Browsing by Subject "Sodium-Hydrogen Antiporter"

Now showing 1 - 7 of 7
  • Thumbnail Image
    ItemOpen Access
    A C-terminal motif found in the beta2-adrenergic receptor, P2Y1 receptor and cystic fibrosis transmembrane conductance regulator determines binding to the Na+/H+ exchanger regulatory factor family of PDZ proteins.
    (Proc Natl Acad Sci U S A, 1998-07-21) Hall, RA; Ostedgaard, LS; Premont, RT; Blitzer, JT; Rahman, N; Welsh, MJ; Lefkowitz, RJ
    The Na+/H+ exchanger regulatory factor (NHERF) binds to the tail of the beta2-adrenergic receptor and plays a role in adrenergic regulation of Na+/H+ exchange. NHERF contains two PDZ domains, the first of which is required for its interaction with the beta2 receptor. Mutagenesis studies of the beta2 receptor tail revealed that the optimal C-terminal motif for binding to the first PDZ domain of NHERF is D-S/T-x-L, a motif distinct from those recognized by other PDZ domains. The first PDZ domain of NHERF-2, a protein that is 52% identical to NHERF and also known as E3KARP, SIP-1, and TKA-1, exhibits binding preferences very similar to those of the first PDZ domain of NHERF. The delineation of the preferred binding motif for the first PDZ domain of the NHERF family of proteins allows for predictions for other proteins that may interact with NHERF or NHERF-2. For example, as would be predicted from the beta2 receptor tail mutagenesis studies, NHERF binds to the tail of the purinergic P2Y1 receptor, a seven-transmembrane receptor with an intracellular C-terminal tail ending in D-T-S-L. NHERF also binds to the tail of the cystic fibrosis transmembrane conductance regulator, which ends in D-T-R-L. Because the preferred binding motif of the first PDZ domain of the NHERF family of proteins is found at the C termini of a variety of intracellular proteins, NHERF and NHERF-2 may be multifunctional adaptor proteins involved in many previously unsuspected aspects of intracellular signaling.
  • Thumbnail Image
    ItemOpen Access
    Cooperativity between the Phosphorylation of Thr(95) and Ser(77) of NHERF-1 in the Hormonal Regulation of Renal Phosphate Transport
    (JOURNAL OF THE ROYAL SOCIETY INTERFACE, 2013-01-06) Weinman, Edward J; Steplock, Deborah; Zhang, Yinghua; Biswas, Rajatsubhra; Bloch, Robert J; Shenolikar, Shirish
    The phosphorylation of the sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) plays a key role in the regulation of renal phosphate transport by parathyroid hormone (PTH) and dopamine. Ser(77) in the first PDZ domain of NHERF-1 is a downstream target of both hormones. The current experiments explore the role of Thr(95), another phosphate acceptor site in the PDZ I domain, on hormone-mediated regulation of phosphate transport in the proximal tubule of the kidney. The substitution of alanine for threonine at position 95 (T95A) significantly decreased the rate and extent of in vitro phosphorylation of Ser(77) by PKC. In NHERF-1-null proximal tubule cells, neither PTH nor dopamine inhibited sodium-dependent phosphate transport. Infection of the cells with adenovirus expressing full-length WT GFP-NHERF-1 increased basal phosphate transport and restored the inhibitory effect of both PTH and dopamine. Infection with full-length NHERF-1 containing a T95A mutation, however, increased basal phosphate transport but not the responsiveness to either hormone. As determined by surface plasmon resonance, the substitution of serine for aspartic acid (S77D) in the PDZ I domain decreased the binding affinity to the sodium-dependent phosphate transporter 2a (Npt2a) as compared with WT PDZ I, but a T95D mutation had no effect on binding. Finally, cellular studies indicated that both PTH and dopamine treatment increased the phosphorylation of Thr(95). These studies indicate a remarkable cooperativity between the phosphorylation of Thr(95) and Ser(77) of NHERF-1 in the hormonal regulation of renal phosphate transport. The phosphorylation of Thr(95) facilitates the phosphorylation of Ser(77). This, in turn, results in the dissociation of NHERF-1 from Npt2a and a decrease in phosphate transport in renal proximal tubule cells.
  • Thumbnail Image
    ItemOpen Access
    Cyclosporine A inhibits Ca2+-dependent stimulation of the Na+/H+ antiport in human T cells.
    (J Cell Biol, 1986-08) Rosoff, PM; Terres, G
    The cyclic undecapeptide cyclosporine A (CsA) is a potent immunosuppressive agent that inhibits the initial activation of T lymphocytes. This agent appears to be most effective in blocking the action of mitogens such as concanavalin A and the calcium ionophore A23187, which cause an influx of Ca2+, but not those that may act by alternate mechanisms. These observations suggest that CsA may block a Ca2+-dependent step in T cell activation. We have shown that stimulation of the T3-T cell receptor complex-associated Ca2+ transporter activates the Na+/H+ antiport (Rosoff, P. M., and L. C. Cantley, 1985, J. Biol. Chem., 260: 14053-14059). The tumor-promoting phorbol esters, which are co-mitogenic for T cells, activate the exchanger by a separate pathway which is mediated by protein kinase C. Both the rise in intracellular Ca2+ and intracellular pH may be necessary for the successful triggering of cellular activation. In this report we show that CsA blocks the T3-T cell receptor-stimulated, Ca2+ influx-dependent activation of Na+/H+ exchange, but not the phorbol ester-mediated pathway in a transformed human T cell line. CsA inhibited mitogen-stimulation of interleukin-2 production in a separate cell line. CsA also inhibited vasopressin stimulation of the antiporter in normal rat kidney fibroblasts, but had no effect on serum or 12-O-tetradecanoyl phorbol 13-acetate stimulation. CsA did not affect serum or vasopressin or serum stimulation of normal rat kidney cell proliferation. CsA also had no effect on lipopolysaccharide or phorbol ester stimulation of Na+/H+ exchange activity or induction of differentiation in 70Z/3 pre-B lymphocytes in which these events are initiated by the protein kinase C pathway. These data suggest that mechanisms of activation of Na+/H+ exchange that involve an elevation in cytosolic Ca2+ are blocked by CsA but that C kinase-mediated regulation is unaffected. The importance of the Na+/H+ antiport in the regulation of growth and differentiation of T cells is discussed.
  • Thumbnail Image
    ItemOpen Access
    Dynamics of PTH-induced disassembly of Npt2a/NHERF-1 complexes in living OK cells.
    (American journal of physiology. Renal physiology, 2011-01) Weinman, Edward J; Steplock, Deborah; Shenolikar, Shirish; Blanpied, Thomas A
    Parathyroid hormone (PTH) inhibits the reabsorption of phosphate in the renal proximal tubule by disrupting the binding of the sodium-dependent phosphate transporter 2A (Npt2a) to the adapter protein sodium-hydrogen exchanger regulatory factor-1 (NHERF-1), a process initiated by activation of protein kinase C (PKC). To gain additional insights into the dynamic sequence of events, the time course of these responses was studied in living opossum kidney (OK) cells. Using a FRET-based biosensor, we found that PTH activated intracellular PKC within seconds to minutes. In cells expressing GFP-Npt2a and mCherry-NHERF, PTH did not affect the relative abundance of NHERF-1 but there was a significant and time-dependent decrease in the Npt2a/NHERF-1 ratio. The half-time to maximal dissociation was 15 to 20 min. By contrast, PTH had no effect on the fluorescence ratio for GFP-ezrin compared with mCherry-NHERF-1 at the apical surface. These experiments establish that PTH treatment of proximal tubule OK cells leads to rapid activation of PKC with the subsequent dissociation of Npt2a/NHERF-1 complexes. The association of NHERF-1 with Ezrin and their localization at the apical membrane, however, was unperturbed by PTH, thereby enabling the rapid recruitment and membrane reinsertion of Npt2a and other NHERF-1 targets on termination of the hormone response.
  • Thumbnail Image
    ItemOpen Access
    Fibroblast growth factor-23-mediated inhibition of renal phosphate transport in mice requires sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) and synergizes with parathyroid hormone.
    (The Journal of biological chemistry, 2011-10) Weinman, Edward J; Steplock, Deborah; Shenolikar, Shirish; Biswas, Rajatsubhra
    Fibroblast growth factor-23 (FGF-23) inhibits sodium-dependent phosphate transport in brush border membrane vesicles derived from hormone-treated kidney slices of the mouse and in mouse proximal tubule cells by processes involving mitogen-activated protein kinase (MAPK) but not protein kinase A (PKA) or protein kinase C (PKC). By contrast, phosphate transport in brush border membrane vesicles and proximal tubule cells from sodium-hydrogen exchanger regulatory factor-1 (NHERF-1)-null mice were resistant to the inhibitory effect of FGF-23 (10(-9) m). Infection of NHERF-1-null proximal tubule cells with wild-type adenovirus-GFP-NHERF-1 increased basal phosphate transport and restored the inhibitory effect of FGF-23. Infection with adenovirus-GFP-NHERF-1 containing a S77A or T95D mutation also increased basal phosphate transport, but the cells remained resistant to FGF-23 (10(-9) m). Low concentrations of FGF-23 (10(-13) m) and PTH (10(-11) m) individually did not inhibit phosphate transport or activate PKA, PKC, or MAPK. When combined, however, these hormones markedly inhibited phosphate transport associated with activation of PKC and PKA but not MAPK. These studies indicate that FGF-23 inhibits phosphate transport in the mouse kidney by processes that involve the scaffold protein NHERF-1. In addition, FGF-23 synergizes with PTH to inhibit phosphate transport by facilitating the activation of the PTH signal transduction pathway.
  • Thumbnail Image
    ItemOpen Access
    Platelet-derived growth factor receptor association with Na(+)/H(+) exchanger regulatory factor potentiates receptor activity.
    (Mol Cell Biol, 2000-11) Maudsley, S; Zamah, AM; Rahman, N; Blitzer, JT; Luttrell, LM; Lefkowitz, RJ; Hall, RA
    Platelet-derived growth factor (PDGF) is a potent mitogen for many cell types. The PDGF receptor (PDGFR) is a receptor tyrosine kinase that mediates the mitogenic effects of PDGF by binding to and/or phosphorylating a variety of intracellular signaling proteins upon PDGF-induced receptor dimerization. We show here that the Na(+)/H(+) exchanger regulatory factor (NHERF; also known as EBP50), a protein not previously known to interact with the PDGFR, binds to the PDGFR carboxyl terminus (PDGFR-CT) with high affinity via a PDZ (PSD-95/Dlg/Z0-1 homology) domain-mediated interaction and potentiates PDGFR autophosphorylation and extracellular signal-regulated kinase (ERK) activation in cells. A point-mutated version of the PDGFR, with the terminal leucine changed to alanine (L1106A), cannot bind NHERF in vitro and is markedly impaired relative to the wild-type receptor with regard to PDGF-induced autophosphorylation and activation of ERK in cells. NHERF potentiation of PDGFR signaling depends on the capacity of NHERF to oligomerize. NHERF oligomerizes in vitro when bound with PDGFR-CT, and a truncated version of the first NHERF PDZ domain that can bind PDGFR-CT but which does not oligomerize reduces PDGFR tyrosine kinase activity when transiently overexpressed in cells. PDGFR activity in cells can also be regulated in a NHERF-dependent fashion by stimulation of the beta(2)-adrenergic receptor, a known cellular binding partner for NHERF. These findings reveal that NHERF can directly bind to the PDGFR and potentiate PDGFR activity, thus elucidating both a novel mechanism by which PDGFR activity can be regulated and a new cellular role for the PDZ domain-containing adapter protein NHERF.
  • Thumbnail Image
    ItemOpen Access
    Role of NHERF and scaffolding proteins in proximal tubule transport.
    (Urological research, 2010-08) Cunningham, Rochelle; Biswas, Rajatsubhra; Steplock, Deborah; Shenolikar, Shirish; Weinman, Edward
    Eukaryotic cells coordinate specific responses to hormones and growth factors by spatial and temporal organization of "signaling components." Through the formation of multiprotein complexes, cells are able to generate "signaling components" that transduce hormone signals through proteins, such as PSD-95/Dlg/ZO-1(PDZ)-containing proteins that associate by stable and dynamic interactions. The PDZ homology domain is a common protein interaction domain in eukaryotes and with greater than 500 PDZ domains identified, it is the most abundant protein interaction domain in eukaryotic cells. The NHERF (sodium hydrogen exchanger regulatory factor) proteins are PDZ domain-containing proteins that play an important role in maintaining and regulating cell function. NHERF-1 was initially identified as a brush border membrane-associated phosphoprotein essential for the cAMP/PKA-induced inhibition of the sodium hydrogen exchanger isoform 3 (NHE3). Mouse, rabbit and human renal proximal tubules also express NHERF-2 (E3KARP), a structurally related protein, which in model cell systems also binds NHE3 and mediates its inhibition by cAMP. PDZK1 (NHERF-3) and IKEPP (NHERF-4) were later identified and found to have similar homology domains, leading to their recent reclassification. Although studies have revealed similar binding partners and overlapping functions for the NHERF proteins, it is clear that there is a significant amount of specificity between them. This review focuses primarily on NHERF-1, as the prototypical PDZ protein and will give a brief summary of its role in phosphate transport and the development of some forms of nephrolithiasis.