Method of Identifying Transmembrane Protein-interacting Compounds

Abstract

A method for screening compounds for their ability to interact with transmembrane proteins is provided. Also provided is a method for determining whether proteins such as transmembrane proteins are able to oligomerise.

We claim: 
     
         1 . A method for screening a candidate compound for its ability to interact with at least one transmembrane protein comprising:
 (a) contacting a eurkaryotic cell with a candidate compound, wherein the eukaryotic cell comprises at least one nucleotide sequence encoding a protein comprising a transmembrane protein modified to alter trafficking of the ligand-bound transmembrane protein to the cell membrane and a detectable moiety, and the encoded protein is expressed in the cell; and   (b) determining the distribution of the expressed protein in the eukaryotic cell by detecting the distribution of the detectable moiety in the cell; wherein detection of an altered distribution of the detectable moiety in the cell relative to the distribution of the detectable moiety in a control cell not contacted with the candidate compound indicates that the compound interacts with the transmembrane protein.   
     
     
         2 . The method of  claim 1 , wherein the transmembrane protein is modified to facilitate ligand-independent trafficking of the transmembrane protein away from the cell membrane. 
     
     
         3 . The method of  claim 2 , wherein the transmembrane protein is modified to contain an amino acid sequence that facilitates ligand-independent trafficking of the transmembrane protein away from the cell membrane. 
     
     
         4 . The method of  claim 1 , wherein the eukaryotic cell is selected from the group consisting of a mammalian cell, a yeast cell, an insect cell, a nematode cell, a plant cell and a fungal cell. 
     
     
         5 . The method of  claim 1 , wherein the detectable moiety is a detectable peptide comprising an antigenic portion of the amino acid sequence of the transmembrane protein. 
     
     
         6 . The method of  claim 5 , wherein the detectable moiety is an antigenic peptide and the distribution of the antigenic peptide in the cell is determined by allowing it to bind to an antibody-based detection system comprising an antibody specific for the antigenic peptide. 
     
     
         7 . The method of  claim 6 , wherein the antibody-based detection system comprises a first antibody specific for the antigenic peptide and a second antibody carrying a detectable label and specific for the first antibody. 
     
     
         8 . The method of  claim 6 , wherein the antibody-based detection system comprises a first antibody specific for the antigenic peptide comprising a detectable label. 
     
     
         9 . The method of  claim 1 , wherein the detectable moiety is at least one of an optically detectable label, a luminescent label, or a fluorescent label. 
     
     
         10 . The method of  claim 1 , wherein the detectable moiety is an enzyme which can be reacted to give a detectable end point. 
     
     
         11 . The method of  claim 10 , wherein the enzyme is β-galactosidase. 
     
     
         12 . The method of  claim 1 , wherein the detectable moiety is a polypeptide selected from the group consisting of green fluorescent protein, red fluorescent protein and modified variants thereof. 
     
     
         13 . The method of  claim 1 , wherein the transmembrane protein is selected from the group consisting of a G protein-coupled receptor (GPCR), a transporter, a cytokine receptor, a tyrosine kinase receptor and a low-density lipoprotein (LDL) receptor. 
     
     
         14 . The method of  claim 13 , wherein the transmembrane protein is a GPCR. 
     
     
         15 . The method of  claim 14 , wherein the GPCR is selected from the group consisting of a dopamine D1 receptor, a dopamine D2 receptor, a dopamine D3 receptor, a dopamine D5 receptor, a histamine 1 receptor, a cysteinyl leukotriene receptor 1, a cysteinyl leukotriene receptor 2, an opioid receptor, a muscarinic receptor, a serotonin receptor, a beta2-adrenergic receptor and a metabotropic glutamate 4 receptor. 
     
     
         16 . The method of  claim 3 , wherein the nucleotide sequence encoding the transmembrane protein is modified within a region encoding an intracellular domain of the protein to contain an amino acid sequence that facilitates ligand-independent transfer of the transmembrane protein away from the cell membrane. 
     
     
         17 . The method of  claim 3 , wherein the nucleotide sequence encoding the transmembrane protein is modified within a region encoding the proximal carboxyl tail to contain an amino acid sequence that facilitates ligand-independent transfer of the transmembrane protein away from the cell membrane. 
     
     
         18 . The method of  claim 3 , wherein the nucleotide sequence encoding the transmembrane protein is modified within a region encoding helix 8 to contain an amino acid sequence that facilitates ligand-independent transfer of the transmembrane protein away from the cell membrane. 
     
     
         19 . The method of  claim 1 , wherein the cell is contacted with a compound known to interact with the at least one transmembrane protein prior to contacting the cell with the candidate compound and wherein detection of an altered distribution of the detectable moiety in the cell relative to the distribution of the detectable moiety in a control cell contacted with the compound known to interact with the transmembrane protein but not contacted with the candidate compound indicates that the candidate compound interacts with the transmembrane protein.