NHE3 Regulatory Factor 1 (NHERF1) Modulates Intestinal Sodium-dependent Phosphate Transporter (NaPi-2b) Expression in Apical Microvilli

• Published in: The Journal of biological chemistry Vol. 287; no. 42; pp. 35047 - 35056
• Main Authors: Giral, Hector, Cranston, DeeAnn, Lanzano, Luca, Caldas, Yupanqui, Sutherland, Eileen, Rachelson, Joanna, Dobrinskikh, Evgenia, Weinman, Edward J., Doctor, R.Brian, Gratton, Enrico, Levi, Moshe
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 12.10.2012; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Caco-2 Cells; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cytoskeletal Proteins; Enterocytes - cytology; Enterocytes - metabolism; Fluorescence Resonance Energy Transfer (FRET); Gene Expression Regulation - physiology; HEK293 Cells; Humans; Intestinal Epithelium; Intracellular Signaling Peptides and Proteins - genetics; Intracellular Signaling Peptides and Proteins - metabolism; Male; Membrane Biology; Membrane Proteins; Membrane Transport; Mice; Mice, Knockout; Microvilli - genetics; Microvilli - metabolism; NaPi; PDZ; Phosphate; Phosphoproteins - genetics; Phosphoproteins - metabolism; Plasma Membrane; Protein Structure, Tertiary; Protein-Protein Interactions; Rats; Rats, Sprague-Dawley; Sodium-Hydrogen Exchangers - genetics; Sodium-Hydrogen Exchangers - metabolism; Sodium-Phosphate Cotransporter Proteins, Type IIb - biosynthesis; Sodium-Phosphate Cotransporter Proteins, Type IIb - genetics; Transporters; Plasma Membrane; Phosphate; Membrane Transport; PDZ; Transporters; NaPi; Fluorescence Resonance Energy Transfer (FRET); Intestinal Epithelium; Protein-Protein Interactions
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.392415

Pi uptake in the small intestine occurs predominantly through the NaPi-2b (SLC34a2) co-transporter. NaPi-2b is regulated by changes in dietary Pi 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-2BBE 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 Pi diet. Together these studies demonstrate that NHERF1 associates with NaPi-2b in enterocytes and regulates NaPi-2b adaptation. Background: The type 2b sodium-dependent phosphate co-transporter (NaPi-2b) is the main mediator of intestinal active Pi absorption. Results: NaPi-2b interacts with the PDZ domain of NHE3 regulatory factor 1 (NHERF1). Conclusion: NHERF1 is an important regulator of NaPi-2b apical membrane targeting in response to a low Pi diet. Significance: Understanding of NaPi-2b adaptive mechanisms can help to design new therapies against hypo- and hyperphosphatemic disorders.