    Since PC12 cells endogenously express Rim2 but not CAMP-GEFII, the exogenously introduced CAMP-GEFII may form a complex with endogenous Rim2.  
         [0073]    [0073]FIG. 9 is a graph showing the time course of high K + -induced GH secretion from PC12 cells cotransfected with GH and CAMP-GEFII. FIG. 10 is a graph showing the effect of forskolin on GH secretion from the transfected PC12 cells. Forskolin (50 μM) was added 10 min before the incubation with a low K +  (4.7 mM) or high K +  (60 mM) solution. The meaning of the symbols are as follows: For basal (low K + -induced) secretion: cAMP-GEFII-transfectant (filled triangles); β-galactosidase-transfectant (control)(open circles); high K + -induced secretion: cAMP-GEFII-transfectant (filled circles); β-galactosidase-transfectant (control) (open circles). The values represent the percent GH amounts released into the medium relative to the total cellular GH amounts.  
         [0074]    In the cotransfected PC12 cells, as shown in FIG. 9, cAMP-GEFII did not alter Ca 2+ -dependent (60 mM K + ) secretion of cotransfected GH, compared to the control, but significantly enhanced forskolin (50 μM)-induced, Ca 2+ -dependent GH secretion (FIG. 10). Forskolin acts mainly on adenylate cyclase, serving to increase cAMP levels in the cells. cAMP-GEFII also enhanced 8-Br-cAMP (1 mM)-induced, Ca 2+ -dependent GH secretion (cAMP-GEFII-transfectant, 34.9±1.3%; control, 25.1±1.8%, n=9, P&lt;0.001).  
         [0075]    [0075]FIG. 11 is a graph showing forskolin-induced GH secretion from PC12 cells transfected with various mutant CAMP-GEFII, in which the increment of forskolin (50 μM)-induced GH secretion (in the presence of high K + ) above the basal level during a 15-min incubation for each mutant CAMP-GEFII is expressed as percentage relative to the wild-type CAMP-GEFII (100%). In the figure: WT=wild-type CAMP-GEFII, T810A=mutant CAMP-GEFII (T810A); G114E, G422D=double mutant CAMP-GEFII (G114, G422D).  
         [0076]    The forskolin-induced GH secretion was not affected in the mutant CAMP-GEFII (T810A) in which a potential PKA phosphorylation site is disrupted by substitution of one of its amino acids (FIG. 11). In addition, the forskolin-induced GH secretion in the mutant CAMP-GEFII (G114E, G422D) in which both of the CAMP binding sites are disputed was reduced to ˜40% of that in the wild-type.  
         [0077]    These results indicate that CAMP promotes Ca 2+ -dependent GH secretion by binding to CAMP-GEFII, without involving its phosphorylation by PKA.  
         [0078]    [0078]FIG. 12 is a graph showing the effect of H-89 on forskolin-induced GH secretion from cAMP-GEFII-transfected PC12 cells. H-89 (10 μM) was added to the incubation buffer 10 min before forskolin (50 μM) treatment. The treatment with H-89 (10 μM) reduced high K + -induced GH secretion in both of the CAMP-GEFII-transfected and β-galactosidase-transfected PC12 cells. The data were obtained from 3-5 independent experiments (A-D). The values are means ± SEM (P&lt;0.01).  
         [0079]    Importantly, the forskolin-induced, Ca2+-dependent GH secretion from the cAMP-GEFII-transfected PC12 cells treated with the PKA inhibitor H-89 was significantly higher than that from the control cells. This indicates that cAMP-GEFII mediates cAMP-dependent and PKA-independent exocytosis.  
         [0080]    To ascertain the physiological relevance of cAMP-GEFII, the present inventors investigated the role of endogenous cAMP-GEFII in secretion. In insulin secretion from pancreatic β-cells, cAMP is proposed to stimulate exocytosis by PKA-dependent as well as PKA-independent mechanisms [M. Prentki, F. M. Matschinsky, Physiol. Rev. 67:1185 (1987)/P. M. Jones, S. J. Persaud, Endocrine. Rev. 19:429 (1998)].  
         [0081]    In the high glucose condition of 16.7 mM, 8-Br-cAMP-induced insulin secretion from MIN6 cells treated with antisense oligonucleotides against cAMP-GEFII was significantly reduced (87.5±2.3% of the secretion from MIN6 cells treated with a control oligonucleotide, n=27, P&lt;0.005) (See “Study of the Role of cAMP-GEFII in cAMP-dependent Exocytosis”), suggesting that cAMP-GEFII participates in cAMP-dependent exocytosis in native cells.  
         [0082]    Rab3 is associated with the final step of exocytosis. The structurally-related proteins rabphilin3 [H. Shirataki et al., Mol. Cell. Biol. 13,2061 (1993)] and Rim1 both bind to Rab3A, suggesting that multiple Rab3A effectors could operate in triggering docking and fusion of the vesicles to the plasma membrane.  
         [0083]    In the process toward the present invention, it was found that the cAMP sensor, cAMP-GEFII, mediates cAMP-induced, Ca 2+ -dependent exocytosis by interacting with a Rab3 effector Rim2.  
         [0084]    In addition to its role in PKA phosphorylation of proteins associated with secretory processes, previous studies have suggested that cAMP may act directly on the exocytosis [G. Lonart, et al., Neuron 21:1141 (1998); E. Renstrom, et al., J. Physiol. 502:105(1997); K. Yoshimura et al., Biochim. Biophys. Acta 1402:171(1998)]. In pancreatic β-cells, too, PKA-dependent as well as PKA-independent stimulation of insulin release by cAMP has been proposed [E. Renstrom, et al., J. Physiol. 502:105 (1997)]. It is thought that cAMP probably directly stimulates amylase release in parotid acinar cells [G. Lonart, et al., Neuron 21:1141 (1998)]. In addition, a recent study suggests that cAMP enhances glutamate release in the brain partly by a direct action on the exocytotic machinery [G. Lonart, et al., Neuron 21,1141 (1998)].  
         [0085]    However, while both rabphilin3 and Rim1 are ubiquitously expressed in most of the synapses in the brain[C. Li et al., Neuron 13:885 (1994)], cAMP-enhanced glutamate release occurs in synaptosomes from the CA3 region in the hippocampus, not from the CA1 region, a finding consistent with cAMP-GEFII and Rim1 being coexpressed predominantly in CA3.  
         [0086]    Accordingly, it is considered that, in addition to PKA-dependent phosphorylation in the secretory processes, cAMP promotes regulated exocytosis in a PKA-independent manner by acting directly on a complex of cAMP-GEFII (a cAMP sensor) and Rim (a Rab3 effector) in some neurons and neuroendocrine and endocrine cells, as schematically illustrated in FIG. 13.  
         [0087]    These findings indicates that Rim2 of the present invention also plays an important role in the regulation of exocytosis in neurons and endocrine cells.  
       EXAMPLES  
       [0088]    The present invention will be described below in further detail by presenting specific procedures in the present invention with reference to an example.  
         [0089]    Sequencing of CAMPS (cAMP-GEFII) cDNA  
         [0090]    A plasmid cDNA library has been made from a mouse insulin-secreting cell line, MIN6, in the vector pVP16. A yeast two-hybrid bait vector was constructed in plasmid pBTM 116 using a DNA fragment encoding partial rat SUR1 (amino acid residues 598-1003)(GenBank accession number L40624), a subunit of the pancreatic β-cell K ATP  channel.  
         [0091]    Yeast two-hybrid screen of the plasmid MIN6 cDNA library was performed as described in K. Kotake et al., J. -Biol. Chem. 272:29407 (1997). A prey clone encoding a partial CAMPS, a cAMP sensor, (residues 187-730) was isolated. A full-length mouse CAMPS cDNA was obtained from the A MIN6 cDNA library [N. Inagaki et al., Proc. Natl. Acad. Sci. U.S.A. 91:2679(1994)]. The nucleotide sequence of mouse CAMPS (cAMP-GEFII) has been deposited in Genbank with the accession number of AB021132.  
         [0092]    Preparation and Test of GST fusion Protein  
         [0093]    cAMP-A (amino acid residues 43-153), cAMP-B (amino acid residues 357-469), and rat PKA regulatory subunit (RI a)(full-length) were expressed as GST-fusion proteins using pGEX-4T-1 (Amersham-Pharmacia) and purified according to the manufacturer&#39;s instructions. CAMP binding assay was performed as described in R. A. Steiberg, et al., J. Biol. Chem. 262:2664(1987) with slight modifications.  
         [0094]    Briefly, GST-fusion protein (1 μg) was incubated in binding buffer (200 μl) containing various concentrations of [ 3 H]cAMP, 50 mM potassium phosphate buffer (pH 6.8), 150 mM NaCl, 1 mM EDTA, 5 mM  2 -mercaptoethanol, and 0.5 mg/ml bovine serum albumin with or without 40 mM unlabeled CAMP for 2 hrs on ice.  
         [0095]    Identification of Interacting Molecules by YTH Method  
         [0096]    Yeast two-hybrid bait vector was constructed in plasmid pBTM116 using a full-length mouse CAMP-GEFII cDNA. A prey clone encoding a partial sequence of Rim2 (amino acid residues 53-863) was isolated from the plasmid MIN6 cDNA library. A full-length cDNA for Rim2 was obtained from the A MIN6 cDNA library.  
         [0097]    Study on Interaction between Rim2 and CAMP-GEFII: I  
         [0098]    Rim2 (amino acid residues 538-863) was expressed as a GST fusion protein and purified according to the method described in “Preparation and Test of GST fusion Protein”. A full-length CAMP-GEFII cDNA was subcloned into plasmid pFLAG-CMV-2 (Sigma). The resultant construct was transfected into COS-1 cells, using LipofectAMINE (Life Technologies). The lysate of the COS-1 cells was incubated with GST-Rim2 immobilized on glutathione beads for 2 hrs at 4° C. The complex thus obtained was washed with distilled water, separated by SDS-PAGE, and immunoblotted with an anti-FLAG M2 antibody (Sigma).  
         [0099]    Study on Interaction between Rim2 and cAMP-GEFII: II  
         [0100]    The lysate of MIN6 cells was incubated with GST-Rim2 and interaction between cAMP-GEFII and Rim2 was evaluated according to the method described in “Study on Interaction between Rim2 and cAMP-GEFII: I”, using a IgG antibody raised against the C-terminus (amino acid residues 1001-1011, Gln-Met-Ser-His-Arg-Leu-Glu-Pro-Arg-Arg-Pro) (SEQ ID NO: 5) of mouse CAMP-GEFII.  
         [0101]    Study on Interaction between Rim1 and CAMP-GEFII  
         [0102]    According to the method described in “Preparation and Test of GST fusion Protein”, Rim1 partial sequence (530-806) was expressed as a GST fusion protein and then purified. The brain homogenate from three mice was incubated with GST-Rim1 immobilized on glutathione beads overnight at 4° C. CAMP-GEFII was detected as described in “Study on Interaction between Rim2 and CAMP-GEFII: II”.  
         [0103]    Northern Blotting in Rat Tissues  
         [0104]    Northern Blotting was performed for various tissues of rat using, as probes, mouse cAMP-GEFII (nucleic acids 606-2237), rat Rim1 (1035-1491), and mouse Rim2 (586-1490) cDNA  
         [0105]    In situ Hybridization in Mouse Brain  
         [0106]    In situ hybridization in mouse brain was performed as described in J. Tanaka, M. Murate, C. Z. Wang, S. Seino, T. Iwanaga, Arch. Histol. Cytol. 59:485 (1996).  
         [0107]    Antisense oligonucleotide probes (45 mer) used for mouse cAMP-GEFII and Rim2 correspond to the regions of the nucleic acids 2746-2790 and 1376-1420, respectively.  
         [0108]    For the antisense oligonucleotide for Rim1, Rim1 cDNA was partially cloned from mouse brain: the probe used in this was 5′-ttgcgctcactcttctggcctcccttgccattctgctctgaaagc-3′ (SEQ ID NO: 3).  
         [0109]    Study on Interaction between Rim2 and Rab3A  
         [0110]    According to the method described in “Identification of Interacting molecules by YTH Method”, the full-length cDNA&#39;s for wild type mouse Rab3A and constitutively active bovine Rab3A (Q81L) were cloned into the yeast bait vector pBTM116.  
         [0111]    The nucleotide sequence of zinc finger domains of bovine rabphilin3 (amino acid residues 1-283), rat Rim1 (amino acid residues 1-204) and mouse Rim2 (amino acid residues 1-345 were cloned into the prey vector pVP16. Liquid culture assay of B -galactosidase activities was performed according to the manufacturer&#39;s instructions (Clontech). The activity values were obtained from 3 independent clones for each transformant and normalized by cell numbers determined as OD 600 .  
         [0112]    Lipid-modified Rab3A was purified from the membrane fraction of Sf9 cells expressing Rab3A. Rat Rim1 (amino acid residues 1-204) and mouse Rim2 (amino acid residues 1-345) were expressed as GST fusion proteins and purified. The GTP γS- or GDPβS-bound form of Rab3A was incubated for 90 min at 4° C. with GST-Rim1, or GST-Rim2 (30 pmol for each) immobilized on glutathione beads in reaction buffer. Rab3A was detected by immunoblotting with anti-Rab3A antibody.  
         [0113]    Study on GH secretion from Transfected PC 12 Cells GH secretion from transfected PC12 cells was performed as described in K. Korake et al., J. Biol. Chem., 272:29407(1997). Expression plasmid vectors (pSR α) for wild-type cAMP-GEFII, mutant cAMP-GEFII (T810A), and the double mutant cAMP-GEFII (G114E, G422D) were prepared. As a control, β-galactosidase (β-gal) was used. PC cells were transfected with GH expression vector (pXGH5: Nichols Institute) plus each vector described above, using LipofectAMINE.  
         [0114]    PC12 cell were incubated with a low K 30   (4.7 mM) or high K +  (60 mM) solution, in the presence or absence of forskolin (50 μM) or 8-bromoadenosine 3′,5′ cyclic monophosphate (8-Br-cAMP)(1 mM). Forskolin or 8-Br-cAMP was added 10 min before the incubation with a low or high K + solution. In some experiments, the PKA inhibitor H-89 (10 μM) was added 10 min before forskolin stimulation.  
         [0115]    Study of the Role of cAMP-GEFII in cAMP-dependent Exocytosis  
         [0116]    To interfere with the synthesis of cAMP-GEFII in MIN6 cells, antisense phosphorothioate-substituted oligoDNA (16 mer) against mouse cAMP-GEFII (the region corresponding to nucleic acids 104-119) and control oligoDNA (5′-acctacgtgactacgt-3′) (SEQ ID NO: 4) were synthesized (BIOGNOSTIK).  
         [0117]    MIN6 cells were treated with 4 μM of the antisense oligoDNA or control oligoDNA 24 hours before insulin secretion experiments. The efficacy of antisense oligoDNA was evaluated by immunoblot analysis of the antisense oligoDNA-treated MIN6 cells over-expressing cAMP-GEFII by transient transfection, using anti-cAMP-GEFII antibody. The level of cAMP-GEFII was markedly lowered in the antisense oligoDNA-treated MIN6 cells. Insulin secretory response to 8-Br-cAMP (1 mM) of these MIN6 cells was assessed in the presence of high glucose (16.7 mM). Five separate experiments were performed, in which insulin was measured as described in T. Gonoi et al., J. Biol. Chem. 269:16989 (1994).  
 
     
       
       
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                                                SEQUENCE LISTING  
               
               
                   
               
               
                   
               
               
                 &lt;151&gt; PRIOR FILING DATE: 1999-10-08&lt;160&gt;  5  
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 1  
               
               
                 &lt;211&gt; LENGTH: 1590  
               
               
                 &lt;212&gt; TYPE: PRT  
               
               
                 &lt;213&gt; ORGANISM: Mus musculus  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 1  
               
               
                   
               
               
                 Met Ser Ala Pro Leu Gly Pro Arg Gly Arg Pro Ala Pro Thr Pro Ala  
               
               
                 1               5                   10                  15  
               
               
                   
               
               
                 Ala Ser Gln Pro Pro Pro Gln Pro Glu Met Pro Asp Leu Ser His Leu  
               
               
                             20                  25                  30  
               
               
                   
               
               
                 Thr Glu Glu Glu Arg Lys Ile Ile Leu Ala Val Met Asp Arg Gln Lys  
               
               
                         35                  40                  45  
               
               
                   
               
               
                 Lys Glu Glu Glu Lys Glu Gln Ser Val Leu Lys Ile Lys Glu Glu His  
               
               
                     50                  55                  60  
               
               
                   
               
               
                 Lys Ala Gln Pro Thr Gln Trp Phe Pro Phe Ser Gly Ile Thr Glu Leu  
               
               
                 65                  70                  75                  80  
               
               
                   
               
               
                 Val Asn Asn Val Leu Gln Pro Gln Gln Lys Gln Pro Asn Glu Lys Glu  
               
               
                                 85                  90                  95  
               
               
                   
               
               
                 Pro Gln Thr Lys Leu His Gln Gln Phe Glu Met Tyr Lys Glu Gln Val  
               
               
                             100                 105                 110  
               
               
                   
               
               
                 Lys Lys Met Gly Glu Glu Ser Gln Gln Gln Gln Glu Gln Lys Gly Asp  
               
               
                         115                 120                 125  
               
               
                   
               
               
                 Ala Pro Thr Cys Gly Ile Cys His Lys Thr Lys Phe Ala Asp Gly Cys  
               
               
                     130                 135                 140  
               
               
                   
               
               
                 Gly His Asn Cys Ser Tyr Cys Gln Thr Lys Phe Cys Ala Arg Cys Gly  
               
               
                 145                 150                 155                 160  
               
               
                   
               
               
                 Gly Arg Val Ser Leu Arg Ser Asn Lys Val Met Trp Val Cys Asn Leu  
               
               
                                 165                 170                 175  
               
               
                   
               
               
                 Cys Arg Lys Gln Gln Glu Ile Leu Thr Lys Ser Gly Ala Trp Phe Tyr  
               
               
                             180                 185                 190  
               
               
                   
               
               
                 Asn Ser Gly Ser Asn Thr Leu Gln Gln Pro Asp Gln Lys Val Pro Arg  
               
               
                         195                 200                 205  
               
               
                   
               
               
                 Gly Leu Arg Asn Glu Glu Ala Pro Gln Glu Lys Lys Ala Lys Leu His  
               
               
                     210                 215                 220  
               
               
                   
               
               
                 Glu Gln Pro Gln Phe Gln Gly Ala Pro Gly Asp Leu Ser Val Pro Ala  
               
               
                 225                 230                 235                 240  
               
               
                   
               
               
                 Val Glu Lys Gly Arg Ala His Gly Leu Thr Arg Gln Asp Thr Ile Lys  
               
               
                                 245                 250                 255  
               
               
                   
               
               
                 Asn Gly Ser Gly Val Lys His Gln Ile Ala Ser Asp Met Pro Ser Asp  
               
               
                             260                 265                 270  
               
               
                   
               
               
                 Arg Lys Arg Ser Pro Ser Val Ser Arg Asp Gln Asn Arg Arg Tyr Glu  
               
               
                         275                 280                 285  
               
               
                   
               
               
                 Gln Ser Glu Glu Arg Glu Asp Tyr Ser Gln Tyr Val Pro Ser Asp Gly  
               
               
                     290                 295                 300  
               
               
                   
               
               
                 Thr Met Pro Arg Ser Pro Ser Asp Tyr Ala Asp Arg Arg Ser Gln Arg  
               
               
                 305                 310                 315                 320  
               
               
                   
               
               
                 Glu Pro Gln Phe Tyr Glu Glu Pro Gly His Leu Asn Tyr Arg Asp Ser  
               
               
                                 325                 330                 335  
               
               
                   
               
               
                 Asn Arg Arg Gly His Arg His Ser Lys Glu Tyr Ile Val Asp Asp Glu  
               
               
                             340                 345                 350  
               
               
                   
               
               
                 Asp Val Glu Ser Arg Asp Glu Tyr Glu Arg Gln Arg Arg Glu Glu Glu  
               
               
                         355                 360                 365  
               
               
                   
               
               
                 Tyr Gln Ala Arg Tyr Arg Ser Asp Pro Asn Leu Ala Arg Tyr Pro Val  
               
               
                     370                 375                 380  
               
               
                   
               
               
                 Lys Pro Gln Pro Tyr Glu Glu Gln Met Arg Ile His Ala Glu Val Ser  
               
               
                 385                 390                 395                 400  
               
               
                   
               
               
                 Arg Ala Arg His Glu Arg Arg His Ser Asp Val Ser Leu Ala Asn Ala  
               
               
                                 405                 410                 415  
               
               
                   
               
               
                 Glu Leu Glu Asp Ser Arg Ile Ser Leu Leu Arg Met Asp Arg Pro Ser  
               
               
                             420                 425                 430  
               
               
                   
               
               
                 Arg Gln Arg Ser Val Ser Glu Arg Arg Ala Ala Met Glu Asn Gln Arg  
               
               
                         435                 440                 445  
               
               
                   
               
               
                 Ser Tyr Ser Met Glu Arg Thr Arg Glu Ala Gln Gly Gln Ser Ser Tyr  
               
               
                     450                 455                 460  
               
               
                   
               
               
                 Pro Gln Arg Thr Ser Asn His Ser Pro Pro Thr Pro Arg Arg Ser Pro  
               
               
                 465                 470                 475                 480  
               
               
                   
               
               
                 Ile Pro Leu Asp Arg Pro Asp Met Arg Arg Ala Asp Ser Leu Arg Lys  
               
               
                                 485                 490                 495  
               
               
                   
               
               
                 Gln His His Leu Asp Pro Ser Ser Ala Val Arg Lys Thr Lys Arg Glu  
               
               
                             500                 505                 510  
               
               
                   
               
               
                 Lys Met Glu Thr Met Leu Arg Asn Asp Ser Leu Ser Ser Asp Gln Ser  
               
               
                         515                 520                 525  
               
               
                   
               
               
                 Glu Ser Val Arg Pro Pro Pro Pro Arg Pro His Lys Ser Lys Lys Gly  
               
               
                     530                 535                 540  
               
               
                   
               
               
                 Gly Lys Met Arg Gln Val Ser Leu Ser Ser Ser Glu Glu Glu Leu Ala  
               
               
                 545                 550                 555                 560  
               
               
                   
               
               
                 Ser Thr Pro Glu Tyr Thr Ser Cys Asp Asp Val Glu Leu Glu Ser Glu  
               
               
                                 565                 570                 575  
               
               
                   
               
               
                 Ser Val Ser Glu Lys Gly Asp Ser Gln Lys Gly Lys Arg Lys Thr Ser  
               
               
                             580                 585                 590  
               
               
                   
               
               
                 Glu Gln Gly Val Leu Ser Asp Ser Asn Thr Arg Ser Glu Arg Gln Lys  
               
               
                         595                 600                 605  
               
               
                   
               
               
                 Lys Arg Met Tyr Tyr Gly Gly His Ser Leu Glu Glu Asp Leu Glu Trp  
               
               
                     610                 615                 620  
               
               
                   
               
               
                 Ser Glu Pro Gln Ile Lys Asp Ser Gly Val Asp Thr Cys Ser Ser Thr  
               
               
                 625                 630                 635                 640  
               
               
                   
               
               
                 Thr Leu Asn Glu Glu His Ser His Ser Asp Lys His Pro Val Thr Trp  
               
               
                                 645                 650                 655  
               
               
                   
               
               
                 Gln Pro Ser Lys Asp Gly Asp Arg Leu Ile Gly Arg Ile Leu Leu Asn  
               
               
                             660                 665                 670  
               
               
                   
               
               
                 Lys Arg Leu Lys Asp Gly Ser Val Pro Arg Asp Ser Gly Ala Met Leu  
               
               
                         675                 680                 685  
               
               
                   
               
               
                 Gly Leu Lys Val Val Gly Gly Lys Met Thr Glu Ser Gly Arg Leu Cys  
               
               
                     690                 695                 700  
               
               
                   
               
               
                 Ala Phe Ile Thr Lys Val Lys Lys Gly Ser Leu Ala Asp Thr Val Gly  
               
               
                 705                 710                 715                 720  
               
               
                   
               
               
                 His Leu Arg Pro Gly Asp Glu Val Leu Glu Trp Asn Gly Arg Leu Leu  
               
               
                                 725                 730                 735  
               
               
                   
               
               
                 Gln Gly Ala Thr Phe Glu Glu Val Tyr Asn Ile Ile Leu Glu Ser Lys  
               
               
                             740                 745                 750  
               
               
                   
               
               
                 Pro Glu Pro Gln Val Glu Leu Val Val Ser Arg Pro Ile Gly Asp Ile  
               
               
                         755                 760                 765  
               
               
                   
               
               
                 Pro Arg Ile Pro Asp Ser Thr His Ala Gln Leu Glu Ser Ser Ser Ser  
               
               
                     770                 775                 780  
               
               
                   
               
               
                 Ser Phe Glu Ser Gln Lys Met Asp Arg Pro Ser Ile Ser Val Thr Ser  
               
               
                 785                 790                 795                 800  
               
               
                   
               
               
                 Pro Met Ser Pro Gly Met Leu Arg Asp Val Pro Gln Phe Leu Ser Gly  
               
               
                                 805                 810                 815  
               
               
                   
               
               
                 Gln Leu Ser Ile Lys Leu Trp Phe Asp Lys Val Gly His Gln Leu Ile  
               
               
                             820                 825                 830  
               
               
                   
               
               
                 Val Thr Ile Leu Gly Ala Lys Asp Leu Pro Ser Arg Glu Asp Gly Arg  
               
               
                         835                 840                 845  
               
               
                   
               
               
                 Pro Arg Asn Pro Tyr Val Lys Ile Tyr Phe Leu Pro Asp Arg Ser Asp  
               
               
                     850                 855                 860  
               
               
                   
               
               
                 Lys Asn Lys Arg Arg Thr Lys Thr Val Lys Lys Thr Leu Glu Pro Lys  
               
               
                 865                 870                 875                 880  
               
               
                   
               
               
                 Trp Asn Gln Thr Phe Ile Tyr Ser Pro Val His Arg Arg Glu Phe Arg  
               
               
                                 885                 890                 895  
               
               
                   
               
               
                 Glu Arg Met Leu Glu Ile Thr Leu Trp Asp Gln Ala Arg Val Arg Glu  
               
               
                             900                 905                 910  
               
               
                   
               
               
                 Glu Glu Ser Glu Phe Leu Gly Glu Ile Leu Ile Glu Leu Glu Thr Ala  
               
               
                         915                 920                 925  
               
               
                   
               
               
                 Leu Leu Asp Asp Glu Pro His Trp Tyr Lys Leu Gln Thr His Asp Val  
               
               
                     930                 935                 940  
               
               
                   
               
               
                 Ser Ser Leu Pro Leu Pro Arg Pro Ser Pro Tyr Leu Pro Arg Arg Gln  
               
               
                 945                 950                 955                 960  
               
               
                   
               
               
                 Leu His Gly Glu Ser Pro Thr Arg Arg Leu Gln Arg Ser Lys Arg Ile  
               
               
                                 965                 970                 975  
               
               
                   
               
               
                 Ser Asp Ser Glu Val Ser Asp Tyr Asp Cys Glu Asp Gly Val Gly Val  
               
               
                             980                 985                 990  
               
               
                   
               
               
                 Val Ser Asp Tyr Arg His Asn Gly  Arg Asp Leu Gln Ser  Ser Thr Leu  
               
               
                         995                 1000                 1005  
               
               
                   
               
               
                 Ser Val  Pro Glu Gln Val Met  Ser Ser Asn His Cys  Ser Pro Ser  
               
               
                     1010                 1015                 1020  
               
               
                   
               
               
                 Gly Ser  Pro His Arg Val Asp  Val Ile Gly Arg Thr  Arg Ser Trp  
               
               
                     1025                 1030                 1035  
               
               
                   
               
               
                 Ser Pro  Ser Ala Pro Pro Pro  Gln Arg Asn Val Glu  Gln Gly His  
               
               
                     1040                 1045                 1050  
               
               
                   
               
               
                 Arg Gly  Thr Arg Ala Thr Gly  His Tyr Asn Thr Ile  Ser Arg Met  
               
               
                     1055                 1060                 1065  
               
               
                   
               
               
                 Asp Arg  His Arg Val Met Asp  Asp His Tyr Ser Ser  Asp Arg Asp  
               
               
                     1070                 1075                 1080  
               
               
                   
               
               
                 Arg Asp  Cys Glu Ala Ala Asp  Arg Gln Pro Tyr His  Arg Ser Arg  
               
               
                     1085                 1090                 1095  
               
               
                   
               
               
                 Ser Thr  Glu Gln Arg Pro Leu  Leu Glu Arg Thr Thr  Thr Arg Ser  
               
               
                     1100                 1105                 1110  
               
               
                   
               
               
                 Arg Ser  Ser Glu Arg Pro Asp  Thr Asn Leu Met Arg  Ser Met Pro  
               
               
                     1115                 1120                 1125  
               
               
                   
               
               
                 Ser Leu  Met Thr Gly Arg Ser  Ala Pro Pro Ser Pro  Ala Leu Ser  
               
               
                     1130                 1135                 1140  
               
               
                   
               
               
                 Arg Ser  His Pro Arg Thr Gly  Ser Val Gln Thr Ser  Pro Ser Ser  
               
               
                     1145                 1150                 1155  
               
               
                   
               
               
                 Thr Pro  Gly Thr Gly Arg Arg  Gly Arg Gln Leu Pro  Gln Leu Pro  
               
               
                     1160                 1165                 1170  
               
               
                   
               
               
                 Pro Lys  Gly Thr Leu Glu Arg  Ser Ala Met Asp Ile  Glu Glu Arg  
               
               
                     1175                 1180                 1185  
               
               
                   
               
               
                 Asn Arg  Gln Met Lys Leu Asn  Lys Tyr Lys Gln Val  Ala Gly Ser  
               
               
                     1190                 1195                 1200  
               
               
                   
               
               
                 Asp Pro  Arg Leu Glu Gln Asp  Tyr His Ser Lys Tyr  Arg Ser Gly  
               
               
                     1205                 1210                 1215  
               
               
                   
               
               
                 Trp Asp  Pro His Arg Gly Ala  Asp Thr Val Ser Thr  Lys Ser Ser  
               
               
                     1220                 1225                 1230  
               
               
                   
               
               
                 Asp Ser  Asp Val Ser Asp Val  Ser Ala Val Ser Arg  Thr Ser Ser  
               
               
                     1235                 1240                 1245  
               
               
                   
               
               
                 Ala Ser  Arg Phe Ser Ser Thr  Ser Tyr Met Ser Val  Gln Ser Glu  
               
               
                     1250                 1255                 1260  
               
               
                   
               
               
                 Arg Pro  Arg Gly Asn Arg Lys  Ile Ser Val Phe Thr  Ser Lys Met  
               
               
                     1265                 1270                 1275  
               
               
                   
               
               
                 Gln Asn  Arg Gln Met Gly Val  Ser Gly Lys Asn Leu  Thr Lys Ser  
               
               
                     1280                 1285                 1290  
               
               
                   
               
               
                 Thr Ser  Ile Ser Gly Asp Met  Cys Ser Leu Glu Lys  Asn Asp Gly  
               
               
                     1295                 1300                 1305  
               
               
                   
               
               
                 Ser Gln  Ser Asp Thr Ala Val  Gly Ala Leu Gly Thr  Ser Gly Lys  
               
               
                     1310                 1315                 1320  
               
               
                   
               
               
                 Lys Arg  Arg Ser Ser Ile Gly  Ala Lys Met Val Ala  Ile Val Gly  
               
               
                     1325                 1330                 1335  
               
               
                   
               
               
                 Leu Ser  Arg Lys Ser Arg Ser  Ala Ser Gln Leu Ser  Gln Thr Glu  
               
               
                     1340                 1345                 1350  
               
               
                   
               
               
                 Gly Gly  Gly Lys Lys Leu Arg  Ser Thr Val Gln Arg  Ser Thr Glu  
               
               
                     1355                 1360                 1365  
               
               
                   
               
               
                 Thr Gly  Leu Ala Val Glu Met  Arg Asn Trp Met Thr  Arg Gln Ala  
               
               
                     1370                 1375                 1380  
               
               
                   
               
               
                 Ser Arg  Glu Ser Thr Asp Gly  Ser Met Asn Ser Tyr  Ser Ser Glu  
               
               
                     1385                 1390                 1395  
               
               
                   
               
               
                 Gly Asn  Leu Ile Phe Pro Gly  Val Arg Leu Ala Ser  Asp Ser Gln  
               
               
                     1400                 1405                 1410  
               
               
                   
               
               
                 Phe Ser  Asp Phe Leu Asp Gly  Leu Gly Pro Ala Gln  Leu Val Gly  
               
               
                     1415                 1420                 1425  
               
               
                   
               
               
                 Arg Gln  Thr Leu Ala Thr Pro  Ala Met Gly Asp Ile  Gln Val Gly  
               
               
                     1430                 1435                 1440  
               
               
                   
               
               
                 Met Met  Asp Lys Lys Gly Gln  Leu Glu Val Glu Ile  Ile Arg Ala  
               
               
                     1445                 1450                 1455  
               
               
                   
               
               
                 Arg Gly  Leu Val Val Lys Pro  Gly Ser Lys Thr Leu  Pro Ala Pro  
               
               
                     1460                 1465                 1470  
               
               
                   
               
               
                 Tyr Val  Lys Val Tyr Leu Leu  Asp Asn Gly Val Cys  Ile Ala Lys  
               
               
                     1475                 1480                 1485  
               
               
                   
               
               
                 Lys Lys  Thr Lys Val Ala Arg  Lys Thr Leu Glu Pro  Leu Tyr Gln  
               
               
                     1490                 1495                 1500  
               
               
                   
               
               
                 Gln Leu  Leu Ser Phe Glu Glu  Ser Pro Gln Gly Arg  Val Leu Gln  
               
               
                     1505                 1510                 1515  
               
               
                   
               
               
                 Ile Ile  Val Trp Gly Asp Tyr  Gly Arg Met Asp His  Lys Ser Phe  
               
               
                     1520                 1525                 1530  
               
               
                   
               
               
                 Met Gly  Val Ala Gln Ile Leu  Leu Asp Glu Leu Glu  Leu Ser Asn  
               
               
                     1535                 1540                 1545  
               
               
                   
               
               
                 Met Val  Ile Gly Trp Phe Lys  Leu Phe Pro Pro Ser  Ser Leu Val  
               
               
                     1550                 1555                 1560  
               
               
                   
               
               
                 Asp Pro  Thr Ser Ala Pro Leu  Thr Arg Arg Ala Ser  Gln Ser Ser  
               
               
                     1565                 1570                 1575  
               
               
                   
               
               
                 Leu Glu  Ser Ser Thr Gly Pro  Ser Tyr Ser Arg Ser  
               
               
                     1580                 1585                 1590  
               
               
                   
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 2  
               
               
                 &lt;211&gt; LENGTH: 4980  
               
               
                 &lt;212&gt; TYPE: DNA  
               
               
                 &lt;213&gt; ORGANISM: Mus musculus  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 2  
               
               
                   
               
               
                 gcttccctag ggtggttcgg ctccgccaaa catgtcggct ccgctcgggc cccggggccg     60  
               
               
                   
               
               
                 cccggctccc accccggcgg cctctcaacc tcctccgcag cccgagatgc cggacctcag    120  
               
               
                   
               
               
                 ccacctcacg gaagaggaga ggaaaatcat cctggctgtc atggatcgtc agaagaaaga    180  
               
               
                   
               
               
                 agaggagaag gagcagtccg tgctcaagat caaagaagaa cacaaagcac aaccgacaca    240  
               
               
                   
               
               
                 gtggtttccc tttagtggga tcactgaact ggtaaataac gttctgcagc cccagcaaaa    300  
               
               
                   
               
               
                 acaacccaat gagaaggagc cccagacaaa gctgcaccaa caatttgaaa tgtataagga    360  
               
               
                   
               
               
                 gcaagtcaag aagatgggag aggaatcgca gcagcagcaa gagcagaagg gtgatgcccc    420  
               
               
                   
               
               
                 gacctgtggc atctgccaca agacaaaatt tgcagatgga tgcggccata attgttccta    480  
               
               
                   
               
               
                 ttgccaaacc aagttctgtg ctcgatgtgg aggtcgagtg tctttacgct caaacaaggt    540  
               
               
                   
               
               
                 tatgtgggtg tgtaatttgt gccgaaaaca acaagaaatc ctcactaaat caggagcatg    600  
               
               
                   
               
               
                 gttttataat agtgggtcta acacactgca gcaacctgat caaaaggttc ctcgagggct    660  
               
               
                   
               
               
                 tcgaaatgag gaagcccctc aggagaagaa agcaaaacta cacgagcagc cccagttcca    720  
               
               
                   
               
               
                 aggagcccca ggtgacttat cagtacctgc agttgagaaa ggccgagctc atgggctcac    780  
               
               
                   
               
               
                 aagacaggat actattaaaa atggatcagg agtgaagcac cagattgcca gtgacatgcc    840  
               
               
                   
               
               
                 ttcagacaga aaacgaagtc catcagtgtc cagggatcaa aatcgaagat acgagcaaag    900  
               
               
                   
               
               
                 tgaagaaaga gaggactact cacagtatgt tccttcagat ggtacaatgc caagatctcc    960  
               
               
                   
               
               
                 ttcggattat gctgatagac gatctcagcg tgagcctcaa ttttatgaag aacctggtca   1020  
               
               
                   
               
               
                 tttaaattac agggattcta acaggagagg ccatagacat tccaaagagt atattgtgga   1080  
               
               
                   
               
               
                 tgatgaagat gtggagagca gagatgaata tgaaagacaa aggagagagg aggaatacca   1140  
               
               
                   
               
               
                 ggcacgctac agaagtgatc caaatctggc ccggtatccc gtaaagccac aaccctacga   1200  
               
               
                   
               
               
                 agaacaaatg cgcatccacg ctgaggtgtc cagggcacga catgagagaa ggcacagtga   1260  
               
               
                   
               
               
                 tgtttctttg gcaaacgctg aactagaaga ttccaggatt tctctgctaa ggatggatag   1320  
               
               
                   
               
               
                 accatcaagg caaagatctg tatctgaacg tagagctgca atggaaaacc aacgatcgta   1380  
               
               
                   
               
               
                 ttcaatggaa agaactcgag aggctcaggg acaaagttct tatccacaaa ggacctcaaa   1440  
               
               
                   
               
               
                 tcatagtcct cccacccctc ggcggagccc tataccgctt gatagaccag acatgaggcg   1500  
               
               
                   
               
               
                 cgctgactcc ctacggaaac agcaccactt agatcccagc tctgctgtga ggaaaacgaa   1560  
               
               
                   
               
               
                 gcgagaaaaa atggaaacca tgttaaggaa tgattctttg agttcagacc agtccgagtc   1620  
               
               
                   
               
               
                 agtgaggccg cccccaccaa ggcctcataa atccaagaaa ggaggtaaaa tgcgccaggt   1680  
               
               
                   
               
               
                 ttcactgagc agctcggagg aggagctggc atccacacct gagtatacaa gctgtgatga   1740  
               
               
                   
               
               
                 tgtggagctg gaaagcgaga gtgtgagtga gaaaggggac agtcaaaagg gaaaaagaaa   1800  
               
               
                   
               
               
                 aactagtgag cagggagttt tgtcggattc taacaccagg tctgagagac aaaagaaaag   1860  
               
               
                   
               
               
                 gatgtactat ggtggccact ctttggaaga ggatttggaa tggtctgagc ctcagattaa   1920  
               
               
                   
               
               
                 ggactctggg gtagatacct gtagtagcac aacccttaac gaggagcata gccatagtga   1980  
               
               
                   
               
               
                 taagcaccct gtgacctggc agccatccaa agatggagat cgcctaattg gtcgtatttt   2040  
               
               
                   
               
               
                 attaaataag cgtttaaaag atgggagtgt acctcgagac tcaggagcaa tgctgggctt   2100  
               
               
                   
               
               
                 aaaggttgta ggaggaaaga tgactgaatc aggtcgactt tgtgcattta ttaccaaagt   2160  
               
               
                   
               
               
                 aaaaaaagga agtttagctg atactgtagg acatcttaga ccaggtgatg aagtcttgga   2220  
               
               
                   
               
               
                 atggaatggg aggctattgc aaggagccac atttgaggaa gtttacaaca ttattctaga   2280  
               
               
                   
               
               
                 atccaaacct gaaccacaag ttgagcttgt tgtttcaagg ccaattggag atattcctag   2340  
               
               
                   
               
               
                 aatacctgat agcacgcatg cacaactgga atccagttct agctcatttg aatctcaaaa   2400  
               
               
                   
               
               
                 aatggaccgt ccttctatat ccgttacctc acccatgagt cctggcatgc tgagggatgt   2460  
               
               
                   
               
               
                 cccgcagttc ttatctggac agctttcaat aaaactatgg tttgacaagg ttggtcacca   2520  
               
               
                   
               
               
                 gttgatagtt acaattttgg gagcaaagga tctcccttcc agggaagatg ggaggccaag   2580  
               
               
                   
               
               
                 gaatccttat gttaagattt acttccttcc agatagaagt gataaaaata agagaagaac   2640  
               
               
                   
               
               
                 aaaaacagtc aagaaaactt tggaacccaa atggaaccag actttcattt attctcctgt   2700  
               
               
                   
               
               
                 ccaccgaaga gaattccgtg aacgaatgct ggaaattacc ctttgggatc aagctagagt   2760  
               
               
                   
               
               
                 tcgagaagaa gagagcgaat tcttaggaga gattttaatt gaattggaaa cagctttgct   2820  
               
               
                   
               
               
                 agatgatgag ccgcactggt ataagctgca gacccatgat gtctcctcat tgccactccc   2880  
               
               
                   
               
               
                 tcgcccttcc ccatatctgc cccggaggca gctccatgga gagagcccaa cgcgcaggct   2940  
               
               
                   
               
               
                 gcaaaggtcg aagagaataa gtgacagtga agtgtctgac tacgactgcg aggatggcgt   3000  
               
               
                   
               
               
                 gggagtagtg tcagattatc gacacaatgg ccgcgatctt caaagctcca cgttgtcggt   3060  
               
               
                   
               
               
                 gccagaacaa gtcatgtcat caaatcattg ctcaccatca gggtctcctc atcgagtaga   3120  
               
               
                   
               
               
                 tgttatagga aggacaaggt catggtcgcc tagtgcccct cctcctcaaa ggaatgtgga   3180  
               
               
                   
               
               
                 acaggggcac cgagggacac gtgctactgg ccattacaac acaattagcc gaatggatag   3240  
               
               
                   
               
               
                 acaccgtgtc atggatgacc actactcttc agatagagac agggattgtg aagcagcaga   3300  
               
               
                   
               
               
                 tagacagcca tatcacagat ccagatcaac agaacaacgg cctctcctag agcggaccac   3360  
               
               
                   
               
               
                 cacccgctcc agatcctctg aacgtcctga tacaaacctc atgaggtcga tgccttcatt   3420  
               
               
                   
               
               
                 aatgactgga agatctgccc ctccttcacc tgccttatcg aggtctcacc ctcgtaccgg   3480  
               
               
                   
               
               
                 gtctgtccag acaagcccat caagtactcc gggaacagga cgaaggggcc gacagcttcc   3540  
               
               
                   
               
               
                 acagcttcca ccaaagggaa cattggagag aagtgctatg gatatagagg agagaaatcg   3600  
               
               
                   
               
               
                 ccaaatgaaa cttaacaaat acaaacaggt agccggatca gaccccagac tggagcaaga   3660  
               
               
                   
               
               
                 ttaccattcg aagtatcgct caggatggga tccacataga ggggcagata ctgtttccac   3720  
               
               
                   
               
               
                 taaatcctcg gacagtgatg taagtgatgt atctgcggtt tcaaggacta gtagtgcttc   3780  
               
               
                   
               
               
                 tcgtttcagc agcacaagct acatgtccgt ccaatcagag cggccgagag gaaacaggaa   3840  
               
               
                   
               
               
                 aatcagtgtc tttacatcca aaatgcaaaa cagacagatg ggcgtgtcgg ggaagaactt   3900  
               
               
                   
               
               
                 gaccaaaagc accagcatca gtggagacat gtgctcactg gagaagaatg acggcagcca   3960  
               
               
                   
               
               
                 gtccgacact gcagtgggcg ccctgggtac cagtggcaag aagcggcgat ctagcattgg   4020  
               
               
                   
               
               
                 ggccaaaatg gtagctattg ttggtctctc acggaaaagt cgcagtgcct ctcaactcag   4080  
               
               
                   
               
               
                 ccaaaccgaa ggaggaggta aaaagctacg gagcactgtt cagagaagca cggagaccgg   4140  
               
               
                   
               
               
                 gctagcagtg gagatgagga actggatgac ccgccaggcc agccgggaat ccacagatgg   4200  
               
               
                   
               
               
                 cagcatgaac agctatagct cggaaggaaa tctgatcttc cctggggtcc gcctggcctc   4260  
               
               
                   
               
               
                 tgacagccag ttcagtgatt tcctggatgg cctgggccct gctcagctag tgggacgcca   4320  
               
               
                   
               
               
                 gaccctggct actcctgcaa tgggtgacat tcaggtggga atgatggata aaaagggaca   4380  
               
               
                   
               
               
                 gctggaggta gaaatcatcc gggcgcgcgg ccttgtggta aaaccaggtt ccaagacact   4440  
               
               
                   
               
               
                 gccagcaccg tatgtcaagg tgtatctgtt agacaacgga gtctgcatag ccaaaaagaa   4500  
               
               
                   
               
               
                 aaccaaggtg gcgagaaaga ccctggagcc cctgtaccag cagctcttgt ccttcgagga   4560  
               
               
                   
               
               
                 gagcccccag gggagggtgt tacagatcat tgtctgggga gattatggtc gtatggatca   4620  
               
               
                   
               
               
                 caaatccttt atgggagtgg cccagatact cttagatgaa ctggaactat ccaacatggt   4680  
               
               
                   
               
               
                 gattggatgg ttcaaactct tccctccttc ctccctagta gatccaacct cggcacctct   4740  
               
               
                   
               
               
                 gacaagaaga gcttcccaat cgtctctgga aagttctacc ggaccttctt actctcgttc   4800  
               
               
                   
               
               
                 atagcaacta taaaactgtt gtcacaacaa ccagcgatac aaaaaccaga agaaaacgca   4860  
               
               
                   
               
               
                 caggtggaag cccctggtaa cactgcatgc ttgatgttgt gtctacagag cccacgtcta   4920  
               
               
                   
               
               
                 gggataccaa gcagtcctgt gttctcagag gaagtcgtac acattgtgcc ctagcaaagg   4980  
               
               
                   
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 3  
               
               
                 &lt;211&gt; LENGTH: 45  
               
               
                 &lt;212&gt; TYPE: DNA  
               
               
                 &lt;213&gt; ORGANISM: Mus musculus  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 3  
               
               
                   
               
               
                 ttgcgctcac tcttctggcc tcccttgcca ttctgctctg aaagc                     45  
               
               
                   
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 4  
               
               
                 &lt;211&gt; LENGTH: 16  
               
               
                 &lt;212&gt; TYPE: DNA  
               
               
                 &lt;213&gt; ORGANISM: Mus musculus  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 4  
               
               
                   
               
               
                 acctacgtga ctacgt                                                     16  
               
               
                   
               
               
                   
               
               
                 &lt;210&gt; SEQ ID NO 5  
               
               
                 &lt;211&gt; LENGTH: 11  
               
               
                 &lt;212&gt; TYPE: PRT  
               
               
                 &lt;213&gt; ORGANISM: Mus musculus  
               
               
                   
               
               
                 &lt;400&gt; SEQUENCE: 5  
               
               
                   
               
               
                 Gln Met Ser His Arg Leu Glu Pro Arg Arg Pro  
               
               
                 1               5                   10

Technology Category: c