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An essential role in phosphate homeostasis is played by Na/Pi cotransporter IIa that is localized in the brush borders of renal proximal tubular cells. Recent studies identified several PDZ proteins interacting with the COOH-terminal tail of NaPi-IIa, such as PDZK1 and NHERF-1. Here, by using yeast two-hybrid screen of mouse kidney cDNA library, we attempted to find proteins interacting with the N[H.sub.2]-terminal part of NaPi-IIa. We identified MAP17, a 17-kDa membrane protein that has been described to be associated with various human carcinomas, but it is also expressed in normal kidneys. Results obtained by various in vitro analyses suggested that MAP17 interacts with the fourth domain of PDZK1 but not with other PDZ proteins localized in proximal tubular brush borders. As revealed by immunofluorescence, MAP17 was abundant in S1 but almost absent in S3 segments. No alterations of the apical abundance of MAP17 were observed after maneuvers undertaken to change the content of NaPi-IIa (parathyroid hormone treatment, different phosphate diets). In agreement, no change in the amount of MAP17 mRNA was observed. Results obtained from transfection studies using opossum kidney cells indicated that the apical localization of MAP17 is independent of PDZK1 but that MAP17 is required for apical localization of PDZK1. In summary, we conclude that MAP17 1) interacts with PDZK1 only, 2) associates with the N[H.sub.2] terminus of NaPi-IIa within the PDZK1/NaPi-IIa/ MAP17 complex, and 3) acts as an apical anchoring site for PDZK1. interacting proteins; Na/Pi cotransport; PDZ proteins; NHERF-1; opossum kidney cells

The epithelial-Na+-channel (αβγENaC) regulates kidney salt-transport and blood pressure. Each ENaC subunit contains a PY motif (PPxY) and its mutation in β/γENaC causes Liddle syndrome, a hereditary hypertension. These (extended) PY motifs (PP616xY618xxL621) serve as binding sites for the ubiquitin ligase Nedd4-2, which decreases cell-surface expression of ENaC by unknown route(s). Using polarized kidney epithelia [Madin-Darby canine kidney I (MDCK-I)] cells stably expressing extracellularly myc-tagged wild type (WT) or PY-motif mutants of βENaC (P616A, Y618A or L621A, with WT-αγENaC), and live-imaging plus enzyme-linked immunosorbent assay (ELISA)-type assays to analyze routes/rates of ENaC internalization/recycling, we show here that cell-surface half-life of all PY mutants was fourfold longer than WT-ENaC (∼120 versus 30 minutes), reflecting primarily reduced channel internalization but also attenuated replenishment of cell-surface ENaC from a large subapical pool. The Y618A mutant revealed more severe internalization and replenishment defects than the other PY mutants. Internalized WT-ENaC was detected in sorting/recycling and late endosomes/lysosomes, while the Y618A mutant accumulated in the former. Nedd4-2 ubiquitinated ENaC at the apical membrane causing channel internalization and degradation. Cyclic AMP (cAMP) accelerated mobilization of subapical ENaC to the cell surface and long-term ENaC recycling, but only mobilization, not recycling, was inhibited in the PY mutants. These results suggest that the ENaC PY motifs (and Nedd4-2) primarily regulate channel internalization but also affect cAMP-dependent replenishment, providing important insight into the Liddle syndrome defects. [ABSTRACT FROM AUTHOR]

Inorganic phosphate (Pi) reabsorption in the renal proximal tubule occurs mostly via the Na+/Pi cotransporter type IIa (NaPi-IIa) located in the brush-border membrane (BBM) and is regulated, among other factors, by dietary Pi intake and parathyroid hormone (PTH). The PTH-induced inhibition of Pi reabsorption is mediated by endocytosis of Na/Pi-IIa from the BBM and subsequent lysosomal degradation. Megalin is involved in receptor-mediated endocytosis of proteins from the urine in the renal proximal tubule. The recently identified receptor-associated protein (RAP) is a novel type of chaperone responsible for the intracellular transport of endocytotic receptors such as megalin. Gene disruption of RAP leads to a decrease of megalin in the BBM and to a disturbed proximal tubular endocytotic machinery. Here we investigated whether the distribution of NaPi-IIa and/or its regulation by dietary Pi intake and PTH is affected in the proximal tubules of RAP-deficient mice as a model for megalin loss. In RAP-deficient mice megalin expression was strongly reduced and restricted to a subapical localization. NaPi-IIa protein distribution and abundance in the kidney was not altered. The localization and abundance of the NaPi-IIa interacting proteins MAP17, PDZK-1, D-AKAP2, and NHE-RF1 were also normal. Other transport proteins expressed in the BBM such as the Na+/H+ exchanger NHE-3 and the Na+/sulphate cotransporter NaSi were normally expressed. In whole animals and in isolated fresh kidney slices the PTH-induced internalization of NaPi-IIa was strongly delayed in RAP-deficient mice. PTH receptor expression in the proximal tubule was not affected by the RAP knock-out. cAMP, cGMP or PKC activators induced internalization which was delayed in RAP-deficient mice. In contrast, both wildtype and RAP-deficient mice were able to adapt to high-, normal, and low-Pi diets appropriately as indicated by urinary Pi excretion and NaPi-IIa protein abundance. [ABSTRACT FROM AUTHOR]