Rabconnectin-3-binding protein

Abstract

A protein useful for clarifying the regulation mechanism of Ca 2+ -dependent exocytosis in particular, the activation and inactivation of Rab3A, and a method of screening for a material useful for regulating Ca 2+ -dependent exocytosis in particular, the activation and inactivation of Rab3A, using the above protein. By using the coimmunoprecipitation with an anti-Rab3A GEP antibody, a protein participated in the regulation of activation or inactivation of Rab3A is determined. As the protein binds to rabconnectin-3 and GDP/GTP exchange protein, it can be used for screening for a material that increases or decreases the binding.

1 . A protein as defined in (a) or (b): 
 (a) a protein comprising the amino acid sequence of SEQ ID NO: 2; and    (b) a protein comprising the amino acid sequence of SEQ ID NO: 2 with one or several amino acids deleted, replaced, or added, and having an activity of binding rabconnectin-3 and a GDP/GTP exchange protein.    
     
     
         2 . The protein of  claim 1 , which has the amino acid sequence of SEQ ID NO: 2.  
     
     
         3 . A polynucleotide that encodes a protein as defined in  claim 1 .  
     
     
         4 . The polynucleotide of  claim 3 , comprising the nucleotide sequence of SEQ ID NO: 1.  
     
     
         5 . A polynucleotide as defined in (a) or (b): 
 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1; and    (b) a polynucleotide which hybridizes with a polynucleotide comprising a nucleotide sequence that is complementary to the nucleotide sequence of SEQ ID NO: 1 under a stringent condition, and encodes a protein having an activity of binding rabconnectin-3 and a GDP/GTP exchange protein.    
     
     
         6 . A polynucleotide as defined in (a) or (b): 
 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1; and    (b) a polynucleotide comprising a nucleotide sequence whose homology to the nucleotide sequence of SEQ ID NO: 1 is 80% or higher and encoding a protein having an activity of binding rabconnectin-3 and a GDP/GTP exchange protein.    
     
     
         7 . A recombinant vector comprising a polynucleotide as defined in any one of  claims 3  to  6 .  
     
     
         8 . A transformant obtained by transforming a host with a polynucleotide as defined in any one of  claims 3  to  6 .  
     
     
         9 . A method of producing a protein having an activity of binding rabconnectin-3 and a GDP/GTP exchange protein, comprising: 
 culturing a transformant as defined in  claim 8  in a culture; and    collecting, from the culture, a protein having an activity of binding rabconnectin-3 and the GDP/GTP exchange protein.    
     
     
         10 . (Canceled)  
     
     
         11 . A method of analyzing a first polynucleotide as defined in any one of  claims 3  to  6 , comprising hybridizing a probe or a primer with the first polynucleotide, wherein the probe or primer includes a second polynucleotide having at least 15 nucleotides complementary to the first polynucleotide.  
     
     
         12 . The analyzing method of  claim 11 , wherein the first polynucleotide is present in a tissue or a cell.  
     
     
         13 . A method of analyzing a first polynucleotide encoding a protein as defined in  claim 1  or  2 , comprising hybridizing a probe or a primer with the first polynucleotide, wherein the probe or primer includes a second polynucleotide having at least 15 nucleotides complementary to the first polynucleotide.  
     
     
         14 . The analyzing method of  claim 13 , wherein the first polynucleotide is present in a tissue or a cell.  
     
     
         15 . A method comprising amplifying an mRNA in a tissue or a cell by an RT-PCR method with a primer that includes a polynucleotide having at least 15 nucleotides complementary to a polynucleotide as defined in any one of  claims 3  to  6 .  
     
     
         16 . An antisense polynucleotide which hybridizes with an mRNA encoding a protein as defined in  claim 1  or  2 .  
     
     
         17 . A ribozyme for cutting an mRNA encoding a protein as defined in  claim 1  or  2 .  
     
     
         18 . A double-stranded RNA for cutting an mRNA encoding a protein as defined in  claim 1  or  2  by RNA interference.  
     
     
         19 . An antibody against a protein as defined in  claim 1  or  2 .  
     
     
         20 . A method of immunohistologically analyzing a protein as defined in  claim 1  or  2 , comprising contacting the protein with an antibody as defined in  claim 19 .  
     
     
         21 . The method of  claim 20 , further comprising determining the location of the protein.  
     
     
         22 . The method of  claim 20 , further comprising determining the amount of expression of the protein.  
     
     
         23 . A method of screening for a material that promotes or inhibits binding between a protein as defined in  claim 1  or  2  or a heterogeneous homologous protein thereof, and rabconnectin-3, comprising the steps of: 
 contacting a protein as defined in  claim 1  or  2  or a heterogeneous homologous protein thereof with rabconnectin-3 in the presence and absence of candidate materials, and    selecting a material which increases or decreases binding between the protein and rabconnectin-3.    
     
     
         24 . A method of screening for a material that promotes or inhibits binding between a protein as defined in  claim 1  or  2  or a heterogeneous homologous protein thereof, and a Rab 3 GDP/GTP exchange protein, comprising the steps of: 
 contacting a protein as defined in  claim 1  or  2  or a heterogeneous homologous protein thereof with Rab3 GDP/GTP exchange protein in the presence and absence of candidate materials, and    selecting a material which increases or decreases binding between the protein and Rab3 GDP/GTP exchange protein.    
     
     
         25 . A polynucleotide that encodes a protein as defined in  claim 2 .  
     
     
         26 . A recombinant vector comprising a polynucleotide as defined in  claim 25 .  
     
     
         27 . A transformant obtained by transforming a host with a polynucleotide as defined in  claim 25 .  
     
     
         28 . A method of producing a protein having an activity of binding rabconnectin-3 and a GDP/GTP exchange protein, comprising: 
 culturing a transformant as defined in  claim 27  in a culture; and    collecting, from the culture, a protein having an activity of binding rabconnectin-3 and the GDP/GTP exchange protein.    
     
     
         29 . A method of analyzing a first polynucleotide as defined in  claim 25 , comprising hybridizing a probe or a primer with the first polynucleotide, wherein the probe or primer includes a second polynucleotide having at least 15 nucleotides complementary to the first polynucleotide.  
     
     
         30 . The analyzing method of  claim 11 , wherein the first polynucleotide is present in a tissue or a cell.  
     
     
         31 . A method comprising amplifying an mRNA in a tissue or a cell by an RT-PCR method with a primer that includes a polynucleotide having at least 15 nucleotides complementary to a polynucleotide as defined in  claim 25.