DESENSITIZATION AND DOWN REGULATION OF CALCIUM RECEPTORS

汽车感觉到[钙离子]的变化，并激活下游效应器。有证据表明，在甲状旁腺和肾脏等组织中，足够数量的CARS的表达是精确感知钙离子所必需的。1个CAR基因杂合子的患者有轻微的高钙血症和低钙尿，而纯合子患者严重影响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CAR蛋白表达减少。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节靶细胞对激动剂反应的机制。内化通常伴随着这些事件，并用于从细胞表面移除活跃的信号受体。我们发现，用蛋白激酶C激动剂处理表达CAR cRNA的甲状旁腺细胞和非洲爪哇卵母细胞，从甲状旁腺激素(PTH)的释放和高[Ca2+]调节的信号转导方面，使这些细胞对随后的高[Ca2+]-攻击不敏感。我们将测试这样的假设，即CAR激动剂促进受体脱敏和内化；CAR羧基末端尾巴中的序列参与这些途径；CAR蛋白表达水平在确定细胞对[Ca~(2+)]的反应中至关重要。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甲状旁腺素的分泌和信号反应，以及通过表面生物素化试验定量CARS的内化，来确定CAR激动剂是否促进CARS的脱敏和内化。在共聚焦显微镜下，将在表达绿色荧光蛋白标记的CARS的HEK-293细胞中跟踪CARS的细胞内转运。(2)我们将使用尾部截断突变和定点突变来确定可能的正或负内吞基序，以评估CAR的C末端尾巴在脱敏和内化中的作用。完整的信号通路在内化中的作用将被讨论。(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，并检测其对高(Ca~(2+))调节的降钙素分泌和细胞增殖的影响。我们的研究应该为深入了解CAR在分泌和生长过程中的反应机制提供依据。

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.