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.
Highlights
The type 2b sodium-dependent phosphate co-transporter (NaPi-2b) is the main mediator of intestinal active Pi absorption
NHERF1 in regulating epithelial sodiumdependent Pi transporters (NaPi)-2b Has a PDZ Binding Motif on the C-terminal Tail— Many of the PDZ domain containing proteins that have documented within the apical membrane of epithelial cells are type I domains
Immunofluorescence imaging of NaPi-2b, NHE3 regulatory factor 1 (NHERF1), and PDZK1 showed that all three proteins were expressed within the apical microvilli domain, putting each of the PDZ domain containing proteins in approximate position to interact with NaPi-2b (Fig. 1B)
Summary
The type 2b sodium-dependent phosphate co-transporter (NaPi-2b) is the main mediator of intestinal active Pi absorption. 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. In the renal proximal tubule, NaPi-2a (SLC34a1) and NaPi-2c (SLC34a3) are expressed in the apical membrane of the proximal tubule where they perform the bulk of the renal Pi reabsorption Both renal transporters are regulated in response to chronic and acute changes in dietary Pi content, metabolic conditions, and hormones, including parathyroid hormone and fibroblast growth factor 23 (FGF23) [7,8,9]. Mice chronically fed a low Pi diet have an increased apical expression of NaPi-2b transporter in enterocytes and an upregulation of Pi uptake [13].
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