NHE3 regulatory factor 1 (NHERF1) modulates intestinal sodium-dependent phosphate transporter (NaPi-2b) expression in apical microvilli.
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.
Department of Medicine, University of Colorado Denver, Aurora, Colorado and the Veterans Affairs Eastern Colorado Health Care System, Denver, Colorado 80045.
Hector
H
Giral
Department of Medicine, University of Colorado Denver, Aurora, Colorado and the Veterans Affairs Eastern Colorado Health Care System, Denver, Colorado 80045.
DeeAnn
D
Cranston
Department of Biomedical Engineering, Laboratory for Fluorescence Dynamics, University of California, Irvine, California 92697.
Luca
L
Lanzano
Department of Medicine, University of Colorado Denver, Aurora, Colorado and the Veterans Affairs Eastern Colorado Health Care System, Denver, Colorado 80045.
Yupanqui
Y
Caldas
Department of Medicine, University of Colorado Denver, Aurora, Colorado and the Veterans Affairs Eastern Colorado Health Care System, Denver, Colorado 80045.
Eileen
E
Sutherland
Department of Medicine, University of Colorado Denver, Aurora, Colorado and the Veterans Affairs Eastern Colorado Health Care System, Denver, Colorado 80045.
Joanna
J
Rachelson
Department of Medicine, University of Colorado Denver, Aurora, Colorado and the Veterans Affairs Eastern Colorado Health Care System, Denver, Colorado 80045.
Evgenia
E
Dobrinskikh
Department of Medicine and Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201.
Edward J
EJ
Weinman
Department of Medicine, University of Colorado Denver, Aurora, Colorado and the Veterans Affairs Eastern Colorado Health Care System, Denver, Colorado 80045.
R Brian
RB
Doctor
Department of Biomedical Engineering, Laboratory for Fluorescence Dynamics, University of California, Irvine, California 92697.
Enrico
E
Gratton
Department of Medicine, University of Colorado Denver, Aurora, Colorado and the Veterans Affairs Eastern Colorado Health Care System, Denver, Colorado 80045. Electronic address: Moshe.Levi@ucdenver.edu.
Moshe
M
Levi
D000818:Animals; D018938:Caco-2 Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D003598:Cytoskeletal Proteins; D020895:Enterocytes / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins; D051379:Mice; D018345:Mice, Knockout; D008871:Microvilli / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*; D050614:Sodium-Phosphate Cotransporter Proteins, Type IIb / Q000096:biosynthesis* / Q000235:genetics
D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C514896:PDZK1 protein, mouse; C109245:Pdzk1 protein, rat; D010750:Phosphoproteins; C493558:Slc34a2 protein, mouse; C493559:Slc34a2 protein, rat; D017923:Sodium-Hydrogen Exchangers; D050614:Sodium-Phosphate Cotransporter Proteins, Type IIb; C093376:sodium-hydrogen exchanger regulatory factor