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Functional Desensitization of the Extracellular Calcium-Sensing Receptor Is Regulated via Distinct Mechanisms: Role of G Protein-Coupled Receptor Kinases, Protein Kinase C and ß-Arrestins

III. Medical Department (S.L., R.F., R.P.), Leipzig University, D-04109 Leipzig, Germany; Weis Center for Research (G.E.B.), Geisinger Clinic, Danville, Pennsylvania 17822; and Endocrine Unit (S.U.M.), Massachusetts General Hospital, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Susanne U. Miedlich, Endocrine Unit, Massachusetts General Hospital, Bulfinch 327, 55 Fruit Street, Boston, Massachusetts 02114. E-mail: smiedlich{at}partners.org.

The extracellular calcium-sensing receptor (CaR) senses small fluctuations of the extracellular calcium (Ca²⁺_e) concentration and translates them into potent changes in parathyroid hormone secretion. Dissecting the regulatory mechanisms of CaR-mediated signal transduction may provide insights into the physiology of the receptor and identify new molecules as potential drug targets for the treatment of osteoporosis and/or hyperparathyroidism. CaR can be phosphorylated by protein kinase C (PKC) and G protein-coupled receptor kinases (GRKs), and has been shown to bind to ß-arrestins, potentially contributing to desensitization of CaR, although the mechanisms by which CaR-mediated signal transduction is terminated are not known. We used a PKC phosphorylation site-deficient CaR, GRK and ß-arrestin overexpression or down-regulation to delineate CaR-mediated desensitization. Fluorescence-activated cell sorting was used to determine whether receptor internalization contributed to desensitization. Overexpression of GRK 2 or 3 reduced Ca²⁺_e-dependent inositol phosphate accumulation by more than 70%, whereas a GRK 2 mutant deficient in G_q binding (D110A) was without major effect. Overexpression of GRK 4–6 did not reduce Ca²⁺_e-dependent inositol phosphate accumulation. Overexpression of ß-arrestin 1 or 2 revealed a modest inhibitory effect on Ca²⁺_e-dependent inositol phosphate production (20–30%), which was not observed for the PKC phosphorylation site-deficient CaR. Agonist-dependent receptor internalization (10–15%) did not account for the described effects. Thus, we conclude that PKC phosphorylation of CaR contributes to ß-arrestin-dependent desensitization of CaR coupling to G proteins. In contrast, GRK 2 predominantly interferes with G protein-mediated inositol-1,4,5-trisphosphate formation by binding to G_q.

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