NHERFs Specify PTH Receptor Signaling

描述（由申请人提供）：钠-氢交换调节因子2（NHERF 2）通过PDZ结构域特异性相互作用组装甲状旁腺激素1受体（PTH 1 R）和PLC β; PL β 1和2与PDZ 1结合，PTH 1 R与NHERF 1和2的PDZ 2结合。PS12 O细胞不表达NHERF、任何NHE或PTH 1 R，因此为研究PTH 1 R-NHERF相互作用提供了极好的模型。表达PTH 1 R和NHERF 2的PS12 O细胞的PTH刺激显著增强通过PLC β的信号传导并阻断通过腺苷酸环化酶的信号传导。与NHERF结合的PTH 1 R与Gi/o蛋白偶联-激活的Gi/o蛋白释放β-γ亚基，其刺激抑制腺苷酸环化酶的PLC β和α亚基。这种机制是新颖的;它为细胞提供了一种调节PTH信号传导的机制，该机制对细胞是特异性的，也对组装NHERF-PTH 1 R的极化细胞膜是特异性的。NHERF-PTH 1 R结合改变了我们对PTH信号传导和反应的看法。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，并且基因组中有超过96种PDZ结构域蛋白。目的确定NHERF与PTH 1 R之间的PLC β亚型。目的II将确定G蛋白，PTH激活时，PTH 1 R是未组装的，也当它与NHERF组装。β 2-肾上腺素能受体结合NHERF PDZ 1，而不是PDZ 2。目的III将确定如何受体结合PDZ 1和2差异指定两个功能已知的NHERF-β 2 AR组件- Na+/H+交换和内吞分选调节。我们还将确定NHERF-PTH 1 R解离在哪里以及通过什么机制进行调节，以及NHERF与细胞骨架结合对PTH信号传导的影响。目的4将定义当PTH 1 R未组装时以及当与NHERF组装时生理PTH靶点-骨、肾近端小管和内皮细胞-中的信号传导。在目标V中，我们将产生表达不能结合NHERF的PTH 1 R和不能结合NHERF并且也具有失能的Gq偶联的PTH 1 R的小鼠。与表达不能偶联至Gq的PTH 1 R的小鼠和野生型同窝小鼠的表型相比，小鼠的表型将允许我们在PTH 1 R偶联至Gi/o、Gq以及Gi/o和Gq两者的情况下对PTH和PTHrP作用进行分类，这将具有重要的生理学意义，并且将定义疾病的一些机制，例如甲状旁腺功能亢进和骨质疏松症。

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.