Method for improving the function of heterologous G protein-coupled receptors
View Patent ↗This invention relates to mutant G protein-coupled receptors with improved G-protein coupling and receptor response, yeast cells expressing such receptors, vectors useful for making such cells, and methods of making and using same.
1. A yeast cell comprising a nucleic acid sequence encoding a mutated, heterologous G protein-coupled receptor, wherein the mutation is a deletion mutation in (1) the 3 rd intracellular loop resulting in a 44 amino acid third intracellular loop comprising 22 residues proximal to the 5 th and the 6 th transmembrane domains or (2) the C-terminus, said mutation improving the function of said heterologous G protein-coupled receptor by causing it to couple more efficiently with a heterotrimeric G protein compared to a wild-type form of the heterologous G protein-coupled receptor, and wherein the mutated G protein-coupled receptor is selected from the group consisting of a muscarinic acetylcholine receptor, a cholecystokinin CCKB receptor, a somatostatin receptor, and a serotonin receptor.
2. The yeast cell according to claim 1 , wherein the mutation promotes agonist stimulated growth, and wherein the agonist is a G protein-coupled receptor agonist.
3. The yeast cell according to claim 2 , wherein the mutation results in an improved coupling between receptor and a chimeric G protein or failure of the receptor to interact with cell desensitization or sequestration-internalization machinery or proper plasma membrane localization.
4. The yeast cell according to claim 1 , wherein the deletion is a point mutation.
5. The yeast cell according to claim 1 , wherein the G protein-coupled receptor is selected from the group consisting of a muscarinic acetylcholine receptor and a cholecystokinin CCKB receptor.
6. The yeast cell according to claim 1 , wherein the muscarinic acetylcholine receptor is a rat M3 muscarinic acetylcholine receptor or a D. melanogaster muscarinic acetylcholine receptor.
7. A method for screening compounds capable of binding to G protein-coupled receptors comprising:
(a) subjecting the yeast cell according to claim 1 to a test compound; and
(b) measuring the effect of the test compounds on cell growth.
8. A yeast cell comprising a heterologous G protein-coupled receptor, wherein the G protein-coupled receptor has a deletion in the 3 rd intracellular loop resulting in a 44 amino acid third intracellular loop comprising 22 residues proximal to the 5 th and the 6 th transmembrane domains, said deletion improving the function of said heterologous G protein-coupled receptor by causing it to couple more efficiently with a heterotrimeric G protein as compared to a wild-type form of the heterologous G protein-coupled receptor.
9. The yeast cell according to claim 8 , wherein the deletion promotes agonist stimulated growth, and wherein the agonist is a G protein-coupled receptor agonist.
10. The yeast cell according to claim 9 , wherein the deletion results in improved coupling between the heterologous G protein-coupled receptor and a heterotrimeric G protein or failure of the heterologous G protein-coupled receptor to interact with cell desensitization or sequestration-internalization machinery or proper plasma membrane localization.
11. A method for screening compounds capable of binding to G protein-coupled receptors comprising:
(a) subjecting the yeast cell according to claim 8 to a test compound; and
(b) measuring the effect of the test compound on yeast cell growth.