DESENSITIZATION AND DOWN REGULATION OF CALCIUM RECEPTORS

CaRs感知[Ca2+]的变化并激活下游效应物。有证据表明，在甲状旁腺和肾脏等组织中，表达足够数量的CaRs对于精确的Ca2+感应是必要的。1个异常CaR基因杂合子的患者有轻度高钙血症和低钙尿症，而纯合子严重影响危及生命的高钙血症和甲状旁腺功能亢进，支持基因剂量效应。原发性甲状旁腺功能亢进症患者甲状旁腺中CaR蛋白表达降低。受体过度降解或生物合成减少都可以解释这一发现。受体的脱敏和下调是调控靶细胞对激动剂反应的机制。内化通常伴随着这些事件，并从细胞表面去除活跃的信号受体。我们发现，用蛋白激酶C激动剂处理表达CaR - cRNA的甲状旁腺细胞和爪蟾卵母细胞，可以使这些细胞对随后的高[Ca2+]挑战脱敏——就甲状旁腺激素（PTH）释放和高[Ca2+]调节的信号转导而言。我们将检验CaR激动剂促进受体脱敏和内化的假设；CaR的羧基末端尾部的序列参与了这些途径；并且CaR蛋白表达水平在决定细胞对[Ca2+]的反应中是至关重要的。三个目标将解决这一假设。(1)我们将通过评估用激动剂处理细胞后的PTH分泌和信号反应，以及使用表面生物素化试验量化CaR的内化，来确定CaR激动剂是否促进了CaR的脱敏和内化。通过共聚焦显微镜观察表达绿色荧光蛋白标记car的HEK-293细胞内car的细胞内运输情况。(2)我们将评估CaR的c端尾部在脱敏和内化中的作用，使用尾部截断突变体和位点定向诱变来确定假定的阳性或阴性内吞基序。完整的信号通路在内化中的作用将被解决。(3)内化缺陷突变体将在成纤维细胞和降钙素分泌细胞中表达，并将测试其对高（Ca2+）调控的降钙素分泌和细胞增殖的影响。我们的研究应该为CaR在分泌和生长中的反应性机制提供深入的见解。

CaRs sense changes in the [Ca2+] and activate downstream effectors. Evidence indicates that the expression of adequate numbers of CaRs is necessary for precise Ca2+-sensing in tissues like the parathyroid and kidney. Patients heterozygous for 1 abnormal CaR gene have mild hypercalcemia and hypocalciuria, while homozygotes are severely affected with life-threatening hypercalcemia and hyperparathyroidism supporting a gene-dosage effect. CaR protein expression is reduced in parathyroid glands from patients with primary hyperparathyroidism. Either excessive receptor degradation or reduced biosynthesis could explain this finding. Desensitization and downregulation of receptors are mechanisms for regulating target cell responses to agonists. Internalization often accompanies these events and serves to remove active signaling receptors from the cell surface. We found that treating parathyroid cells and Xenopus oocytes expressing CaR cRNA with protein kinase C agonists desensitizes these cells to subsequent challenge with high [Ca2+] -- in terms of parathyroid hormone (PTH) release and high [Ca2+] -regulated signal transduction. We will test the hypothesis that CaR agonists promote receptor desensitization and internalization; that sequences in the CaR's carboxy-terminal tail are involved in these pathways; and that levels of CaR protein expression are critical in determining cellular responses to [Ca2+] . Three aims will address the hypothesis. (1) We will determine whether CaR agonists promote desensitization and internalization of CaRs by assessing PTH secretion and signaling responses after treating cells with agonists and by quantifying internalization of CaRs using a surface biotinylation assay. Intracellular trafficking of CaRs will be followed in HEK-293 cells expressing green fluorescent protein-tagged CaRs by confocal microscopy. (2) We will assess the role of the CaR's C-terminal tail in desensitization and internalization using tail-truncation mutants and site-directed mutagenesis to identify putative positive or negative endocytic motifs. The role of intact signaling pathways in internalization will be addressed. (3) Internalization-defective mutants will be expressed in fibroblasts and calcitonin-secreting cells and the effects on high (Ca2+] -regulated calcitonin secretion and cell proliferation will be tested. Our studies should provide insights into mechanisms underlying CaR responsiveness in secretion and growth.