NHERFs Specify PTH Receptor Signaling

NHERF 指定 PTH 受体信号传导

• 批准号: 6747919
• 负责人: GINO V SEGRE
• 金额: $ 36.51万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6747919
• 关键词: G protein; G protein coupled receptor kinase; SDS polyacrylamide gel electrophoresis; affinity chromatography; biological signal transduction; confocal scanning microscopy; genetically modified animals; hormone receptor; immunocytochemistry; immunoprecipitation; laboratory mouse; parathyroid hormone related protein; parathyroid hormones; phospholipase C; protein isoforms; receptor binding; receptor coupling; receptor expression; sodium hydrogen exchanger; southern blotting; western blottings

DESCRIPTION (provided by applicant): The sodium-hydrogen-exchanger regulatory factor 2 (NHERF2) assembles the parathyroid hormone 1 receptor (PTH1R) and PLCbeta through PDZ domain-specific interactions; PLbeta beta 1 and 2 bind to PDZ1 and PTH1R binds to PDZ2 of both NHERF1 and 2. PS12O cells do not express NHERFs, any NHE, or PTH1R, and thus provide an excellent model to study PTH1R-NHERF interactions. PTH stimulation of PS12O cells which express PTH1R and NHERF2 markedly augments signaling through PLCbeta and blocks signaling through adenylyl cyclase. PTH1R bound to NHERF couples to Gi/o protein(s) - activated Gi/o protein(s) releases beta-gamma subunits that stimulate PLCbeta and alpha subunits that inhibit adenylyl cyclase. This mechanism is novel; it provides cells with a mechanism to regulate PTH signaling that is specific to cells and also specific to membranes of polarized cells where NHERF-PTH1R is assembled. NHERF-PTH1R binding changes the way we think about PTH signaling and responses. It may also impact signaling by other G protein-coupled receptors (GPCR) because approximately 20% of GPCR have PDZ interaction motifs and more than 96 PDZ-domain proteins are in the genome. Aim I will define the PLCbeta isoforms that can be assembled by NHERF with PTH1R. Aim II will identify G proteins that are activated by PTH when PTH1R is unassembled, and also when it is assembled with NHERF. The beta2-adrenergic receptor binds NHERF PDZ1, not PDZ2. Aim III will determine how receptor-binding to PDZ1 and 2 differentially specify two functions known to be regulated by NHERF-beta2AR assembly - Na+/H+ exchange and endocytic sorting. We will also determine where and by what mechanisms NHERF-PTH1R dissociation is regulated, and the effects of NHERF binding to the cytoskeleton on PTH signaling. Aim 4 will define signaling in physiologic PTH targets - bone, renal proximal tubule and endothelial cells - when PTH1R is unassembled, and when assembled with NHERF. In Aim V, we will generate mice that express PTH1R which cannot bind NHERF, and PTH1R which cannot bind NHERF and also have disabled Gq-coupling. Phenotypes of the mice, compared with phenotypes of mice that express PTH1R which cannot couple to Gq and wild-type littermates, will allow us to sort PTH and PTHrP actions in the absence of PTH1R-coupling to Gi/o, Gq and both Gi/o and Gq, will have important physiologic implications, and will define some mechanisms of diseases, such as hyperparathyroidism and osteoporosis.

描述（由申请人提供）：通过PDZ域特异性相互作用组装甲状旁腺激素1受体（PTH1R）和PLCBETA； PLBETA Beta 1和2与pdz1和PTH1R结合，与NHERF1和2的PDZ2结合。PS12O细胞均未表达NHERF，任何NHE或PTH1R，因此为研究PTH1R-NHERF相互作用提供了出色的模型。 PTH刺激PS12O细胞表达PTH1R和NHERF2的PS12O细胞通过PLCBETA明显增强信号传导，并通过腺苷酸环化酶阻止信号传导。 PTH1R与NHERF夫妇结合到GI/O蛋白（S）激活的GI/O蛋白（S）释放β -GAMMA亚基，这些亚基刺激了PLCBETA和Alpha亚基，这些亚基抑制了抑制腺苷酸环化酶的α-蛋白。这种机制是新颖的。它为细胞提供了一种调节特定于细胞的PTH信号传导的机制，也针对组装NHERF-PTH1R的偏振细胞的膜。 NHERF-PTH1R结合改变了我们对PTH信号传导和响应的看法。它也可能影响其他G蛋白偶联受体（GPCR）的信号传导，因为大约20％的GPCR具有PDZ相互作用基序，而96个PDZ核心蛋白在基因组中。目的我将定义可以由NHERF与PTH1R组装的PLCBETA同工型。 AIM II将识别当PTH1R未组装时，以及与NHERF组装时，这些G蛋白会被PTH激活。 β2-肾上腺素受体结合NHERF PDZ1，而不是PDZ2。 AIM III将确定对PDZ1和2的受体结合如何差异指定两个已知的函数，该功能已被NHERF-BETA2AR组装调节-NA+/H+交换和内吞分类。我们还将通过调节NHERF-PTH1R解离的位置和哪些机制，以及NHERF与细胞骨架对PTH信号传导的影响。 AIM 4将定义生理PTH靶标的信号传导 - 骨骼，肾近端小管和内皮细胞 - 当PTH1R未被组装时，以及与NHERF组装时。在AIM V中，我们将生成表达无法绑定nherf的PTH1R的小鼠，而PTH1R无法绑定nherf并且也已禁用GQ耦合。与表达无法将pth1r的小鼠表型相比，小鼠的表型将无法与GQ和野生型窝窝亲，可以使我们能够在没有PTH1R偶联的情况下对PTH和PTHRP作用进行分类和PTHRP动作O和GQ将具有重要的生理意义，并将定义一些疾病的机制，例如甲状旁腺功能亢进和骨质疏松症。