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.