Project Summary/Abstract The goal of this project is to determine how the structural protein NHERF-1, sodium-hydrogen exchanger regulatory factor isoform 1, regulates the trafficking of Npt2a, the type IIa sodium phosphate cotransporter, to the apical membrane of the renal proximal tubule. The expression of Npt2a at the apical membrane is a critical regulatory step because the level of expression and function of Npt2a is the primary regulator of total body phosphate homeostasis. VA-funded research from this laboratory had revealed that the absence of NHERF-1 in a model of proximal renal tubule, OK (opossum kidney) cells, resulted in a marked decrease in Npt2a expression. The decrease in expression was due to two factors 1) decreased transcription of Npt2a, and 2) decreased trafficking of Npt2a to the apical membrane. The mechanisms for the faulty trafficking of Npt2a have not been determined and are the subject of this proposal. Npt2a translated in the NHERF-deficient cells (OK-H) lacked a critical post-translational modification, glycosylation. These proteins, instead of trafficking to apical membrane, accumulated in a perinuclear location. Inefficient apical membrane localization had previously been described in a mouse lacking expression of NHERF-1. Expression of a NHERF-1 construct that lacked a normal PDZ-2 domain did not allow trafficking of Npt2a to the apical membrane. NHERF-1 is a multiple PDZ domain protein which interacts with multiple signaling molecules and transporter proteins. A role for the PDZ-2 domain in Npt2a regulation had not been observed previously. Finally, inhibition of SNARE (SNAP Receptor) protein interaction using a competing peptide introduced into OK cells blocked insertion of Npt2a into the apical membrane, demonstrating that Npt2a is transported to the apical membrane via a vesicular transport mechanism. The preliminary data suggested the hypothesis that NHERF-1 is essential for the forward trafficking of Npt2a from site of synthesis to the apical membrane. First, the role of the post-translational modifications glycosylation and phosphorylation on Npt2a apical membrane trafficking will be examined by determining the cellular localization of transfected Npt2a constructs lacking the motifs required for these post translational modifications in a cell culture model. The glycosylation, phosphorylation, and ubiquitination states of Npt2a will be compared in NHERF-replete and NHERF-deficient OK cells and in wild type and NHERF-1 knock out mouse proximal tubule cells. The effect of inhibitors of glycosylation on the intracellular localization of Npt2a will be determined. Second, the intracellular site or sites of interaction between NHERF-1 and Npt2a will be analyzed. Forward trafficking of Npt2a labeled by a GFP tag and by S35 methionine in OK cells and OK-H cells will be slowed by culture at 20 C. Analysis of Npt2a localization by confocal imaging, density centrifugation, and immunoelectronmicroscopy will be performed at sequential time points until the proteins are detected as biotinylated forms, indicating appropriate insertion into the apical membrane. Trafficking of Npt2a in the two cell culture models will be compared. Proteins associated with Npt2a at each time point will be determined by immunoprecipitation and proteomic analysis. To define which steps in the forward trafficking are NHERF- dependent, Npt2a trafficking will be compared in NHERF-replete and NHERF-deficient cells under conditions of traffic arrest: ezrin deficiency, inhibition of SNARE interaction, and CLC-5 (intracellular chloride channel CLC family isoform 5) deficiency. Third, the sites on Npt2a and NHERF-1 critical for the NHERF-1 effect on Npt2a trafficking will be determined by mutational analysis of both proteins followed by both in vitro analysis of protein interaction and assessment of intracellular interaction by FRET methodology. These studies will define where NHERF-1 acts in Npt2a forward trafficking, define the sites on both proteins responsible for their interaction, and yield mechanistic insights for this unique functional process.