Dataset Information

NHE3 regulatory factor 1 (NHERF1) modulates intestinal sodium-dependent phosphate transporter (NaPi-2b) expression in apical microvilli.

ABSTRACT: P(i) uptake in the small intestine occurs predominantly through the NaPi-2b (SLC34a2) co-transporter. NaPi-2b is regulated by changes in dietary P(i) but the mechanisms underlying this regulation are largely undetermined. Sequence analyses show NaPi-2b has a PDZ binding motif at its C terminus. Immunofluorescence imaging shows NaPi-2b and two PDZ domain containing proteins, NHERF1 and PDZK1, are expressed in the apical microvillar domain of rat small intestine enterocytes. Co-immunoprecipitation studies in rat enterocytes show that NHERF1 associates with NaPi-2b but not PDZK1. In HEK co-expression studies, GFP-NaPi-2b co-precipitates with FLAG-NHERF1. This interaction is markedly diminished when the C-terminal four amino acids are truncated from NaPi-2b. FLIM-FRET analyses using tagged proteins in CACO-2(BBE) cells show a distinct phasor shift between NaPi-2b and NHERF1 but not between NaPi-2b and the PDZK1 pair. This shift demonstrates that NaPi-2b and NHERF1 reside within 10 nm of each other. NHERF1(-/-) mice, but not PDZK1(-/-) mice, had a diminished adaptation of NaPi-2b expression in response to a low P(i) diet. Together these studies demonstrate that NHERF1 associates with NaPi-2b in enterocytes and regulates NaPi-2b adaptation.

SUBMITTER: Giral H

PROVIDER: S-EPMC3471691 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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NHE3 regulatory factor 1 (NHERF1) modulates intestinal sodium-dependent phosphate transporter (NaPi-2b) expression in apical microvilli.

Giral Hector H Cranston DeeAnn D Lanzano Luca L Caldas Yupanqui Y Sutherland Eileen E Rachelson Joanna J Dobrinskikh Evgenia E Weinman Edward J EJ Doctor R Brian RB Gratton Enrico E Levi Moshe M

The Journal of biological chemistry 20120817 42

P(i) uptake in the small intestine occurs predominantly through the NaPi-2b (SLC34a2) co-transporter. NaPi-2b is regulated by changes in dietary P(i) but the mechanisms underlying this regulation are largely undetermined. Sequence analyses show NaPi-2b has a PDZ binding motif at its C terminus. Immunofluorescence imaging shows NaPi-2b and two PDZ domain containing proteins, NHERF1 and PDZK1, are expressed in the apical microvillar domain of rat small intestine enterocytes. Co-immunoprecipitation ...[more]

PMID: 22904329

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Project description:The rat renal brush border membrane sodium/phosphate co-transporter NaPi-2 was analysed in Western blots with polyclonal antibodies raised against its N-terminal and C-terminal segments. Under reducing conditions, proteins of 45-49 and 70-90 kDa (p45 and p70) were detected with N-terminal antibodies, and proteins of 40 and 70-90 kDa (p40 and p70) were detected with C-terminal antibodies. p40 and p45 apparently result from a post-translational cleavage of NaPi-2 but remain linked through one or more disulphide bonds. Glycosidase digestion showed that both polypeptides are glycosylated; the cleavage site could thus be located between Asn-298 and Asn-328, which have been shown to constitute the only two N-glycosylated residues in NaPi-2. In the absence of reducing agents, both N-terminal and C-terminal antibodies detected p70 and a protein of 180 kDa (p180), suggesting the presence of p70 dimers. Much higher concentrations of beta-mercaptoethanol were required to produce a given effect in intact membrane vesicles than in solubilized proteins, indicating that the affected disulphide bonds are not exposed at the surface of the co-transporter. Phosphate transport activity decreased with increasing concentrations of reducing agents [beta-mercaptoethanol, dithiothreitol and tris-(2-carboxyethyl)phosphine] and was linearly correlated with the amount of p180 detected. The target sizes estimated from the radiation-induced loss of intensity of p40, p70 and p180 were all approx. 190 kDa, suggesting that NaPi-2 exists as an oligomeric protein in which the subunits are sufficiently close to one another to allow substantial energy transfer between the monomers. When protein samples were pretreated with beta-mercaptoethanol [2.5% and 5% (v/v) to optimize the detection of p40 and p70] before irradiation, target sizes estimated from the radiation-induced loss of intensity of p40 and p70 were 74 and 92 kDa respectively, showing the presence of disulphide bridges in the molecular structure of NaPi-2.

Project description:Transporters of the SLC34 family (NaPi-IIa,b,c) catalyze uptake of inorganic phosphate (Pi) in renal and intestinal epithelia. The transport cycle requires three Na(+) ions and one divalent Pi to bind before a conformational change enables translocation, intracellular release of the substrates, and reorientation of the empty carrier. The electrogenic interaction of the first Na(+) ion with NaPi-IIa/b at a postulated Na1 site is accompanied by charge displacement, and Na1 occupancy subsequently facilitates binding of a second Na(+) ion at Na2. The voltage dependence of cotransport and presteady-state charge displacements (in the absence of a complete transport cycle) are directly related to the molecular architecture of the Na1 site. The fact that Li(+) ions substitute for Na(+) at Na1, but not at the other sites (Na2 and Na3), provides an additional tool for investigating Na1 site-specific events. We recently proposed a three-dimensional model of human SLC34a1 (NaPi-IIa) including the binding sites Na2, Na3, and Pi based on the crystal structure of the dicarboxylate transporter VcINDY. Here, we propose nine residues in transmembrane helices (TM2, TM3, and TM5) that potentially contribute to Na1. To verify their roles experimentally, we made single alanine substitutions in the human NaPi-IIa isoform and investigated the kinetic properties of the mutants by voltage clamp and (32)P uptake. Substitutions at five positions in TM2 and one in TM5 resulted in relatively small changes in the substrate apparent affinities, yet at several of these positions, we observed significant hyperpolarizing shifts in the voltage dependence. Importantly, the ability of Li(+) ions to substitute for Na(+) ions was increased compared with the wild-type. Based on these findings, we adjusted the regions containing Na1 and Na3, resulting in a refined NaPi-IIa model in which five positions (T200, Q206, D209, N227, and S447) contribute directly to cation coordination at Na1.

Dataset Information

NHE3 regulatory factor 1 (NHERF1) modulates intestinal sodium-dependent phosphate transporter (NaPi-2b) expression in apical microvilli.

Publications

NHE3 regulatory factor 1 (NHERF1) modulates intestinal sodium-dependent phosphate transporter (NaPi-2b) expression in apical microvilli.

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