We hypothesize that PDZK1 and NHERF-1 establish an extended network beneath the apical membrane to which membrane proteins and regulatory components are anchored.

1-channel (abgENaC) regulates kidney salt-transport and blood pressure. Each ENaC subunit contains a PY motif (PPxY) and its mutation in b/gENaC causes Liddle syndrome, a hereditary hypertension. These (extended) PY motifs (PP 616 xY 618 xxL 621 ) 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 bENaC (P616A, Y618A or L621A, with WT-agENaC), 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 cAMPdependent replenishment, providing important insight into the Liddle syndrome defects.

Experimental studies have highlighted the potential in£uence of contaminants on marine mammal immune function and anthropogenic contaminants are commonly believed to in£uence the development of diseases observed in the wild. However, estimates of the impact of contaminants on wild populations are constrained by uncertainty over natural variation in disease patterns under di¡erent environmental conditions. We used photographic techniques to compare levels of epidermal disease in ten coastal populations of bottlenose dolphins (Tursiops truncatus) exposed to a wide range of natural and anthropogenic conditions. Epidermal lesions were common in all populations (a¡ecting 460% of individuals), but both the prevalence and severity of 15 lesion categories varied between populations. No relationships were found between epidermal disease and contaminant levels across the four populations for which toxicological data were available. In contrast, there were highly signi¢cant linear relationships with oceanographic variables. In particular, populations from areas of low water temperature and low salinity exhibited higher lesion prevalence and severity. Such conditions may impact on epidermal integrity or produce more general physiological stress, potentially making animals more vulnerable to natural infections or anthropogenic factors. These results show that variations in natural environmental factors must be accounted for when investigating the importance of anthropogenic impacts on disease in wild marine mammals.

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 NH 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 NH 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 NA-DEPENDENT PHOSPHATE transport protein NaPi-IIa (SLC34A1) is the major mediator in renal reabsorption of inorganic phosphate (P i ) (2; for review, see Ref. 24). In proximal tubules, NaPi-IIa is localized in the brush border membrane and is a part of heteromultimeric complexes scaffolded by the PDZ proteins PDZK1 and NHERF-1 (4,9,11,12,17,19,26,30). Interaction of NaPi-IIa with these PDZ proteins was shown to occur via the COOH-terminal PDZ binding motif (TRL) of NaPi-IIa. Moreover, these interactions occur only with distinct PDZ domains of PDZK1 and NHERF-1 (11). Recent data suggested that these interactions play important roles for the apical positioning of NaPi-IIa. Overexpression of single PDZ domains in opossum kidney (OK) cells resulted in an impairment of apical sorting/positioning of NaPi-IIa cotransporters (12), and the complete lack of NHERF-1, as demonstrated with a mouse knockout, resulted in reduced abundance of NaPi-IIa and urinary wasting of phosphate (26). On the other hand, recent experiments on targeted PDZK1 gene disruption did not result in a significant renal phenotype, suggesting that functional compensation of the lack of PDZK1 might occur by other PDZ proteins (19).To explore the interactions of NaPi-IIa in more detail, we attempted to find proteins that interact with the NH 2 terminus of NaPi-IIa. Results obtained by a yeast two-hybrid screen against a mouse kidney cDNA library suggested that MA...

We conclude that D-AKAP2 anchors protein kinase A (PKA) to PDZK1 and to a lesser extent to NHERF-1. Since PDZK1 and NHERF-1 both sequester NaPi-IIa to the apical membrane, D-AKAP2 may play an important role in the parathyroid hormone (PTH)-mediated regulation of NaPi-IIa by compartmentalization of PKA.

Regulation of renal proximal tubular reabsorption of phosphate (P i ) is one of the critical steps in P i homeostasis. Experimental evidence suggests that this regulation is achieved mainly by controlling the apical expression of the Na + -dependent P i cotransporter type IIa (NaPi-IIa) in proximal tubules. Only recently have we started to obtain information regarding the molecular mechanisms that control the apical expression of NaPi-IIa. The first critical observation was the finding that truncation of only its last three amino acid residues has a strong effect on apical expression. A second major finding was the observation that the last intracellular loop of NaPi-IIa contains sequence information that confers parathyroid hormone (PTH) sensitivity. The use of the above domains of the cotransporter in yeast two-hybrid (Y2H) screening allowed the identification of proteins interacting with NaPi-IIa. Biochemical and morphological, as well as functional, analyses have allowed us to obtain insights into the physiological roles of such interactions, although our present knowledge is still far from complete.

Inorganic phosphate (P i ) reabsorption in the renal proximal tubule occurs mostly via the Na + /P i cotransporter type IIa (NaP i -IIa) located in the brushborder membrane (BBM) and is regulated, among other factors, by dietary P i intake and parathyroid hormone (PTH). The PTH-induced inhibition of P i reabsorption is mediated by endocytosis of Na/P i -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 NaP i -IIa and/ or its regulation by dietary P i 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. NaP i -IIa protein distribution and abundance in the kidney was not altered. The localization and abundance of the NaP i -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 NaP i -IIa was strongly delayed in RAPdeficient 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-P i diets appropriately as indicated by urinary P i excretion and NaP i -IIa protein abundance.

CFTR was reported to regulate ENaC channel opening, decreasing ENaC activity in airways and increasing it in sweat ducts. We generated MDCK-I cell lines stably expressing tagged alphabetagammaENaC+CFTR or ENaC alone, and developed an assay to quantify cell-surface half-life of ENaC. Surprisingly, we found that co-expressed CFTR stabilizes ENaC at the plasma membrane, suggesting that CFTR regulates ENaC stability, not just opening.