G-protein coupled receptors require cytosolic adaptor proteins for proper function and regulation. A prime example is the regulation of type 1 parathyroid hormone receptor (PTH1R) by NaVhT Exchange Regulatory Factor 1 (NHERF1). In non-polarized cells, NHERF1 has been shown to alter the lateral mobility, signaling and internalization of the PTH1R. In this proposal we will investigate the role of NHERF1 in the regulation of PTH1R function in polarized proximal convoluted tubule (PCT) cells. In PCT cells PTH1R is expressed on both the apical and basolateral membrane while NHERF1 localization is confined to the apical membrane. Because of this polarized expression of NHERF1, we hypothesize that PTH1R will behave differently in the two membranes. The effects of NHERF1 on the distribution, mobility, signaling and internalization of apical and basolateral receptors will be studied. We predict that NHERF1 will anchor PTH1R to the apical actin cytoskeleton causing a decrease in the lateral mobility and rate of internalization. Furthermore, this tethering is expected to lead to ligand-induced calcium transients. Disruption of anchoring with NHERF1 knockdowns, or expression of receptor constructs unable to bind NHERF1 or NHERF1 mutants that do not bind the cytoskeleton are expected to reverse the polarizing effects of NHERF1 and lead to uniform PTH1R behavior throughout the cell. These studies will be conducted using various live-cell imaging techniques such as fluorescence recover after photobleaching, quantum dot single molecule tracking, optical measurements of cAMP and calcium and image cross-correlation analysis. These studies will enhance our understanding of the regulation of PTH1R by NHERF1 in the physiologically relevant setting of the proximal convoluted tubule. Since disorders of PTH1R and NHERF1 in the proximal tubules lead to disruption of phosphate homeostasis, better understanding of their physiology may lead to new treatment strategies for phosphatemia and phosphoruria. Because NHERF1 interacts with a wide range of GPCRs, ion channels and tyrosine kinases, extensions of these studies could translate into treatments for numerous disorders.
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