G-Protein-coupled receptor kinases, protein kinase C and beta-arrestins regulate functional desensitization of the extracellular Calcium-sensing Receptor (CaR) via distinct mechanisms

The extracellular Calcium-sensing Receptor (CaR) senses small fluctuations of the extracellular calcium concentration and translates them into potent changes of parathyroid hormone secretion. Dissecting the regulatory mechanisms of CaR-mediated signal transduction may not only provide insights into the physiology of the receptor but may also identify new regulatory molecules as potential drug targets for the treatment of osteoporosis and/or hyperparathyroidism.

Objectives: CaR can be phosphorylated by protein kinase C (PKC), GPCR kinases (GRKs), and has been shown to bind to beta-arrestins contributing to functional desensitization of CaR. However, the mechanisms by which PKC, GRKs and/or beta-arrestins terminate CaR-mediated signal transduction are not known and were investigated.

Methods: We used a PKC phosphorylation site-deficient CaR, GRK and beta-arrestin overexpression to delineate the mechanisms of CaR-mediated functional desensitization. Fluorescence-activated cell sorting was applied to measure receptor internalization.

Results: Overexpression of GRK 2 or 3 reduced Ca2+e-dependent inositol phosphate accumulation by more than 70% whereas overexpression of GRK 4 to 6 did not significantly affect inositol phosphate production. Expression of a GRK 2 mutant deficient in Galphaq binding (D110A) led to a significant recovery of the inositol phosphate signal. Overexpression of beta-arrestin 1 or 2 revealed a minor but significant inhibitory effect on Ca2+e-dependent inositol phosphate production (20–30%) which could not be observed for the PKC phosphorylation site-deficient CaR. Agonist-dependent receptor internalization (<10%) did not account for the above described effects.

Conclusions: We demonstrate here that PKC-mediated phosphorylation of CaR is required for beta-arrestin-dependent desensitization of CaR signaling to G proteins. In contrast, GRK 2-dependent functional desensitization interferes with inositol phosphate accumulation by binding to Galphaq. Receptor internalization does not contribute to functional desensitization.