The renal tubular reabsorption of phosphate is mediated, in large measure, by a sodium- dependent phosphate transporter called Npt2a located in the apical membrane of the cells of the proximal convoluted tubule. Npt2a is tethered to the apical membrane by a PDZ domain adaptor protein isolated and cloned by our laboratory called the Sodium-Hydrogen Exchanger Regulatory Factor-1 (NHERF-1). NHERF-1 null mice demonstrate increased excretion of phosphate, calcium, and uric acid associated with calcium deposition in the renal papilla. In confirmation with the mouse, NHERF-1 mutations in humans have been associated kidney stones. Recent studies from our laboratory have indicated that Parathyroid hormone results in the phosphorylation of the first PDZ domain of NHERF-1 with the subsequent dissociation of Npt2a/NHERF-1 complexes, a decrease in the apical membrane abundance of Npt2a, and a decrease in phosphate transport. We speculate that this unique mechanism may be a general process that regulates NHERF-1 binding to some of its other target proteins. To study the role of NHERF-1 in renal phosphate transport and to determine if the phosphorylation of NHERF-1 is regulated by other hormones, we propose studies to examine the mechanism by which dopamine inhibits renal phosphate transport. Using freshly prepared kidney slices and renal proximal tubule cells in primary culture from wild-type and NHERF-1 null animals, we will first determine if NHERF-1 binds to the dopamine D1-like receptor using immunoprecipitation and confocal microscopy. Using 32P labeled cells, we will then determine if occupancy of the dopamine D1-like receptor results in the phosphorylation of NHERF-1 and, using adenovirus-mediated gene transfer of wild-type and mutated forms of NHERF-1, determine which residues in the first PDZ domain, the binding site of Npt2a, are phosphorylated. Specific interest will focus on serine77 that lies in the Npt2a binding groove of NHERF-1. We will then determine the signaling pathways used by the dopamine D1-like receptor by measuring cAMP accumulation, and PKC activity in wild-type and NHERF-1 null cells. To determine the physiologic role of NHERF-1, we will measure the effect of dopamine on sodium-dependent phosphate transport in wild-type and NHERF-1 null proximal tubule cells. Studies will also be performed in NHERF-1 null cells infected with adenovirus-NHERF-1 to determine the specific role of NHERF-1 in mediating the effect of dopamine and with mutant forms of NHERF-1 to determine the role of specific residues such as serine77. To examine the mechanism of inhibition, we will determine if dopamine dissociates Npt2a/NHERF-1 complexes using quantitative immunoprecipitation, confocal microscopy, and sucrose density gradient ultracentrifugation. Thus, our immediate objectives are to test the hypothesis that NHERF-1 interacts with the dopamine receptor(s) and that dopamine inhibits renal phosphate transport by phosphorylating the Npt2a binding protein NHERF-1 thereby dissociating NHERF-1/Npt2a complexes and decreasing the apical membrane abundance of Npt2a. We anticipate that comparison of hormonal transduction pathways will provide new insights into the biologic role of NHERF-1 on phosphate transport and, further understandings of the role of NHERF-1 in other physiologic and pathophysiologic processes such as nephrolithiasis, schizophrenia, psoriasis, aggressive forms of estrogen receptor positive breast cancer, and nerve deafness (Usher's syndrome). .
Hypophosphatemia as well as kidney stones are relatively common in Veteran patients. Recent studies have identified NHERF-1 as an important modulator of renal phosphate excretion and a target of Parathyroid hormone. Continued study of the role of the NHERF proteins is relevant to these diseases directly and indirectly to NHERF-1 related pathophysiologic conditions that may include kidney stones, schizophrenia, psoriasis, breast cancer, and nerve deafness (Usher's syndrome).