Methods of detecting and modulating oligomerization of G protein-coupled receptors

Patent 6824990 Issued on November 30, 2004. Estimated Expiration Date:

October 5, 2020. Estimated Expiration Date is calculated based on simple USPTO term provisions. It does not account for terminal disclaimers, term adjustments, failure to pay maintenance fees, or other factors which might affect the term of a patent.

Abstract Claims Description Full Text

Inventor

Blumer, Kendall J.

Application

No. 09679852 filed on 10/05/2000

US Classes:

435/7.1, Involving antigen-antibody binding, specific binding protein assay or specific ligand-receptor binding assay435/7.2, Involving a micro-organism or cell membrane bound antigen or cell membrane bound receptor or cell membrane bound antibody or microbial lysate530/300, PEPTIDES OF 3 TO 100 AMINO ACID RESIDUES530/350, PROTEINS, I.E., MORE THAN 100 AMINO ACID RESIDUES530/402Chemical modification or the reaction product thereof, e.g., covalent attachment or coupling, etc.

Examiners

Primary: Landsman, Robert S.

Attorney, Agent or Firm

Needle & Rosenberg, P.C.

International Classes

G01N 3353
G01N 33567
A61K 3800
C07K 100
C07K 1400

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.