DESENSITIZATION AND DOWN REGULATION OF CALCIUM RECEPTORS

钙受体的脱敏和下调

• 批准号: 6178064
• 负责人: DOLORES M. SHOBACK
• 金额: $ 28.26万
• 依托单位: NORTHERN CALIFORNIA INSTITUTE/RES/EDU
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6178064
• 关键词: adenylate cyclase; biological signal transduction; calcitonin; calcium; cell proliferation; confocal scanning microscopy; cyclic AMP; enzyme activity; enzyme inhibitors; fibroblasts; green fluorescent proteins; inositol phosphates; intracellular transport; kidney cell; mutant; protein sequence; receptor binding; receptor expression; receptor sensitivity; site directed mutagenesis; stimulant /agonist; tissue /cell culture

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.

CaR 感知 [Ca2+] 的变化并激活下游效应器。 有证据表明，足够数量的 CaR 表达对于甲状旁腺和肾脏等组织中精确的 Ca2+ 传感是必要的。 1 个异常 CaR 基因的杂合子患者患有轻度高钙血症和低钙尿症，而纯合子则严重受到危及生命的高钙血症和甲状旁腺功能亢进的影响，这支持了基因剂量效应。 原发性甲状旁腺功能亢进症患者的甲状旁腺中 CaR 蛋白表达降低。 受体过度降解或生物合成减少可以解释这一发现。 受体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，并用于从细胞表面去除活性信号受体。 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 羧基末端尾部的序列参与了这些途径； CaR 蛋白表达水平对于确定细胞对 [Ca2+] 的反应至关重要。 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 分泌和信号反应以及使用表面生物素化测定定量 CaR 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跟踪表达绿色荧光蛋白标记的 CaR 的 HEK-293 细胞中 CaR 的细胞内运输。 (2)我们将使用尾部截短突变体和定点诱变来评估CaR C末端尾部在脱敏和内化中的作用，以鉴定假定的阳性或阴性内吞基序。 将讨论完整信号通路在内化中的作用。 (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，并测试对高 (Ca2+] 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对分泌和生长中 CaR 反应性的潜在机制的见解。