MODULATION OF RECEPTOR NUMBER IN CULTURED CELLS

培养细胞中受体数量的调节

• 批准号: 6142183
• 负责人: PATRICIA M. HINKLE
• 金额: $ 2.12万
• 依托单位: UNIVERSITY OF ROCHESTER
• 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 2000-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6142183
• 关键词: G protein; antisense nucleic acid; calcium flux; calcium transporting ATPase; calmodulin; electron microscopy; hormone receptor; hormone regulation /control mechanism; immunofluorescence technique; phospholipase C; phosphorylation; pituitary gland; receptor binding; receptor coupling; receptor expression; receptor sensitivity; thyrotropin releasing hormone; tissue /cell culture; transfection

DESCRIPTION (Adapted from the applicant's abstract): The pituitary receptor for thyrotropin-releasing hormone (TRH) serves as a model for the family of G-protein coupled Ca2+-mobilizing receptors. The proposed studies aim to establish how the [Ca2+]i response to TRH is controlled and how receptor localization affects the TRH response. The first goal is to determine the molecular mechanisms of the entire [Ca2+]i response to TRH. The observation that TRH directly activates Ca2+ efflux from cells forms the basis for the working hypothesis: that the TRH receptor is coupled to a Ca2+ pump, likely through a G-protein, i.e., that a plasma membrane Ca2+-transporting ATPase is a novel effector, and that activation of Ca2+ afflux controls the TRH response. The biochemical basis for TRH activation of Ca2+ afflux will be established by determining if TRH activates a Ca2+ pump directly, how the activation occurs, and which Ca2+ pump is activated. The importance of TRH activation of Ca2+ afflux to the [Ca2+]i and secretory responses will be measured in cells that are over expressing the hormone-responsive Ca2+ pump and in cells in which the Ca2+ pump has been inhibited. A limited analysis of TRH action in normal cells will be performed to determine which cells bind TRH, whether these internalize the TRH receptor; and whether key findings about TRH control of [Ca2+]i apply to normal lactotrophs and thyrotrophs. The second goal is to determine the mechanism of agonist-induced changes in TRH receptor localization and the importance of receptor trafficking to the TRH response. The TRH receptor undergoes extensive internalization when agonist binds and undergoes extensive recycling when agonist is removed. The hypothesis that the cycling of receptors controls the cellular responses to TRH will be tested. Agonist-activated receptor redistribution will be characterized with the objective of learning the mechanism and, most importantly, the consequences of receptor trafficking to the cell. The pathways of ligand-induced sequestration and recycling of TRH receptors will be determined by co-localizing receptor and ligand, localizing receptors at the EM level, and seeing if receptors concentrate in caveolae. The mechanism of receptor sequestration will be determined by altering residues potentially important in targeting, testing the importance of phosphorylation, and measuring the association of wild type and internalization defective receptors with adaptor proteins.

描述（改编自申请人摘要）：垂体 促甲状腺激素释放激素（TRH）受体作为模型， G蛋白偶联钙动员受体家族。拟议 研究旨在确定[Ca 2 +]i对TRH的反应是如何控制的， 以及受体定位如何影响TRH反应。第一个目标 是为了确定整个[Ca 2 +]i反应的分子机制， 到TRH。TRH直接激活Ca 2+流出的观察结果表明， 细胞形成了工作假设的基础：TRH受体 可能通过G蛋白与Ca 2+泵偶联，即，的 质膜Ca 2+转运ATP酶是一种新的效应子， Ca 2+内流的激活控制TRH反应。生化 TRH激活Ca 2+内流的基础将通过以下方式建立： 确定TRH是否直接激活Ca 2+泵， 发生，以及哪个Ca 2+泵被激活。TRH的重要性 激活Ca 2+流入[Ca 2 +]i和分泌反应， 在过度表达钙敏感性Ca 2+的细胞中测量 在细胞中，Ca 2+泵被抑制。有限 将进行正常细胞中TRH作用的分析以确定 哪些细胞结合TRH，这些细胞是否内化TRH受体;以及 关于TRH控制[Ca 2 +]i的关键发现是否适用于正常 催乳素和促甲状腺素。第二个目标是确定 激动剂诱导的TRH受体定位变化的机制 受体运输对TRH反应的重要性。TRH 当激动剂结合时，受体经历广泛的内化， 当激动剂被去除时经历广泛的再循环。的假设 受体的循环控制细胞对TRH的反应 会得到考验激动剂激活的受体再分布将是 以学习机制为目标， 重要的是，受体运输到细胞的后果。的 TRH受体的配体诱导隔离和再循环途径 将通过共定位受体和配体，定位 在EM水平上观察受体是否集中在 小窝受体隔离的机制将被确定 通过改变在靶向中潜在重要的残基，测试 磷酸化的重要性，并测量野生型 型和内化缺陷受体与衔接蛋白。