Lorenz Stephan, Frenzel Romy, Paschke Ralf, Breitwieser Gerda E, Miedlich Susanne U
III. Medical Department, Leipzig University, Germany.
Endocrinology. 2007 May;148(5):2398-404. doi: 10.1210/en.2006-1035. Epub 2007 Jan 25.
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).
细胞外钙敏感受体（CaR）可感知细胞外钙（Ca(2+)(e)）浓度的微小波动，并将其转化为甲状旁腺激素分泌的显著变化。剖析CaR介导的信号转导调控机制可能有助于深入了解该受体的生理学特性，并识别出作为治疗骨质疏松症和/或甲状旁腺功能亢进潜在药物靶点的新分子。CaR可被蛋白激酶C（PKC）和G蛋白偶联受体激酶（GRK）磷酸化，并且已被证明可与β - 抑制蛋白结合，这可能导致CaR脱敏，尽管CaR介导的信号转导终止机制尚不清楚。我们使用PKC磷酸化位点缺陷的CaR、GRK和β - 抑制蛋白的过表达或下调来描述CaR介导的脱敏过程。利用荧光激活细胞分选来确定受体内化是否导致脱敏。GRK 2或3的过表达使Ca(2+)(e)依赖性肌醇磷酸积累减少超过70%，而缺乏Gα(q)结合能力的GRK 2突变体（D110A）则无主要影响。GRK 4 - 6的过表达并未降低Ca(2+)(e)依赖性肌醇磷酸积累。β - 抑制蛋白1或2的过表达对Ca(2+)(e)依赖性肌醇磷酸产生有适度抑制作用（20 - 30%），而PKC磷酸化位点缺陷的CaR未观察到这种作用。激动剂依赖性受体内化（10 - 15%）不能解释上述效应。因此，我们得出结论，CaR的PKC磷酸化有助于CaR与G蛋白偶联的β - 抑制蛋白依赖性脱敏。相反，GRK 2主要通过与Gα(q)结合来干扰G蛋白介导的肌醇 - 1,4,5 - 三磷酸形成。
