NHERFs Specify PTH Receptor Signaling

描述(申请人提供)：钠氢交换调节因子2(NHERF2)通过PDZ结构域的特异性相互作用组装甲状旁腺激素1受体(PTH1R)和PLCbeta；PLbeta 1和2结合PDZ1，PTH1R结合NHERF1和2的PDZ2。PS120O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PS120O细胞显著增强通过PLCβ的信号转导，并阻断通过腺苷环化酶的信号转导。与NHERF结合的PTH1R偶联到Gi/o蛋白(S)激活的Gi/o蛋白(S)释放β-伽马亚基，刺激PLCβ和α亚基抑制腺苷环化酶。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，该机制专用于细胞，也专用于组装NHERF-PTH1R的极化细胞膜。NHERF-PTH1R结合改变了我们思考PTH信号和反应的方式。它也可能影响其他G蛋白偶联受体的信号转导，因为大约20%的G蛋白偶联受体具有PDZ相互作用基序，基因组中有96个以上的PDZ结构域蛋白。目的定义可由NHERF与PTH1R组装的PLCβ亚型。AIM II将在PTH1R解组和与NHERF组装时鉴定PTH激活的G蛋白。β2-肾上腺素能受体结合NHERF PDZ1，而不是PDZ2。目的III将确定与PDZ1和2的受体结合如何不同地指定已知的受NHERF-Beta2AR组装调控的两种功能-Na+/H+交换和内吞分选。我们还将确定NHERF-PTH1R解离的调节位置和机制，以及NHERF与细胞骨架结合对PTH信号的影响。AIM 4将定义当PTH1R未组装以及当与NHERF组装时，生理性PTH靶点-骨、肾近端小管和内皮细胞-中的信号。在目标V中，我们将产生表达PTH1R的小鼠，PTH1R不能结合NHERF，PTH1R不能结合NHERF，也禁用了GQ偶联。与表达PTH1R的小鼠的表型相比，不能与Gq偶联的PTH1R和野生型小鼠相比，将使我们能够在PTH1R偶联到Gi/o、Gq以及Gi/o和Gq的情况下对PTH和PTHrP的作用进行分类，将具有重要的生理意义，并将定义一些疾病的机制，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.