NHERFs Specify PTH Receptor Signaling

描述（由申请人提供）：钠-氢交换调节因子2 （NHERF2）通过PDZ结构域特异性相互作用组装甲状旁腺激素1受体（PTH1R）和PLCbeta；plβ β 1和β 2结合PDZ1， PTH1R结合NHERF1和2的PDZ2。ps120细胞不表达nherf、任何NHE或PTH1R，因此为研究PTH1R- nherf相互作用提供了一个很好的模型。PTH刺激表达PTH1R和NHERF2的ps120细胞可显著增强通过PLCbeta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PTH1R与Gi/o蛋白偶联-激活的Gi/o蛋白释放β - γ亚基，刺激plcβ和α亚基，抑制腺苷酸环化酶。这种机制是新颖的；它为细胞提供了一种调节PTH信号的机制，这种机制是细胞特异性的，也是NHERF-PTH1R聚集的极化细胞膜特异性的。NHERF-PTH1R结合改变了我们对PTH信号和反应的看法。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，基因组中有超过96个PDZ结构域蛋白。目的：我将定义可通过NHERF与PTH1R组装的PLCbeta亚型。Aim II将识别当PTH1R被拆解时以及当PTH1R与NHERF组装时被PTH激活的G蛋白。β -肾上腺素能受体结合NHERF PDZ1，而不是PDZ2。Aim III将确定PDZ1和pdz2的受体结合如何不同地指定已知由NHERF-beta2AR组装调节的两种功能- Na+/H+交换和内噬分选。我们还将确定NHERF- pth1r解离在何处以及通过何种机制受到调节，以及NHERF与细胞骨架结合对PTH信号传导的影响。Aim 4将定义PTH生生性靶点（骨、肾近端小管和内皮细胞）在PTH1R解组装和与NHERF组装时的信号传导。在Aim V中，我们将产生表达不能结合NHERF的PTH1R和不能结合NHERF且gq偶联也失效的PTH1R的小鼠。将这些小鼠的表型与表达PTH1R不能与Gq偶联的小鼠和野生型幼鼠的表型进行比较，将使我们能够在PTH1R与Gi/o、Gq以及Gi/o和Gq均偶联的情况下，对PTH和PTHrP的作用进行分类，具有重要的生理意义，并将明确甲状旁腺功能亢进和骨质疏松等疾病的一些机制。

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.