Regulation of renal proximal tubule transporters, NHE3 (sodium hydrogen exchanger 3) and Npt2 (sodium phosphate cotransporter Ila) involve the PDZ proteins NHERF-1 and NHERF-2 (sodium hydrogen exchanger regulatory factors) that bind to these transporters and transduce hormonal signals that regulate transport activity. The current application explores the hypothesis that the NHERF proteins interact with one another and with other PDZ proteins to regulate the activity and/or cell surface expression of NHE3, Npt2 and other proteins. To this end, a NHERF-1 (-/-) mouse was developed that will prove extremely valuable in defining the functions of NHERF in the mammalian kidney and providing new insights into the defects in electrolyte metabolism associated with human disease.
Specific Aim 1 is to define the structural basis for NHERF localization in cells and its impact on recognition of renal targets. Gel overlays and pulldowns assays will be used to define the regions required for NHERF-1 phosphorylation, dimerization and recognition of targets such as NHE3 and Npt2. Coexpression, coimmunoprecipitation and confocal microscopy will be used to investigate the link between NHERF phosphorylation, localization, target recognition and physiologic effects.
Specific Aim 2 is to define the role of NHERF isoforms in signal complex regulation of NHE3. Acute and chronic hormonal regulation of NHE3 will be determined in wild-type (WT) and NHERF-1 (-/-) null mice using in vivo microperfusion, fluorescence measurements in tubule suspensions and in primary cell cultures, and 22Na+uptake in isolated brush border membrane vesicles (BBM). NHE3 trafficking in WT and NHERF-1 null mice will be studied using immunocytochemistry, cell surface biotinylation and western immunoblotting of subcellular organelles.
Specific Aim 3 will define the role of NHERF-1 in the trafficking and activity of Npt2. Clearance and balance experiments as well as studies using cultured cells and isolated BBM will be used to examine the effects of cAMP, hormones such as PTH and vitamin D and alterations in the dietary intake of phosphorus on Npt2 distribution and activity in WT and NHERF-1 (-/-) animals.
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