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

Abstract

The extracellular calcium-sensing receptor (CaR) senses small fluctuations of the extracellular calcium (Ca(2+)(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 beta-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 beta-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 Ca(2+)(e)-dependent inositol phosphate accumulation by more than 70%, whereas a GRK 2 mutant deficient in G alpha(q) binding (D110A) was without major effect. Overexpression of GRK 4-6 did not reduce Ca(2+)(e)-dependent inositol phosphate accumulation. Overexpression of beta-arrestin 1 or 2 revealed a modest inhibitory effect on Ca(2+)(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 beta-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 alpha(q).

The Mechanism of Chemokine Receptor 9 Internalization Triggered by Interleukin 2 and Interleukin 4

“…To date, GPCR internalization is characterized by the receptor phosphorylation by GRKs and/or second messengerdependent kinases, -arrestin binding, and subsequent receptor sequestration (26). In our experiments, inhibition and activation of PKC could prevent or promote the CCR9 internalization triggered by IL-2 and IL-4, which confirmed the participation of PKC in the process.…”

The GPRC6A receptor displays constitutive internalization and sorting to the slow recycling pathway

“…Although agonist-mediated endocytosis has been observed for several of the mGluRs, mGluR1 and mGluR5 additionally demonstrate constitutive internalization (48,49). CaSR is the closest related mammalian receptor of GPRC6A and has proven highly resistant to desensitization, which most likely reflects a persistent exposure to the endogenous agonist Ca 2ϩ (50,51). Constitutive internalization of CaSR has been observed in some studies (52,53), and moreover, a distinct regulatory mechanism for CaSR has been reported in which agonist stimulation triggers further insertion of receptors in the cell membrane from intracellular stores, thereby increasing the amount of receptors available for activation.…”

Calcimimetic R-568 effects on activity of R990G polymorphism of calcium-sensing receptor

“…The receptor senses changes in extracellular calcium concentration ([Ca 2C ] o ) and activates different intracellular pathways (Ward 2004, Tfelt-Hansen & Brown 2005, Huang & Miller 2007, Lorenz et al 2007). In particular, CASR activation triggers calcium mobilization from intracellular stores through inositol trisphosphate (IP 3 ) receptors and activation of the mitogen activated protein kinase (MAPK) cascade through p44/42 extracellular signaling-regulated kinase (ERK) phosphorylation.…”