Method of identifying transmembrane protein-interacting compounds

1. A method for screening a candidate compound for its ability to interact with at least one transmembrane protein comprising:

contacting a eukaryotic cell with a candidate compound, wherein the eukaryotic cell comprises at least one nucleotide sequence encoding a protein comprising a transmembrane protein containing at least one nuclear localisation sequence (NLS) and a detectable moiety and the encoded protein is expressed in the cell; and

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, wherein the wild-type transmembrane protein lacks an NLS and the nucleotide sequence encoding the transmembrane protein is modified to encode an NLS.

2. 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.

3. The method of claim 1 wherein the nucleotide sequence encodes a fusion protein comprising a transmembrane protein containing at least one NLS and a detectable moiety.

4. The method of claim 1 wherein the nucleotide sequence is modified to encode an NLS selected from Table 1 or wherein the nucleotide sequence is modified to encode an amino acid sequence selected from the group consisting of KKFKR (SEQ ID NO: 157), PKKKRKV (SEQ ID NO: 129) and AFSAKKFKR (SEQ ID NO: 158).

5. 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.

6. The method of claim 3 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 3 wherein the detectable moiety is a polypeptide selected from the group consisting of green fluorescent protein, red fluorescent protein and modified variants thereof.

8. 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.

9. The method of claim 8 wherein the transmembrane protein is a GPCR.

10. The method of claim 9 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.

11. The method of claim 8 wherein the transmembrane protein is a transporter selected from the group consisting of a dopamine transporter and a serotonin transporter, a cytokine receptor selected from the group consisting of an erythropoietin receptor and an insulin receptor, a tyrosine kinase receptor selected from the group consisting of an epidermal growth factor receptor and an insulin receptor or a low density lipoprotein receptor.

12. The method of claim 1 wherein the cell comprises a plurality of nucleotide sequences, each of said sequences encoding a protein comprising a different NLS-containing transmembrane protein and a detectable moiety, wherein each of said nucleotide sequences encodes a protein having a different detectable moiety or wherein at least one detectable moiety is common to at least two encoded proteins.

13. 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.

14. The method of claim 1 wherein detection of an altered distribution of the detectable moiety comprises detection of a reduced level or an increased level of the detectable moiety associated with the cell membrane.

15. The method of claim 1 wherein detection of an altered distribution of the detectable moiety comprises detection of a reduced level or an increased level of the detectable moiety in the nucleus of the cell.

16. The method of claim 5 , wherein the mammalian cell is selected from the group consisting of HEK, COS and CHO cells.

17. 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.

18. The method of claim 6 , wherein the antibody-based detection system comprises a first antibody specific for the antigenic peptide comprising a detectable label.

19. The method of claim 18 , wherein the detectable label is at least one of an optically detectable label, a luminescent label or a fluorescent label.