Calcineurin homologous protein as an essential cofactor for Na+/H+ exchangers

The Na+/H+ exchangers (NHEs) comprise a family of transporters that catalyze cell functions such as regulation of the pH and volume of a cell and epithelial absorption of Na+ and bicarbonate. Ubiquitous calcineurin B homologous protein (CHP or p22) is co-localized and co-immunoprecipitated with expressed NHE1, NHE2, or NHE3 independently of its myristoylation and Ca2+ binding, and its binding site was identified as the juxtamembrane region within the carboxyl-terminal cytoplasmic domain of exchangers. CHP binding-defective mutations of NHE1-3 or CHP depletion by injection of the competitive CHP-binding region of NHE1 into Xenopus oocytes resulted in a dramatic reduction (>90%) in the Na+/H+ exchange activity. The data suggest that CHP serves as an essential cofactor, which supports the physiological activity of NHE family members.     

Multiple domains of Ruk/CIN85/SETA/CD2BP3 are involved in interaction with p85alpha regulatory subunit of PI 3-kinase

Ruk/CIN85/SETA/CD2BP3 and CD2AP/CMS/METS-1 comprise a new family of proteins involved in such fundamental processes as clustering of receptors and rearrangement of the cytoskeleton in regions of specialised cell-cell contacts, ligand-activated internalisation and targeting to lysosome degradation pathway of receptor tyrosine kinases, and apoptotic cell death. As typical adapter proteins they execute these functions by interacting with other signalling molecules via multiple protein-protein interaction interfaces: SH3 domains, Pro-rich region and coiled-coil domain. It has been previously demonstrated that Ruk is able to interact with the p85alpha regulatory subunit of PI 3-kinase and that the SH3 domain of p85alpha is required for this interaction. However, later observations hinted at a more complex mechanism than simple one-way SH3-Pro-rich interaction. Because interaction with p85alpha was suggested to be important for pro-apoptotic activity of the long isoform of Ruk, Ruk(l)/CIN85, we carried out detailed studies of the mechanism of this interaction and demonstrated that multiple domains are involved; SH3 domains of Ruk are required and sufficient for efficient interaction with full-length p85alpha but the SH3 domain of p85alpha is vital for their "activation" by ousting them from intramolecular interaction with the Pro-rich region of Ruk. Our data also suggest that homodimerisation via C-terminal coiled-coil domain affects both intra- and intermolecular interactions of Ruk proteins.     

Two Histidine Residues in the Juxta-Membrane Cytoplasmic Domain of Na+/H+ Exchanger Isoform 3 (NHE3) Determine the Set Point

Histidine residues in Na+/H+ exchangers are believed to participate in proton binding and influence the Na+/H+ exchanger activity. In the present study, the function of three highly conserved histidines in the juxtamembrane cytoplasmic domain of NHE3 was studied. His-479, His-485, and His-499 were mutated to Leu, Gln or Asp and expressed in an Na+/H+ exchanger null cell line and functional consequences on Na+/H+ exchange kinetics were characterized. None of the histidines were essential for NHE3 activity, with all mutated NHE3 resulting in functional exchangers. However, the mutation in His-475 and His-499 significantly lowered NHE3 transport activity, whereas the mutation in H485 showed no apparent effect. In addition, the pH profiles of the H479 and H499 mutants were shifted to a more acidic region, and lowered its set point, the intracellular pH value above which the Na+/H+ exchanger becomes inactive, by approximately 0.3-0.6 pH units. The changes in set point by the mutations were further shifted to more acidic values by ATP depletion, indicating that the mechanism by which the mutations on the histidine residues altered the NHE3 set point differs from that caused by ATP depletion. We suggest that His-479 and His-499 are part of the H+ sensor, which is involved in determining the sensitivity to the intracellular H+ concentration and Na+/H+ exchange rate.     

Grb10, a Positive, Stimulatory Signaling Adapter in Platelet-Derived Growth Factor BB-, Insulin-Like Growth Factor I-, and Insulin-Mediated Mitogenesis

Grb10 has been described as a cellular partner of several receptor tyrosine kinases, including the insulin receptor (IR) and the insulin-like growth factor I (IGF-I) receptor (IGF-IR). Its cellular role is still unclear and a positive as well as an inhibitory role in mitogenesis depending on the cell context has been implicated. We have tested other mitogenic receptor tyrosine kinases as putative Grb10 partners and have identified the activated forms of platelet-derived growth factor (PDGF) receptor beta (PDGFRbeta), hepatocyte growth factor receptor (Met), and fibroblast growth factor receptor as candidates. We have mapped Y771 as a PDFGRbeta site that is involved in the association with Grb10 via its SH2 domain. We have further investigated the putative role of Grb10 in mitogenesis with four independent experimental strategies and found that all consistently suggested a role as a positive, stimulatory signaling adaptor in normal fibroblasts. (i) Complete Grb10 expression from cDNA with an ecdysone-regulated transient expression system stimulated PDGF-BB-, IGF-I, and insulin- but not epidermal growth factor (EGF)-induced DNA synthesis in an ecdysone dose-responsive fashion. (ii) Microinjection of the (dominant-negative) Grb10 SH2 domain interfered with PDGF-BB- and insulin-induced DNA synthesis. (iii) Alternative experiments were based on cell-permeable fusion peptides with the Drosophila antennapedia homeodomain which effectively traverse the plasma membrane of cultured cells. A cell-permeable Grb10 SH2 domain similarly interfered with PDGF-BB-, IGF-I-, and insulin-induced DNA synthesis. In contrast, a cell-permeable Grb10 Pro-rich putative SH3 domain binding region interfered with IGF-I- and insulin- but not with PDGF-BB- or EGF-induced DNA synthesis. (iv) Transient overexpression of complete Grb10 increased whereas cell-permeable Grb10 SH2 domain fusion peptides substantially decreased the cell proliferation rate (as measured by cell numbers) in normal fibroblasts. These experimental strategies independently suggest that Grb10 functions as a positive, stimulatory, mitogenic signaling adapter in PDGF-BB, IGF-I, and insulin action. This function appears to involve the Grb10 SH2 domain, a novel sequence termed BPS, and the Pro-rich putative SH3 domain binding region in IGF-I- and insulin-mediated mitogenesis. In contrast, PDGF-BB-mediated mitogenesis appears to depend on the SH2 but not on the Pro-rich region and may involve other, unidentified Grb10 domains. Distinct protein domains may help to define specific Grb10 functions in different signaling pathways.     

Interaction of the metalloprotease disintegrins MDC9 and MDC15 with two SH3 domain-containing proteins, endophilin I and SH3PX1.

Metalloprotease disintegrins (a disintegrin and metalloprotease (ADAM) and metalloprotease, disintegrin, cysteine-rich proteins (MDC)) are a family of membrane-anchored glycoproteins that function in diverse biological processes, including fertilization, neurogenesis, myogenesis, and ectodomain processing of cytokines and other proteins. The cytoplasmic domains of ADAMs often include putative signaling motifs, such as proline-rich SH3 ligand domains, suggesting that interactions with cytoplasmic proteins may affect metalloprotease disintegrin function. Here we report that two SH3 domain-containing proteins, endophilin I (SH3GL2, SH3p4) and a novel SH3 domain- and phox homology (PX) domain-containing protein, termed SH3PX1, can interact with the cytoplasmic domains of the metalloprotease disintegrins MDC9 and MDC15. These interactions were initially identified in a yeast two-hybrid screen and then confirmed using bacterial fusion proteins and co-immunoprecipitations from eukaryotic cells expressing both binding partners. SH3PX1 and endophilin I both preferentially bind the precursor but not the processed form of MDC9 and MDC15 in COS-7 cells. Since rat endophilin I is thought to play a role in synaptic vesicle endocytosis and SH3PX1 has sequence similarity to sorting nexins in yeast, we propose that endophilin I and SH3PX1 may have a role in regulating the function of MDC9 and MDC15 by influencing their intracellular processing, transport, or final subcellular localization.     

C-terminal domains of Na(+)/H(+) exchanger isoform 3 are involved in the basal and serum-stimulated membrane trafficking of the exchanger

When expressed either in polarized epithelial cells or in fibroblasts, two Na(+)/H(+) exchanger isoforms, NHE1 and NHE3, have different subcellular distributions. Using a quantitative cell surface biotinylation technique, we found PS120 cells target approximately 90% of mature NHE1 but only 14% of NHE3 to the cell surface, and this pattern occurs irrespective of NHE protein expression levels. In this study, we examined surface fractions of NHE3 C-terminal truncation mutants to identify domains involved in the targeting of NHE3. Removing the C-terminal 76 amino acids doubled surface fractions to 30% of total and doubled the V(max) from 1300 to 2432 microM H(+)/s. Removal of another 66 amino acids increased surface levels to 55% of total with an increase in the V(max) to 5794 microM H(+)/s. Surface fractions did not change with a further 105 amino acid truncation. We postulated that inhibition of the basal recycling of NHE3 could result in the surface accumulation of the NHE3 truncations. Accordingly, we found that, unlike wild-type NHE3, the truncations were shown to internalize poorly and were not affected by PI3 kinase inhibition. However, while the truncations demonstrated reduced basal recycling, they retained the same serum response as full-length NHE3, with a mobilization of approximately 10% of total NHE to the surface. We conclude that basal recycling of NHE3 is controlled by endocytic determinants contained within its C-terminal 142 amino acids and that serum-mediated exocytosis is independently regulated through a different part of the protein.     

Localization and interaction of NHERF isoforms in the renal proximal tubule of the mouse

In expression systems and in yeast, Na/H exchanger regulatory factor (NHERF)-1 and NHERF-2 have been demonstrated to interact with the renal brush border membrane proteins NHE3 and Npt2. In renal tissue of mice, however, NHERF-1 is required for cAMP regulation of NHE3 and for the apical targeting of Npt2 despite the presence of NHERF-2, suggesting another order of specificity. The present studies examine the subcellular location of NHERF-1 and NHERF-2 and their interactions with target proteins including NHE3, Npt2, and ezrin. The wild-type mouse proximal tubule expresses both NHERF-1 and NHERF-2 in a distinct pattern. NHERF-1 is strongly expressed in microvilli in association with NHE3, Npt2, and ezrin. Although NHERF-2 can be detected weakly in the microvilli, it is expressed predominantly at the base of the microvilli in the vesicle-rich domain. NHERF-2 appears to associate directly with ezrin and NHE3 but not Npt2. NHERF-1 is involved in the apical expression of Npt2 and the presence of other Npt2-binding proteins does not compensate totally for the absence of NHERF-1 in NHERF-1-null mice. Although NHERF-1 links NHE3 to the actin cytoskeleton through ezrin, the absence of NHERF-1 does not result in a generalized disruption of the architecture of the cell. Thus the mistargeting of Npt2 seen in NHERF-1-null mice likely represents a specific disruption of pathways mediated by NHERF-1 to achieve targeting of Npt2. These findings suggest that the organized subcellular distribution of the NHERF isoforms may play a role in the specific interactions mediating physiological control of transporter function.     

The effect of environmental hypercapnia and salinity on the expression of NHE-like isoforms in the gills of a euryhaline fish (Fundulus heteroclitus)

The current models for branchial acid excretion in fishes include Na(+)/H(+) exchange and the electrogenic excretion of H+ via H+-ATPase. The predominant route of acid excretion in some freshwater fishes is thought to be via the H+-ATPase/Na+ channel system. The euryhaline Fundulus heteroclitus may not fit this profile even when adapted to freshwater (FW). We hypothesize that the Na+/H+ exchanger (NHE) in this species may play a predominant role in acid-base regulation for both marine and FW adapted animals. Acidosis induced by ambient hypercapnia (1% CO2 in air), resulted in an increase in net H+ excretion to the water in F. heteroclitus pre-adapted to FW, brackish (isoosmotic; BW) and seawater (SW). Both FW and SW adapted mummichogs were tested for NHE protein expression using mammalian NHE antibodies, and we identified NHE-like immunoreactive proteins in gill membrane preparations from both groups. Hypercapnia induced a approximately three-fold elevation in gill NHE2-like protein in FW animals but SW adapted fish showed inconsistent NHE3-like protein expression. There was no change in NHE-1 levels in FW fish. In contrast, SW animals demonstrated a significant increase in both NHE1 and NHE3-like proteins following hypercapnia but limited expression of the NHE2 protein. We hypothesize that different isoforms of NHE may be preferentially expressed depending on the salinity to which the animals are adapted. Net H+ transfers during acidosis may be driven, at least in part by the action of these transporters.     

Solution structure of the cytoplasmic region of Na+/H+ exchanger 1 complexed with essential cofactor calcineurin B homologous protein 1

Na+/H+ exchanger 1 (NHE1) regulates intracellular pH, Na+ content, and cell volume. Calcineurin B homologous protein 1 (CHP1) serves as an essential cofactor that facilitates NHE1 exchange activity under physiological conditions by direct binding to the cytoplasmic juxtamembrane region of NHE1. Here we describe the solution structure of the cytoplasmic juxtamembrane region of NHE1 complexed with CHP1. The region of NHE1 forms an amphipathic helix, which is induced by CHP1 binding, and CHP1 possesses a large hydrophobic cleft formed by EF-hand helices. The apolar side of the NHE1 helix participates in extensive hydrophobic interactions with the cleft of CHP1. We suggest that helix formation of the cytoplasmic region of NHE1 by CHP1 is a prerequisite for generating the active form of NHE1. The molecular recognition detailed in this study also provides novel insight into the target binding mechanism of EF-hand proteins.     

Modulation of Na+/H+ exchange activity by Cl-

Na+/H+ exchanger (NHE) activity is exquisitely dependent on the intra- and extracellular concentrations of Na+ and H+. In addition, Cl- ions have been suggested to modulate NHE activity, but little is known about the underlying mechanism, and the Cl- sensitivity of the individual isoforms has not been established. To explore their Cl- sensitivity, types 1, 2, and 3 Na+/H+ exchangers (NHE1, NHE2, and NHE3) were heterologously expressed in antiport-deficient cells. Bilateral replacement of Cl- with nitrate or thiocyanate inhibited the activity of all isoforms. Cl- depletion did not affect cell volume or the cellular ATP content, which could have indirectly altered NHE activity. The number of plasmalemmal exchangers was unaffected by Cl- removal, implying that inhibition was due to a decrease in the intrinsic activity of individual exchangers. Analysis of truncated mutants of NHE1 revealed that the anion sensitivity resides, at least in part, in the COOH-terminal domain of the exchanger. Moreover, readdition of Cl- into the extracellular medium failed to restore normal transport, suggesting that intracellular Cl- is critical for activity. Thus interaction of intracellular Cl- with the COOH terminus of NHE1 or with an associated protein is essential for optimal activity.     

Different ionic conditions prompt NHE2 and NHE3 translocation to the plasma membrane

We tested whether NHE3 and NHE2 Na(+)/H(+) exchanger isoforms were recruited to the plasma membrane (PM) in response to changes in ion homeostasis. NHE2-CFP or NHE3-CFP fusion proteins were functional Na(+)/H(+) exchangers when transiently expressed in NHE-deficient PS120 fibroblasts. Confocal morphometry of cells whose PM was labeled with FM4-64 measured the fractional amount of fusion protein at the cell surface. In resting cells, 10-20% of CFP fluorescence was at PM and stable over time. A protocol commonly used to activate the Na(+)/H(+) exchange function (NH(4)-prepulse acid load sustained in Na(+)-free medium), increased PM percentages of PM NHE3-CFP and NHE2-CFP. Separation of cellular acidification from Na(+) removal revealed that only NHE3-CFP translocated when medium Na(+) was removed, and only NHE2-CFP translocated when the cell was acidified. NHE2/NHE3 chimeric proteins demonstrate that the Na(+)-removal response element resides predominantly in the NHE3 cytoplasmic tail and is distinct from the acidification response sequence of NHE2.     

NHE3 in an ancestral vertebrate: primary sequence, distribution, localization, and function in gills

In mammals, the Na+/H+ exchanger 3 (NHE3) is expressed with Na+/K+-ATPase in renal proximal tubules, where it secretes H+ and absorbs Na+ to maintain blood pH and volume. In elasmobranchs (sharks, skates, and stingrays), the gills are the dominant site of pH and osmoregulation. This study was conducted to determine whether epithelial NHE homologs exist in elasmobranchs and, if so, to localize their expression in gills and determine whether their expression is altered by environmental salinity or hypercapnia. Degenerate primers and RT-PCR were used to deduce partial sequences of mammalian NHE2 and NHE3 homologs from the gills of the euryhaline Atlantic stingray (Dasyatis sabina). Real-time PCR was then used to demonstrate that mRNA expression of the NHE3 homolog increased when stingrays were transferred to low salinities but not during hypercapnia. Expression of the NHE2 homolog did not change with either treatment. Rapid amplification of cDNA was then used to deduce the complete sequence of a putative NHE3. The 2,744-base pair cDNA includes a coding region for a 2,511-amino acid protein that is 70% identical to human NHE3 (SLC9A3). Antisera generated against the carboxyl tail of the putative stingray NHE3 labeled the apical membranes of Na+/K+-ATPase-rich epithelial cells, and acclimation to freshwater caused a redistribution of labeling in the gills. This study provides the first NHE3 cloned from an elasmobranch and is the first to demonstrate an increase in gill NHE3 expression during acclimation to low salinities, suggesting that NHE3 can absorb Na+ from ion-poor environments.     

Acute hypotension induced by aortic clamp vs. PTH provokes distinct proximal tubule Na+ transporter redistribution patterns

Renal parathyroid hormone (PTH) action is often studied at high doses (100 microg PTH/kg) that lower mean arterial pressure significantly, albeit transiently, complicating interpretation of studies. Little is known about the effect of acute hypotension on proximal tubule Na(+) transporters. This study aimed to determine the effects of acute hypotension, induced by aortic clamp or by high-dose PTH (100 microg PTH/kg), on renal hemodynamics and proximal tubule Na/H exchanger isoform 3 (NHE3) and type IIa Na-P(i) cotransporter protein (NaPi2) distribution. Subcellular distribution was analyzed in renal cortical membranes fractionated on sorbitol density gradients. Aortic clamp-induced acute hypotension (from 100 +/- 3 to 78 +/- 2 mmHg) provoked a 62% decrease in urine output and a significant decrease in volume flow from the proximal tubule detected as a 66% decrease in endogenous lithium clearance. There was, however, no significant change in glomerular filtration rate (GFR) or subcellular distribution of NHE3 and NaPi2. In contrast, high-dose PTH rapidly (<2 min) decreased arterial blood pressure to 51 +/- 3 mmHg, decreased urine output, and shifted NHE3 and NaPi2 out of the low-density membranes enriched in apical markers. PTH at much lower doses (<1.4 microg.kg(-1).h(-1)) did not change blood pressure and was diuretic. In conclusion, acute hypotension per se increases proximal tubule Na(+) reabsorption without changing NHE3 or NaPi2 subcellular distribution, indicating that trafficking of transporters to the surface is not the likely mechanism; in comparison, hypotension secondary to high-dose PTH blocks the primary diuretic effect of PTH but does not inhibit the PTH-stimulated redistribution of NHE3 and NaPi2 to the base of the microvilli.     

Calcineurin B homologous protein 3 promotes the biosynthetic maturation, cell surface stability, and optimal transport of the Na+/H+ exchanger NHE1 isoform

Calcineurin B homologous protein (CHP) 1 and 2 are Ca(2+)-binding proteins that modulate several cellular processes, including cytoplasmic pH by positively regulating plasma membrane-type Na(+)/H(+) exchangers (NHEs). Recently another CHP-related protein, termed tescalcin or CHP3, was also shown to interact with the ubiquitous NHE1 isoform, but seemingly suppressed its activity. However, the precise physical and functional nature of this association was not examined in detail. In this study, biochemical and cellular studies were undertaken to further delineate this relationship. Glutathione S-transferase-NHE1 fusion protein pulldown assays revealed that full-length CHP3 binds directly to the proximal juxtamembrane C-terminal region (amino acids 505-571) of rat NHE1 in the same region that binds CHP1 and CHP2. The interaction was further validated by coimmunoprecipitation and coimmunolocalization experiments using full-length CHP3 and wild-type NHE1 in transfected Chinese hamster ovary AP-1 cells. Simultaneous mutation of four hydrophobic residues within this region ((530)FLDHLL(535)) to either Ala, Gln, or Arg (FL-A, FL-Q, or FL-R) abrogated this interaction both in vitro and in intact cells. The NHE1 mutants were sorted properly to the cell surface but showed markedly reduced (FL-A) or minimal (FL-R and FL-Q) activity. Interestingly, and contrary to an earlier finding, ectopic coexpression of CHP3 up-regulated the cell surface activity of wild-type NHE1. This stimulation was not observed with the CHP3 binding-defective mutants. Mechanistically, overexpression of CHP3 did not alter the H(+) sensitivity of wild-type NHE1 but rather promoted its biosynthetic maturation and half-life at the cell surface, thereby increasing the steady-state abundance of functional NHE1 protein.     

Mutagenic analysis of the v-crk oncogene: requirement for SH2 and SH3 domains and correlation between increased cellular phosphotyrosine and transformation.

We have constructed a series of mutants with deletion, linker insertion, and point mutations in the v-crk oncogene of avian sarcoma virus CT10. The v-crk gene contains no apparent catalytic domain, but does contain two blocks of homology to putative regulatory domains, termed SH2 and SH3, found in a variety of proteins implicated in signal transduction. Infection with CT10 causes a dramatic increase in the level of tyrosine phosphorylation of several cellular proteins. We found that mutation of either the SH2 or SH3 domain of v-crk reduced or eliminated transforming activity, whereas mutation of regions outside the conserved domains had no effect. Deletion of amino-terminal gag sequences caused a partial loss of transforming activity and a change in subcellular distribution of the crk protein. In all cases, there was an absolute correlation between increased cellular phosphotyrosine and transformation.