This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hasbi, A. Articles by Zingg, H. H. Search for Related Content PubMed PubMed Citation Articles by Hasbi, A. Articles by Zingg, H. H.

Real-Time Detection of Interactions between the Human Oxytocin Receptor and G Protein-Coupled Receptor Kinase-2

Laboratory of Molecular Endocrinology, McGill University Health Centre, Montreal, Quebec, Canada H3A 1A1

Address all correspondence and requests for reprints to: Hans H. Zingg, M.D., Ph.D., Laboratory of Molecular Endocrinology, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec, Canada H3A 1A1.

Although the oxytocin receptor (OTR) mediates many important functions including uterine contractions, milk ejection, and maternal behavior, the mechanisms controlling agonist-induced OTR desensitization have remained unclear, and attempts to demonstrate involvement of a G protein-coupled receptor kinase (GRK) have so far failed. Using the OTR as a model, we demonstrate here directly for the first time the dynamics of agonist-induced interactions of a GRK with a G protein-coupled receptor in real time, using time-resolved bioluminescence resonance energy transfer. GRK2/receptor interactions started within 4 sec, peaked at 10 sec, and decreased to less than 40% within 8 min. By contrast, ß-arrestin/OTR interactions initiated only at 10 sec, reached plateau levels at 120 sec, but remained stable with little decrease thereafter. Physical GRK2/OTR association was further demonstrated by coimmunoprecipitation of endogenous GRK2 with activated OTR. In COS-7 cells, which express low levels of GRK2 and ß-arrestin, overexpression of GRK2 and ß-arrestin increased receptor phosphorylation, desensitization, and internalization to the high levels observed in human embryonic kidney 293 cells. By contrast, specific inhibition of endogenous GRK2 by dominant-negative mutants robustly inhibited OTR phosphorylation and internalization as well as arrestin/OTR interactions. These data characterize the temporal and causal relationship of GRK-2/OTR and ß-arrestin/OTR interactions and establish GRK/OTR interaction as a prerequisite for ß-arrestin-mediated OTR desensitization.

This article has been cited by other articles:

				J. M. Willets, P. J. Brighton, R. Mistry, G. E. Morris, J. C. Konje, and R. A. J. Challiss Regulation of Oxytocin Receptor Responsiveness by G Protein-Coupled Receptor Kinase 6 in Human Myometrial Smooth Muscle Mol. Endocrinol., August 1, 2009; 23(8): 1272 - 1280. [Abstract] [Full Text] [PDF]

				F. Conti, S. Sertic, A. Reversi, and B. Chini Intracellular trafficking of the human oxytocin receptor: evidence of receptor recycling via a Rab4/Rab5 "short cycle" Am J Physiol Endocrinol Metab, March 1, 2009; 296(3): E532 - E542. [Abstract] [Full Text] [PDF]

				R. Jorgensen, N. D. Holliday, J. L. Hansen, M. Vrecl, A. Heding, T. W. Schwartz, and C. E. Elling Characterization of G-Protein Coupled Receptor Kinase Interaction with the Neurokinin-1 Receptor Using Bioluminescence Resonance Energy Transfer Mol. Pharmacol., February 1, 2008; 73(2): 349 - 358. [Abstract] [Full Text] [PDF]

				R. Jorgensen, V. Kubale, M. Vrecl, T. W. Schwartz, and C. E. Elling Oxyntomodulin Differentially Affects Glucagon-Like Peptide-1 Receptor beta-Arrestin Recruitment and Signaling through G{alpha} J. Pharmacol. Exp. Ther., July 1, 2007; 322(1): 148 - 154. [Abstract] [Full Text] [PDF]

				R. Z. Fardoun, M. Asghar, and M. Lokhandwala Role of oxidative stress in defective renal dopamine D1 receptor-G protein coupling and function in old Fischer 344 rats Am J Physiol Renal Physiol, November 1, 2006; 291(5): F945 - F951. [Abstract] [Full Text] [PDF]

				A. Marwaha and M. F. Lokhandwala Tempol reduces oxidative stress and restores renal dopamine D1-like receptor- G protein coupling and function in hyperglycemic rats Am J Physiol Renal Physiol, July 1, 2006; 291(1): F58 - F66. [Abstract] [Full Text] [PDF]

				M. P. Smith, V. J. Ayad, S. J. Mundell, C. A. McArdle, E. Kelly, and A. Lopez Bernal Internalization and Desensitization of the Oxytocin Receptor Is Inhibited by Dynamin and Clathrin Mutants in Human Embryonic Kidney 293 Cells Mol. Endocrinol., February 1, 2006; 20(2): 379 - 388. [Abstract] [Full Text] [PDF]

				F. F. Hamdan, M. Audet, P. Garneau, J. Pelletier, and M. Bouvier High-Throughput Screening of G Protein-Coupled Receptor Antagonists Using a Bioluminescence Resonance Energy Transfer 1-Based {beta}-Arrestin2 Recruitment Assay J Biomol Screen, August 1, 2005; 10(5): 463 - 475. [Abstract] [PDF]

				A. Reversi, V. Rimoldi, T. Marrocco, P. Cassoni, G. Bussolati, M. Parenti, and B. Chini The Oxytocin Receptor Antagonist Atosiban Inhibits Cell Growth via a "Biased Agonist" Mechanism J. Biol. Chem., April 22, 2005; 280(16): 16311 - 16318. [Abstract] [Full Text] [PDF]

				C. Krasel, M. Bunemann, K. Lorenz, and M. J. Lohse {beta}-Arrestin Binding to the {beta}2-Adrenergic Receptor Requires Both Receptor Phosphorylation and Receptor Activation J. Biol. Chem., March 11, 2005; 280(10): 9528 - 9535. [Abstract] [Full Text] [PDF]