G-protein-conjugated receptor having altered ligand affinity, and use thereof

Abstract

A modified G-protein-coupled receptor (GPCR), having modified ligand affinity is provided by binding a G-protein-coupled receptor to a polypeptide consisting of an amino acid sequence of SEQ ID NO: 1. Furthermore, agonists for or antagonists against the modified GPCR are screened using a transformant in which the modified GPCR has been expressed. This makes it possible to provide a technique for analyzing the function of many putative GPCRs whose entities have not been clarified.

What is claimed is: 
     
         1 . A protein complex of a GPCR and a polypeptide,
 the polypeptide being:   (A) a polypeptide consisting of an amino-acid sequence of SEQ ID NO: 1;   (B) a polypeptide (i) consisting of an amino-acid sequence of SEQ ID NO: 1 with a deletion, insertion, substitution, or addition of one or several amino acids, and (ii) having activity to modify ligand affinity of a GPCR with which the polypeptide has formed a complex;   (C) a polypeptide encoded by a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2;   (D) a polypeptide (i) encoded by a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2 with a deletion, insertion, substitution, or addition of one or several nucleotides, and (ii) having activity to modify ligand affinity of a GPCR with which the polypeptide has formed a complex;   (E) a polypeptide (i) encoded by a polynucleotide capable of hybridizing under stringent conditions with a polynucleotide consisting of a sequence complementary to a nucleotide sequence of SEQ ID NO: 2 and (ii) having activity to modify ligand affinity of a GPCR with which the polypeptide has formed a complex; or   (F) a polypeptide (i) coded for by a polynucleotide having a sequence identity of 70% or higher with a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2 and (ii) having activity to modify ligand affinity of a GPCR with which the polypeptide has formed a complex.   
     
     
         2 . The protein complex as set forth in  claim 1 , wherein the GPCR is an adrenergic receptor, a dopamine receptor, a muscarinic receptor, or an endothelin receptor. 
     
     
         3 . The protein complex as set forth in  claim 2 , wherein the adrenergic receptor is α 1 -receptor or a β 1 -receptor. 
     
     
         4 . The protein complex as set forth in  claim 3 , wherein the α 1 -receptor is an α 1A -receptor. 
     
     
         5 . The protein complex as set forth in  claim 2 , wherein the dopamine receptor is a D2 receptor. 
     
     
         6 . A lipid membrane containing a protein complex as set forth in  claim 1 . 
     
     
         7 . A method for producing a lipid membrane as set forth in  claim 6 , comprising the step of causing a GPCR and a polypeptide to coexist on a lipid membrane, the polypeptide being:
 (A) a polypeptide consisting of an amino-acid sequence of SEQ ID NO: 1;   (B) a polypeptide (i) consisting of an amino-acid sequence of SEQ ID NO: 1 with a deletion, insertion, substitution, or addition of one or several amino acids, and (ii) having activity to modify ligand affinity of a GPCR with which the polypeptide has formed a complex;   (C) a polypeptide encoded by a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2;   (D) a polypeptide (i) encoded by a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2 with a deletion, insertion, substitution, or addition of one or several nucleotides, and (ii) having activity to modify ligand affinity of a GPCR with which the polypeptide has formed a complex;   (E) a polypeptide (i) encoded by a polynucleotide capable of hybridizing under stringent conditions with a polynucleotide consisting of a sequence complementary to a nucleotide sequence of SEQ ID NO: 2 and (ii) having activity to modify ligand affinity of a GPCR with which the polypeptide has formed a complex; or   (F) a polypeptide (i) coded for by a polynucleotide having a sequence identity of 70% or higher with a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2 and (ii) having activity to modify ligand affinity of a GPCR with which the polypeptide has formed a complex.   
     
     
         8 . A transformant expressing a protein complex as set forth in  claim 1 . 
     
     
         9 . A method for producing a transformant as set forth in  claim 8 , comprising the step of coexpressing a GPCR and a polypeptide in a cell,
 the polypeptide being:   (A) a polypeptide consisting of an amino-acid sequence of SEQ ID NO: 1;   (B) a polypeptide (i) consisting of an amino-acid sequence of SEQ ID NO: 1 with a deletion, insertion, substitution, or addition of one or several amino acids, and (ii) having activity to modify ligand affinity of a G-protein-coupled receptor with which the polypeptide has formed a complex;   (C) a polypeptide encoded by a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2;   (D) a polypeptide (i) encoded by a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2 with a deletion, insertion, substitution, or addition of one or several nucleotides, and (ii) having activity to modify ligand affinity of a GPCR with which the polypeptide has formed a complex;   (E) a polypeptide (i) encoded by a polynucleotide capable of hybridizing under stringent conditions with a polynucleotide consisting of a sequence complementary to a nucleotide sequence of SEQ ID NO: 2 and (ii) having activity to modify ligand affinity of a GPCR with which the polypeptide has formed a complex; or   (F) a polypeptide (i) coded for by a polynucleotide having a sequence identity of 70% or higher with a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2 and (ii) having activity to modify ligand affinity of a GPCR with which the polypeptide has formed a complex.   
     
     
         10 .- 12 . (canceled) 
     
     
         13 . A method for producing a transformant expressing a G-protein-coupled receptor having modified ligand affinity, comprising the step of inhibiting expression of a polypeptide in a cell in which a GPCR has been expressed,
 the polypeptide being:   (A) a polypeptide consisting of an amino-acid sequence of SEQ ID NO: 1;   (B) a polypeptide (i) consisting of an amino-acid sequence of SEQ ID NO: 1 with a deletion, insertion, substitution, or addition of one or several amino acids, and (ii) having activity to modify ligand affinity of a GPCR with which the polypeptide has formed a complex;   (C) a polypeptide encoded by a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2;   (D) a polypeptide (i) encoded by a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2 with a deletion, insertion, substitution, or addition of one or several nucleotides, and (ii) having activity to modify ligand affinity of a GPCR with which the polypeptide has formed a complex;   (E) a polypeptide (i) encoded by a polynucleotide capable of hybridizing under stringent conditions with a polynucleotide consisting of a sequence complementary to a nucleotide sequence of SEQ ID NO: 2 and (ii) having activity to modify ligand affinity of a GPCR with which the polypeptide has formed a complex; or   (F) a polypeptide (i) coded for by a polynucleotide having a sequence identity of 70% or higher with a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2 and (ii) having activity to modify ligand affinity of a GPCR with which the polypeptide has formed a complex.   
     
     
         14 . The method as set forth in  claim 13 , the polypeptide is an endogenous protein. 
     
     
         15 . The method as set forth in  claim 13 , wherein the step of inhibiting the expression of the polypeptide is performed according to an RNAi method. 
     
     
         16 . The method as set forth in  claim 15 , wherein the RNAi method is performed by inserting an oligonucleotide consisting of a nucleotide sequence of SEQ ID NO: 5. 
     
     
         17 . The method as set forth in  claim 13 , wherein the cell is a transformant expressing an exogenous GPCR. 
     
     
         18 . A method for modifying ligand affinity of a GPCR, comprising the step of inhibiting expression of a polypeptide in a cell in which the GPCR has been expressed,
 the polypeptide being:   (A) a polypeptide consisting of an amino-acid sequence of SEQ ID NO: 1;   (B) a polypeptide (i) consisting of an amino-acid sequence of SEQ ID NO: 1 with a deletion, insertion, substitution, or addition of one or several amino acids, and (ii) having activity to modify ligand affinity of a GPCR with which the polypeptide has formed a complex;   (C) a polypeptide encoded by a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2;   (D) a polypeptide (i) encoded by a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2 with a deletion, insertion, substitution, or addition of one or several nucleotides, and (ii) having activity to modify ligand affinity of a GPCR with which the polypeptide has formed a complex;   (E) a polypeptide (i) encoded by a polynucleotide capable of hybridizing under stringent conditions with a polynucleotide consisting of a sequence complementary to a nucleotide sequence of SEQ ID NO: 2 and (ii) having activity to modify ligand affinity of a GPCR with which the polypeptide has formed a complex; or   (F) a polypeptide (i) coded for by a polynucleotide having a sequence identity of 70% or higher with a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2 and (ii) having activity to modify ligand affinity of a GPCR with which the polypeptide has formed a complex.   
     
     
         19 . A method for screening an agonist for or antagonist against a GPCR having modified ligand affinity, comprising the steps of:
 inhibiting expression of a polypeptide in a cell in which a GPCR has been expressed; and   incubating the cell together with a candidate factor,   the polypeptide being:   (A) a polypeptide consisting of an amino-acid sequence of SEQ ID NO: 1;   (B) a polypeptide (i) consisting of an amino-acid sequence of SEQ ID NO: 1 with a deletion, insertion, substitution, or addition of one or several amino acids, and (ii) having activity to modify ligand affinity of a GPCR with which the polypeptide has formed a complex;   (C) a polypeptide encoded by a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2;   (D) a polypeptide (i) encoded by a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2 with a deletion, insertion, substitution, or addition of one or several nucleotides, and (ii) having activity to modify ligand affinity of a GPCR with which the polypeptide has formed a complex;   (E) a polypeptide (i) encoded by a polynucleotide capable of hybridizing under stringent conditions with a polynucleotide consisting of a sequence complementary to a nucleotide sequence of SEQ ID NO: 2 and (ii) having activity to modify ligand affinity of a GPCR with which the polypeptide has formed a complex; or   (F) a polypeptide (i) coded for by a polynucleotide having a sequence identity of 70% or higher with a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2 and (ii) having activity to modify ligand affinity of a GPCR with which the polypeptide has formed a complex.   
     
     
         20 . The method as set forth in  claim 19 , further comprising the step of measuring an intracellular Ca 2+  concentration or the step of measuring metabolism of intracellular inositol phosphate.