Functional desensitization of the extracellular calcium-sensing receptor is regulated via distinct mechanisms: role of G protein-coupled receptor kinases, protein kinase C and beta-arrestins.
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).
Lorenz
Stephan
S
III. Medical Department, Leipzig University, Germany.
Frenzel
Romy
R
Paschke
Ralf
R
Breitwieser
Gerda E
GE
Miedlich
Susanne U
SU
D019390:Arrestins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D054769:G-Protein-Coupled Receptor Kinase 2; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D016296:Mutagenesis; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D034741:RNA, Small Interfering; D044169:Receptors, Calcium-Sensing / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D051552:beta-Adrenergic Receptor Kinases / Q000235:genetics / Q000378:metabolism*; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins
C000605253:ARRB1 protein, human; D019390:Arrestins; D007295:Inositol Phosphates; D034741:RNA, Small Interfering; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins; D011493:Protein Kinase C; C496567:GRK2 protein, human; D051552:beta-Adrenergic Receptor Kinases; D054769:G-Protein-Coupled Receptor Kinase 2; D043802:GTP-Binding Protein alpha Subunits, Gq-G11