DESENSITIZATION AND DOWN REGULATION OF CALCIUM RECEPTORS

汽车感知[Ca 2 +]的变化并激活下游效应器。 有证据表明，足够数量的汽车的表达对于甲状旁腺和肾脏等组织中的精确Ca 2+传感是必要的。 1个异常CaR基因的杂合子患者有轻度高钙血症和低钙尿，而纯合子患者严重影响危及生命的高钙血症和高甲状旁腺激素支持基因剂量效应。 原发性甲状旁腺功能亢进患者甲状旁腺中CaR蛋白表达减少 过度的受体降解或减少的生物合成可以解释这一发现。 受体的脱敏和下调是调节靶细胞对激动剂应答的机制。内化通常伴随着这些事件，并从细胞表面去除活性信号受体。 我们发现用蛋白激酶C激动剂处理甲状旁腺细胞和表达CaR cRNA的非洲爪蟾卵母细胞使这些细胞对随后的高[Ca 2 +]挑战脱敏--就甲状旁腺激素（PTH）释放和高[Ca 2 +]调节的信号转导而言。 我们将测试的假设，钙调素受体激动剂促进受体脱敏和内化，在钙调素受体的羧基末端尾序列参与这些途径，钙调素受体蛋白的表达水平是决定细胞反应的关键[Ca 2 +]。 三个目标将解决这个假设。 (1)我们将通过评估用激动剂处理细胞后的PTH分泌和信号传导反应以及通过使用表面生物素化测定定量汽车的内化来确定CaR激动剂是否促进CaR的脱敏和内化。将通过共聚焦显微镜在表达绿色荧光蛋白标记的CaR的HEK-293细胞中跟踪CaR的细胞内运输。 (2)我们将评估的作用，钙调素受体的C-末端尾巴脱敏和内化尾截短突变体和定点诱变，以确定推定的积极或消极的内吞基序。 完整的信号转导通路在内化中的作用将得到解决。 (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，并将检测对高（Ca 2+）调节的降钙素分泌和细胞增殖的影响。 我们的研究应该提供深入了解CaR反应的分泌和生长的机制。

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.