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.

FIGURE 1.

NaPi-2b, NHERF1, and PDZK2 are expressed in the enterocyte microvilli. A, isolated rat enterocytes analyzed by Western blot showed expression in the small intestine of the proteins under study. NaPi-2b expression was greater in the duodenum followed by jejunum and negligible levels on the ileal section. Both NHERF1 and PDZK1 proteins showed similar expression levels in all three segments. B, immunofluorescence microscopy of rat duodenal sections demonstrated that NaPi-2b, NHERF1, and PDZK1 (green) are expressed in the apical membrane of enterocytes, co-localizing with the actin signal (red) in the microvilli. Bar, 10 μm.

FIGURE 2.

NaPi-2b associates with NHERF1 in rat duodenal enterocytes. Rat duodenal enterocyte lysates were subjected to immunoprecipitation (IP) and Western blot analysis of co-precipitating proteins. No appreciable levels of any of the proteins studied appeared within the protein A/G pre-cleared samples. In NHERF1 immunoprecipitates, NHERF1 was precipitated and NaPi-2b was readily detected in the immunoprecipitated sample (top panel). In PDZK1 immunoprecipitates, PDZK1 was precipitated but NaPi-2b was not observed in the immunoprecipitated sample (middle panel). In NaPi-2b immunoprecipitates, NHERF1 but not PDZK1 was found in immunoprecipitated samples (bottom panel). Interference of the IgG bands precluded the confirmation that NaPi-2b was precipitated in these samples.

FIGURE 3.

C-tail truncation impacts NaPi-2b binding to NHERF1. A, HEK cells were co-transfected with mCherry-NHERF1 and either GFP-NaPi-2b or GFP-NaPi-2b-4aa. Confocal fluorescence imaging shows that mCherryNHERF1 co-localizes with both GFP-NaPi-2b and GFP-NaPi-2b-4aa. Each of the three proteins is distributed predominantly at the plasma membrane. Bar, 5 μm. B, HEK cells were transfected with GFP-NaPi-2b or GFP-NaPi-2b-4aa, either alone or with FLAG-NHERF1. In the upper panel, Western blotting of the total cell lysates showed similar expression levels of each protein. In the lower panel, Western blotting showed GFP-NaPi-2b, but not GFP-NaPi-2b-4aa, was readily co-precipitated with FLAG-NHERF1.

FIGURE 4.

The enterocyte cell model CACO-2_BBE correctly expresses NaPi-2b, NHERF1, and PDZK1. A, immunofluorescence confocal microscopy of EYFP-NaPi-2b in transfected CACO-2_BBE cells showed that the transporter was correctly expressed in numerous microvilli on the apical membrane. Lower panel shows cross-section of a cell from the apical to the basolateral membrane. B, modulation tracking microscopy was used to image EYFP-NaPi-2b, EYFP-NHERF1, and EYFP-PDZK1 transfected in CACO-2_BBE cells. This technique allows us to track and scan individual microvilli of the apical membrane. Therefore we confirmed proper expression of all three proteins in the microvilli of CACO-2_BBE cells. Color scale represents fluorescence intensity.

FIGURE 5.

FLIM-FRET studies in CACO-2_BBE cells confirmed interaction of NaPi-2b with NHERF1. CACO-2_BBE cells transfected with Cu-NaPi-2b (A) or co-transfected with Cu-NaPi-2b and EYFP-NHERF1 (B) were studied. The phasor diagram showed a displacement of fluorescence lifetime in the cells co-transfected (green triangles) compared with cells transfected only with Cu-NaPi-2b (C). The reduction in donor lifetime fluorescence marks the occurrence of FRET and, therefore that both proteins are within 10 nm. Fluorescence lifetime analysis was repeated for the NaPi-2b/PDZK1 pair (D and E). In this case, there was no significant variation in the fluorescence lifetime of co-transfected cells (blue triangles) compared with the solo transfection of Cu-NaPi-2b (F) meaning no occurrence of FRET. In this phasor diagram, average lifetime of the NaPi-2b/NHERF1 pair (green triangle) is included for comparison (F).

FIGURE 6.

NHERF1 KO mice have impaired adaptation of NaPi-2b protein in response to a low P_i diet. Mice ileal BBMs were analyzed by Western blot to detect NaPi-2b expression. Wild type animals fed a low P_i diet showed a significant increase on the expression of NaPi-2b compared with animals fed a high P_i diet. However, NHERF1^−/− mice had a 39 ± 11% decrease in the adaptive response of NaPi-2b under the same conditions.

FIGURE 7.

Expression of other apical membrane-associated proteins is not modified in the NHERF1^−/− mice. Western blot analysis of ileal BBM of wild type or NHERF1^−/− animals fed low P_i diets. Two apical membrane-associated proteins, Galectin-4 and Na⁺-d-glucose cotransporter (SGLT1), showed no differential expression between wild type and NHERF1^−/− animals unlike NaPi-2b that showed a significant reduction in NHERF1^−/− mice under this dietary condition (n = 8–10). ns, not significant.

FIGURE 8.

PDZK1 KO mice have a normal adaptation to low P_i diet intake. In contrast to the impaired adaptation in the NHERF1^−/− mice, wild type and PDZK1^−/− mice showed no differences in the adaptive response of NaPi-2b apical expression. Ileal BBMs NaPi-2b levels were increased to the same extent in wild type and PDZK1^−/− mice fed a low P_i diet.