Edward J. Weinman - US grants

The apical membrane of the proximal convoluted tubule of the kidney contains a Na+-H+ exchange transport system. This transporter is important in reclamation of filter bicarbonate and, hence, the acid base status of the organism. A Na+-H+ exchanger is also present in other cells and activation of this transporter has been implicated in a number of cell processes including growth, tumor formation, and defense of cell volume. At the present time, the transporter has been defined functionally and is known to be regulated. Little is known, however, about the nature of the transport protein(s) itself. The present application proposes studies of the Na+-H+ exchanger from the brush border membrane of the proximal tubule of the rabbit. There are three specific aims. 1) Solubilization and reconstitution of renal brush border Na+-H+ exchanger. Characterization of the Na+-H+ would be facilitated by development of the methodology to solubilize the transporter from renal brush border membranes and to assay its activity in a reconstituted system using artificial phospholipid vesicles. Detergent extracts of brush border membrane proteins will be reconstituted into proteoliposomes and tested for the presence of proton gradient stimulated, amiloride inhibitable sodium uptake. The transport studies will determine if the transport characteristics in the proteoliposomes are similar to those in natural membranes. 2) Isolation and characterization of renal brush border Na+-H+ exchanger. Protein separation techniques will be used to fractionate, purify, and characterize the Na+-H+ exchanger. Protein fractions will be reconstituted into phospholipid vesicles and Na+-H+ exchange activity assayed. The nature of the proteins will be determined by SDS-PAGE. 3) Regulation of renal brush border Na+-H+ exchanger by protein kinases. The regulation of the Na+-H+ exchanger by cAMP dependent protein kinase, protein kinase C, and calcium-calmodulin dependent protein kinase will be determined by phosphorylating the solubilized membrane proteins and purified protein fractions with the specific protein kinases. The effect of phosphorylation of selected proteins on Na+-H+ exchange transport will be determined in reconstituted proteoliposomes. SDS-PAGE and autoradiography will be used to determine the substrates of the protein kinases.

NHE-RF is a new discovered PDZ motif protein that is a necessary co- factor in cAMP-dependent protein kinase (PKA) inhibition of the renal Na/H exchanger, NHE3. NHE-RF binds to NHERF, to NHE3, and to ezrin suggesting that NHE-RF interacts uniquely with its cellular targets forming signaling-complexes. The present grant focuses on the biochemical requirements for and physiologic significance of the physical interaction between proteins moieties involved in PKA mediated inhibition of NHE3. Specific Aim 1: To determine the functional role of NHE-RF dimerization. NHE-RF binds to NHE-RF forming head-to-head dimers but the mechanism of and the physiologic effect of dimerization are unexplored. Biochemical (Far Westerns) and biophysical (biosensor) assays will be used to define the kinetics of NHE-RF/NHE-RF binding. Yeast two-hybrid screening of mutant mini-libraries will be used to determine the binding amino acid sequences and to select mutants that fail to dimerize. The physiologic effect of dimerization on the effect of cAMP on Na/H exchange will be studied using fluorescence techniques in PS120 cells expressing NHE3 and NHE-RF mutants that do not dimerize. Specific Aim 2: To delineate the molecular basis for NHE3 regulation by NHE-RF. NHE-RF binds to NHE3 and the two proteins co-immunoprecipitate. Biochemical and biophysical assays will be used to define the kinetics of NHE-RF/NHE3 binding, and yeast two-hybrid screening of mutant mini- libraries to determine the binding amino acids sequences and to select loss-of-function mutants. The effect of cAMP on Na/H transport, the phosphorylation state of NHE3, and the co-immunoprecipitation of NHE-RF and NHE3 will be examined in PS120 cells expressing mutant NHE-RF and/or NHE3 which do not bind. Specific Aim 3. To define the physiologic importance of NHE-RF in the PKA regulation of NHE3. New data suggests NHE-RF binds to the PKA anchor protein, ezrin. Biochemical and biophysical assays will be used to study the kinetics of NHE-RF/ezrin binding and yeast two-hybrid studies to define the binding sequences. The physiologic effect of NHE- RF/ezrin binding will be studied in PS120 cells expressing loss-of- function NHE-RF mutants or polypeptides representing the ERM region of ezrin.

[unreadable] DESCRIPTION (provided by applicant): The Sodium-Hydrogen Exchanger Regulatory Factor-1 (NHERF-1) is an adaptor protein containing two protein-interactive PDZ domains and a C-terminal ERM binding domain that localizes to the brush border membrane of renal proximal convoluted tubule cells and binds to Npt2a, the major sodium-dependent phosphate transporter. Our recent studies have indicated that sodium-dependent phosphate transport in proximal tubule cells from NHERF-1-/- kidneys are resistant to the inhibitory effect of Parathyroid Hormone (PTH). In this current application, we explore the hypothesis that PTH mediates the phosphorylation of specific residues in PDZ I of NHERF-1 thereby regulating Npt2a/NHERF-1 complexes, the abundance of Npt2a in the apical membrane of renal proximal tubule cells, and as a consequence, the tubular reabsorption of phosphate. Elucidation of the factors that regulate the binding of target proteins to PDZ I of NHERF-1 may also provide broader insights into how regulation of PDZ domains of adaptor proteins impact on biologic responses to hormones and on the pathophysiology of NHERF-1 related diseases. In intact animals and cultured proximal tubule cells, we will use physiologic, biochemical, and cell biologic assays to determine how PTH-mediated phosphorylation of PDZ I of NHERF-1 regulateS the binding affinity of target proteins such as Npt2a and the proximal tubule reabsorption of phosphate. We propose three specific aims. First, we will map the serine and/or threonine residues in PDZ I of NHERF-1 that are phosphorylated in response to PTH and downstream protein kinases. Second, we propose to study the association and dissociation of Npt2a/NHERF-1 complexes in response to PTH-mediated phosphorylation of NHERF-1 using in-vitro and in-vivo assays. Third, we will determine the physiologic role of PTH-mediated NHERF-1 phosphorylation on the regulation of phosphate transport in the proximal tubule of the kidney. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE [unreadable] NHERF-1 is an adaptor protein that binds multiple transporters in the kidney including Npt2a, the major renal proximal tubule phosphate transporter. We will study the hypothesis that Parathyroid Hormone-mediated regulation of renal phosphate transport involves regulation of the binding of Npt2a to NHERF-1 by site-specific phosphorylation of the PDZ I domain of NHERF-1. These observations may provide mechanistic insights into the processes that regulate the binding of target proteins to adaptors such as NHERF-1 and provide broader insights into the pathophysiology of NHERF-1 related diseases. [unreadable] [unreadable] [unreadable]