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Abstract

The present invention provides a method of detecting oligomerization of G protein coupled receptors comprising: a) obtaining a first G protein coupled receptor fusion protein containing a fluorescence donor; b) obtaining a second C protein coupled receptor fusion protein containing a fluorescence acceptor; c) transfecting a cell with the G protein coupled receptor fusion proteins of a) and b); d) exciting the fluorescence donor at a particular wavelength; e) detecting fluorescence emission of the acceptor (FRET), such that if this emission is greater than the emission detected in control cells expressing only the acceptor, oligomerization of the G protein coupled receptors has been detected. The invention further provides methods of detecting agonists and antagonists of G protein coupled receptors by utilizing FRET.

Other References

• Gilman AG. et al., The Pharmacological Basis of Therapeutics, 8th ed. McGraw Hill, New York, pp. 33 and 45, 1993.*
• Miyawaki A. et al., Nature 388:882-887, 1997.*
• Gama L. et al., J. Biol. Chem. 273:29712-29718, 1998.*
• Pace et al. “Dimerization of the Calcium-Sensing Receptor Occurs within the Extraceullular Domain and is Eliminated by Cys> Ser Mutations at Cys101 and Cys236” J Biol Chem 274(17):11629-11634, Apr. 23, 1999.
• Jordan et al. “G-Protein-Coupled Receptor Heterodimerization Modulates Receptor Function” Nature 399:697-700, Jun. 1999.
• Maggio et al. “G Protein-Linked Receptors: Pharmacological Evidence for the Formation of Heterodimers” J Pharmacol Exp Ther 291(1):251-257, 1999.
• Kuner et al. “Role of Heteromer Formation in GABAB Receptor Function” Science 283:74-77, Jan. 1999.
• Marshall et al. “GABAB Receptors—The First 7TM Heterodimers” Trends Pharmacol Sci 20:396-399, Oct. 1999.
• Jones et al. “GABAB Receptors Function as a Heteromeric Assembly of the Subunits GABABR1 and GABABR2” Nature 396:674-679, Dec. 17, 1998.
• White et al. “Heterodimerization is Required for the Formation of a Functional GABAB Receptor” Nature 396:679-682, Dec. 17, 1998.
• Kaupmann et al. “GABAB-Receptor Subtypes Assemble into Functional Heteromeric Complexes” Nature 396:683-687, Dec. 17, 1998.
• Hebert et al. “Structural and Functional Aspects of G Protein-Coupled Receptor Oligomerization” Biochem Cell Biol 76:1-11, 1998.
• Stefan et al. “Mechanisms Governing the Activation and Trafficking of Yeast G Protein-Coupled Receptors” Mol Biol Cell 9:885-899, Apr. 1998.
• Hebert et al. “A Peptide Derived from a β2-Adrenergic Receptor Transmembrane Domain Inhibits Both Receptor Dimerization and Activation” J Biol Chem 271(27):16384-16392, Jul. 5, 1996.
• Ng et al. “Dopamine D2 Receptor Dimers and Receptor-Blocking Peptides” Biochem Biophys Res Commun 227:200-204, 1996.
• Lemmon et al. “Regulation of Signal Transduction and Signal Diversity by Receptor Oligomerization” Trends Biochem Sci 19:459-463, Nov. 1994.
• Wade et al. “Multisite Interactions of Receptors and G Proteins: Enhanced Potency of Dimeric Receptor Peptides in Modifying G Protein Function” Mol Pharmacol 45:1191-1197, 1994.
• Blumer et al. “The STE2 Gene Product is the Ligand-Binding Component of the α-Factor Receptor of Saccharomyces cerevisiae” J Biol Chem 263(22):10836-10842, Aug. 5, 1988.
• Reneke et al. “The Carboxy-Terminal Segment of the Yeast α-Factor Receptor is a Regulatory Domain” Cell 55:221-234, Oct. 21, 1988.