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Link to original content: https://pubmed.ncbi.nlm.nih.gov/18635543
Phosphorylation and ankyrin-G binding of the C-terminal domain regulate targeting and function of the ammonium transporter RhBG - PubMed Skip to main page content
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. 2008 Sep 26;283(39):26557-67.
doi: 10.1074/jbc.M803120200. Epub 2008 Jul 17.

Phosphorylation and ankyrin-G binding of the C-terminal domain regulate targeting and function of the ammonium transporter RhBG

Fabien Sohet  1 , Yves Colin, Sandrine Genetet, Pierre Ripoche, Sylvain Métral, Caroline Le Van Kim, Claude Lopez
Affiliations

Phosphorylation and ankyrin-G binding of the C-terminal domain regulate targeting and function of the ammonium transporter RhBG

Fabien Sohet et al. J Biol Chem. .

Abstract

RhBG, a human member of the Amt/Mep/Rh/superfamily of ammonium transporters, has been shown to facilitate NH(3) transport and to be anchored to the basolateral plasma membrane of kidney epithelial cells, via ankyrin-G. We showed here that triple alanine substitution of the (419)FLD(421) sequence, which links the cytoplasmic C-terminal domain of RhBG to ankyrin-G, not only disrupted the interaction of RhBG with the spectrin-based skeleton but also delayed its cell surface expression, decreased its plasma membrane stability, and abolished its NH(3) transport function in epithelial cell lines. Similarly, we demonstrated that both anchoring to the membrane skeleton and ammonium transport activity are regulated by the phosphorylation status of the C-terminal tail of RhBG. Tyrosine 429, which belongs to the previously reported YED basolateral targeting signal of RhBG, was demonstrated to be phosphorylated in vitro using purified Src and Syk kinases and ex vivo by analyzing the effect of pervanadate treatment on wild-type RhBG or Y429A mutants. Then, we showed that Y429D and Y429E mutations, mimicking constitutive phosphorylation, abolished NH(3) transport and enhanced Triton X-100 solubilization of RhBG from the cell membrane. In contrast, the nonphosphorylated/nonphosphorylatable Y429A and Y429F mutants behaved the same as wild-type RhBG. Conversely, Y/A or Y/F but not Y/E or Y/D mutations of residue 429 abolished the exclusive basolateral localization of RhBG in polarized epithelial cells. All these results led to a model in which targeting and ammonium transport function of RhBG are regulated by both phosphorylation and membrane skeleton binding of the C-terminal cytoplasmic domain.

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Figures

FIGURE 1.
FIGURE 1.
Amino acid sequence of the cytoplasmic C terminus of human RhBG (RhBG-Cter). The FLD ankyrin-G interaction domain and the YED targeting signal were previously identified (31). Terminal DTQA sequence conforms to a possible type I PDZ-binding motif. Potential phosphorylation sites are denoted by asterisks. TM-12, polytopic structure of RhBG with 12 transmembrane domains upstream the cytoplasmic C terminus.
FIGURE 2.
FIGURE 2.
Immunofluorescence microscopy analysis of the expression of the RhBG C terminus mutants in HEK293 cells. HEK293 clones transfected with an empty pcDNA3 vector (Empty), or expressing wild-type (RhBG), or mutant RhBG were cultured on poly-l-lysine coverslips and fixed in 4% paraformaldehyde. A, cells were permeabilized in 1% SDS before staining with rabbit anti-RhBG-Cter and Alexa Fluor 488 goat anti-rabbit IgG. B, cells were directly labeled with murine ascites A-08 and Alexa Fluor 488 goat anti-mouse IgG. S422D, S426A, S426D, Y429A, Y429D, T456A, and T456D (not shown) exhibited similar plasma membrane and internal staining in A, and exclusive surface labeling in B. Scale bar, 15 μm.
FIGURE 3.
FIGURE 3.
Typical time course of fluorescence changes in HEK293 cells expressing RhBG C terminus mutants and exposed to ammonium gradients. Cells loaded with the fluorescent pH-sensitive probe BCECF-AM were exposed to an iso-osmotic inwardly directed 20 mEq NH+4 gradient at 15 °C. pHi-dependent fluorescence changes were monitored using a stopped-flow spectrofluorometer. Data from seven to nine time courses were averaged and fitted to a mono-exponential function using the simplex procedure of Biokine software (Bio-Logic), from which alkalinization rate constants (k, s-1) were calculated. WT, parental HEK293 cells. S422D, S426A, S426D, Y429F, 454Stop, T456A, and T456D (not shown) exhibited time courses similar to S422A and Y429A. The Y429D profile (not shown) resembled those of Y429E and F419A/L420A/D421A. All experiments were repeated at least three or four times to obtain the k values in Table 1.
FIGURE 4.
FIGURE 4.
Phosphorylation of RhBG at Tyr429. A, top panel, purified recombinant GST, GST-Cter, and GST-Cter Y429A were produced in E. coli TKB1. GST protein phosphorylation was analyzed by Western blot using anti-phosphotyrosine P-Tyr-100 antibody. Bottom panel, Ponceau Red staining of the different GST constructs. B, top panel, GST proteins were phosphorylated in the presence of [γ-32P]ATP by purified Src or Syk kinases. Phosphorylation was analyzed by autoradiography (32P). Bottom panel, Ponceau Red staining. C, effect of pervanadate on NH3 transport efficiency in HEK293 cells expressing RhBG or Y429A. Transport efficiency was expressed as relative values of alkalinization rate constants corrected for membrane expression of native RhBG, like in Table 1.
FIGURE 5.
FIGURE 5.
Targeting of Tyr429 mutants of RhBG in polarized MDCK cells. A, confocal microscopy sections. MDCK clones expressing wild-type (RhBG) or mutant RhBG were filter-grown for 7 days and processed for indirect immunofluorescence microscopy. Cells were fixed and permeabilized before adding rabbit anti-RhBG-Cter and mouse anti-ZO-1 antibodies. ZO-1 (zonula occludens) is a marker for tight junctions, which delimit apical and basolateral domains. Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 568 goat antimouse IgG were used as second antibodies, respectively. Top panels, XY horizontal middle sections (en face views) showing RhBG labeling in native and mutant clones. Bottom panels, XZ sections (side views) showing both RhBG (green) and ZO-1 (red) stainings. Y429A and Y429D (not shown) exhibited the same RhBG distribution as Y429F and Y429E, respectively. Scale bar, 15 μm. B, cell surface distribution detected by domain-selective biotinylation. Filter-grown MDCK cells were metabolically labeled with [35S]methionine/[35S]cysteine and surface biotinylated from either the apical (Ap) or the basolateral (Bl) side. After lysis, total RhBG proteins were immunoprecipitated using anti-RhBG-Cter, and biotinylated proteins were isolated with avidin beads. The biotinylated RhBG proteins were subjected to SDS-PAGE, electrotransferred to nitrocellulose membranes, and autoradiographed. Y429A (Ap 35%/Bl 65%) and Y429D (Ap 8%/Bl 92%) (not shown) displayed a distribution similar to Y429F and Y429E, respectively. Percentage values are averages from two independent experiments.
FIGURE 6.
FIGURE 6.
Triton X-100 extractability of RhBG, Y429F, Y429E, and F419A/L420A/D421A from HEK293 and polarized MDCK cells. HEK293 (A) or polarized MDCK (B) cells expressing wild-type (RhBG) or mutant RhBG were lysed in the presence of increasing concentrations (%) of Triton X-100 as indicated. Total extracted proteins were analyzed by Western blot. Top panels, RhBG proteins (50–55 kDa) revealed using anti-RhBG A-08. Bottom panels, endogenous cytoplasmic Erk1 and Erk2 proteins (42–44 kDa).
FIGURE 7.
FIGURE 7.
Delivery and turnover of RhBG, Y429F, Y429E, and F419A/L420A/D421A at the surface of HEK293 cells. HEK293 cells expressing RhBG mutants of Tyr429 or ankyrin-G binding site were pulse-chase-labeled ([35S]methionine/[35S]cysteine), and total membrane proteins were biotinylated and immunoprecipitated. A, newly delivered RhBG proteins at the cell surface were monitored by autoradiography of biotinylated proteins. Total amounts of RhBG proteins expressed at the plasma membrane at each time of chase were determined by Western blot using anti-RhBG A-08. B, the curves show the ratio of radiolabeled biotinylated RhBG/total biotinylated RhBG at the membrane reflecting the turnover of newly delivered native or mutant RhBG at the cell surface as a function of time. The curves represent mean values from three experiments. ♦, RhBG; ▪ Y429F; ⋄, Y429E; ▵, F419A/L420A/D421A.
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