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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.

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