NHERF-Mediated PTH Signaling in Mineral Ion Homeostasis

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In its biological actions on bone and kidney and overall mineral ion homeostasis, PTH stimulates the PTH/PTHrP receptor and activates distinctive signalling pathways within target cells (including adenylyl cyclase, phospholipase C, phospholipase-C independent protein kinase C and calcium channels). Growing evidence that some of these distinctive pathways have discernable differences in bone and renal cell function has led to increased study of signal selectivity and its cellular role. Na+/H+exchange regulatory factors (NHERFs) (components of signal modulating intracellular scaffold proteins) have recently been shown by our group to act as "molecular switches" that increase activation by PTH of the PLC pathway and suppress the adenylyl cyclase pathway. A gene knockout of NHERF-1, performed by collaborators, has an osteopenic skeletal phenotype. NHERFs have now been identified in bone cell lines. An opossum kidney cell line (OK) which is deficient in NHERF lacks the usual PTH stimulated blockade of phosphate uptake. The deficiency is reversed and wild type OK cell line function restored by NHERF transfection. This cell line serves as a model of PTH-mediated inhibition of tubular re- absorption of phosphate. Our specific aims are therefore: AIM I. Analyze the functional role of NHERFs with respect to PTH signaling and cell morphology in an opossum kidney cell model, comparing cell lines that are NHERF intact, defective and repleted. AIM II. Study the role of the NHERF/PTH1R complex in PTH signaling in bone cell lines in vitro. AIM III. Study the role of NHERF-1 in mediating the actions of PTH in bone, both in vitro and in vivo, working with the NHERF- 1 -/- mice provided by our colleagues for assessment of cellular mechanisms responsible for the skeletal phenotype.