EphA4 Polypeptide Having a Novel Activity and Use Thereof

Abstract

The present invention provides a polynucleotide which consists of a partial sequence of a polynucleotide encoding the amino acid sequence as shown in SEQ ID NO:2, wherein the former polynucleotide encodes a polypeptide consisting of a partial amino acid sequence of SEQ ID NO:2 whose N-terminus starts between positions 564 and 621 of SEQ ID NO:2.

1 . A polynucleotide consisting of a partial sequence of a polynucleotide encoding the amino acid sequence as shown in SEQ ID NO: 2, wherein the former polynucleotide is any one of the following (a) to (c):
 (a) a nucleotide sequence that encodes a polypeptide consisting of a partial amino acid sequence of SEQ ID NO: 2 whose N-terminus starts between positions 564 and 621 of SEQ ID NO: 2;   (b) a nucleotide sequence having at least 95% or more homology to a nucleotide sequence encoding a polypeptide consisting of a partial amino acid sequence of SEQ ID NO: 2 whose N-terminus starts between positions 564 and 621 of SEQ ID NO: 2; or   (c) a nucleotide sequence which hybridizes to the complementary strand of a nucleotide sequence encoding a polypeptide consisting of a partial amino acid sequence of SEQ ID NO: 2 whose N-terminus starts between positions 564 and 621 of SEQ ID NO: 2 under stringent conditions,   and wherein the former polynucleotide yet encodes the amino acid sequence of a polypeptide having the following activity (d) or (e):   (d) promoting spine formation; or   (e) activating Rac.   
     
     
         2 . A polynucleotide selected from the following (a), (b) and (c):
 (a) a polynucleotide encoding the amino acid sequence as shown in SEQ ID NO: 2n where n represents an integer from 45 to 83;   (b) a polynucleotide consisting of the nucleotide sequence as shown in SEQ ID NO: 2n−1 where n represents an integer from 45 to 83; and   (c) a polynucleotide which hybridizes to the complementary strand of the polynucleotide (a) or (b) under stringent conditions and yet encodes a polypeptide having the following activity (d) or (e):   (d) promoting spine formation; or   (e) activating Rac.   
     
     
         3 . The polynucleotide according to  claim 2 , having the nucleotide sequence as shown in SEQ ID NO: 89, 91 or 195. 
     
     
         4 . The polynucleotide according to  claim 1 , wherein the polynucleotide encodes an amino acid sequence of a fusion protein. 
     
     
         5 . A polypeptide consisting of a partial sequence of a polypeptide having the amino acid sequence as shown in SEQ ID NO: 2, having the amino acid sequence selected from any one of following (a) to (c):
 (a) an amino acid sequence with the N-terminus starting between positions 564 and 621 of SEQ ID NO: 2;   (b) an amino acid sequence having at least 95% or more homology to a partial amino acid sequence of SEQ ID NO: 2 whose N-terminus starts between positions 564 and 621 of SEQ ID NO: 2; or   (c) an amino acid sequence encoded by a polynucleotide which hybridizes to the complementary strand of a polynucleotide encoding a partial amino acid sequence of SEQ ID NO: 2 whose N-terminus starts between positions 564 and 621 of SEQ ID NO: 2 under stringent conditions,   and wherein the polypeptide has the following activity (d) or (e):   (d) promoting spine formation; or   (e) activating Rac.   
     
     
         6 . A polypeptide selected from the following (a) and (b):
 (a) a polypeptide consisting of the amino acid sequence as shown in SEQ ID NO: 2n where n represents an integer from 45 to 83;   (b) a polypeptide which has at least 80% or more homology to a polypeptide consisting of the amino acid sequence as shown in SEQ ID NO: 2n where n represents an integer from 45 to 83 and yet has the following activity (c) or (d):   (c) promoting spine formation; or   (d) activating Rac.   
     
     
         7 . The polypeptide according to  claim 6 , having the amino acid sequence as shown in SEQ ID NO: 90, 92 or 196. 
     
     
         8 . The polypeptide according to  claim 5 , wherein the polypeptide has an amino acid sequence of a fusion protein. 
     
     
         9 . An antibody to the polypeptide according to  claim 5 . 
     
     
         10 . A kit comprising the polynucleotide according to  claim 1  and an antibody to a polypeptide consisting of a partial sequence of a polypeptide having the amino acid sequence as shown in SEQ ID NO: 2, having the amino acid sequence selected from any one of following (a) to (c):
 (a) an amino acid sequence with the N-terminus starting between positions 564 and 621 of SEQ ID NO: 2; 
 (b) an amino acid sequence having at least 95% or more homology to a partial amino acid sequence of SEQ ID NO: 2 whose N-terminus starts between positions 564 and 621 of SEQ ID NO: 2; or 
 (c) an amino acid sequence encoded by a polynucleotide which hybridizes to the complementary strand of a polynucleotide encoding a partial amino acid sequence of SEQ ID NO: 2 whose N-terminus starts between positions 564 and 621 of SEQ ID NO: 2 under stringent conditions, 
 and wherein the polypeptide has the following activity (d) or (e): 
 (d) promoting spine formation; or 
 (e) activating Rac. 
 
     
     
         11 . A kit comprising the polypeptide according to  claim 5  and an antibody thereto. 
     
     
         12 . A method of screening for compounds which affect spine formation by γ-secretase-processed EphA4, comprising the following steps:
 (i) culturing Rac expressing cells which have expressed intracellular EphA4 cleaved by γ-secretase; 
 (ii) measuring Rac activity in the Rac expressing cells cultured in step (i) in the presence and absence of a candidate compound; 
 (iii) selecting those candidate compounds which affect Rac activity based on the measuring in step (ii); and 
 (iv) identifying the candidate compounds selected in step (iii) as compounds which affect spine formation by γ-secretase-processed EphA4. 
 
     
     
         13 . The method according to  claim 12 , comprising evaluating a candidate compound as a compound which activates spine formation through γ-secretase-cleaved EphA4 when the Rac activity in the presence of said candidate compound was increased relative to the Rac activity in the absence of said candidate compound in the measurement of Rac activity in step (ii). 
     
     
         14 . The method according to  claim 12 , comprising evaluating a candidate compound as a compound which inhibits spine formation through EphA4 when the Rac activity in the presence of said candidate compound was decreased relative to the Rac activity in the absence of said candidate compound in the measurement of Rac activity in step (ii). 
     
     
         15 . A method of screening for compounds which affect Rac activity mediated by γ-secretase-processed EphA4, comprising the following steps:
 (i) culturing Rac expressing cells which have expressed intracellular EphA4 cleaved by γ-secretase; 
 (ii) measuring Rac activity in the Rac expressing cells cultured in step (i) in the presence and absence of a candidate compound; 
 (iii) selecting those candidate compounds which affect Rac activity based on the measuring in step (ii); and 
 (iv) identifying the candidate compounds selected in step (iii) as compounds which affect Rac activity mediated by γ-secretase-processed EphA4. 
 
     
     
         16 . The method according to  claim 15 , comprising evaluating a candidate compound as a compound which activates Rac through γ-secretase-cleaved EphA4 when the Rac activity in the presence of said candidate compound was increased relative to the Rac activity in the absence of said candidate compound in the measurement of Rac activity in step (ii). 
     
     
         17 . The method according to  claim 15 , comprising evaluating a candidate compound as a compound which inhibits Rac activity through EphA4 when the Rac activity in the presence of said candidate compound was decreased relative to the Rac activity in the absence of said candidate compound in the measurement of Rac activity in step (ii).