The plasma membrane Na+/H+ exchanger NHE1 has an established function in intracellular pH and cell volume homeostasis by catalyzing electroneutral influx of extracellular Na+ and efflux of intracellular H+. A second function of NHE1 as a structural anchor for actin filaments through its direct binding of the ezrin, radixin, and moesin (ERM) family of actin-binding proteins was recently identified. ERM protein binding and actin anchoring by NHE1 are necessary to retain the localization of NHE1 in specialized plasma membrane domains and to promote cytoskeleton-dependent processes, including actin filament bundling and cell-substrate adhesions. This review explores a third function of NHE1, as a plasma membrane scaffold in the assembly of signaling complexes. Through its coordinate functions in H+ efflux, actin anchoring, and scaffolding, we propose that NHE1 promotes protein interactions and activities, assembles signaling complexes in specialized plasma membrane domains, and coordinates divergent signaling pathways.
- hydrogen ion efflux
- intracellular pH
- molecular scaffold
the cytoplasmic domains of a number of integral plasma membrane proteins, including growth factor receptors, adhesion receptors, and ion transport proteins, serve as structural platforms for assembling kinases and phosphatases, cytoskeletal tethers, and adaptor proteins. The assembly of signaling proteins or scaffolding by transmembrane proteins has multiple functions, including inducing conformational and phosphorylation states that regulate activity of the transmembrane protein, retaining proteins in specialized membrane domains, and promoting the specificity and amplification of signal relay. This review examines collective evidence from several laboratories suggesting that the Na+/H+ exchanger isoform 1 (NHE1) acts as a plasma membrane scaffold. The COOH-terminal cytoplasmic domain of NHE1 associates with a number of functionally distinct signaling proteins that coordinately regulate Na+/H+ exchange, restrict the localization of NHE1 to specialized membrane domains, and, we now propose, promote signal relay to diverse effector pathways.
OVERVIEW OF NHE1 STRUCTURE AND INTERACTING SIGNALING MOLECULES
NHE1 is a ubiquitously expressed integral plasma membrane protein containing two functional domains: an NH2-terminal transmembrane ion translocation domain and a COOH-terminal cytoplasmic regulatory domain (Fig. 1). The ion translocation domain is predicted to include 12 transmembrane-spanning α-helices that function in catalyzing electroneutral exchange of extracellular Na+ for intracellular H+. The regulatory domain modulates transport activity, most likely by altering affinity of a H+ transport site in the transmembrane domain (41, 49). The regulatory domain associates with a number of functionally distinct signaling molecules (Fig. 1). At the cytoplasmic juxtamembrane region, NHE1 binds to phosphatidylinositol 4,5-bisphosphate (PIP2) (1), calcineurin homologous protein (CHP)1 (33, 42), and actin-binding proteins of the ezrin, radixin, moesin (ERM) family (16). At the distal COOH terminus NHE1 contains a number of serine residues that are phosphorylated by serine/threonine kinases acting downstream of distinct signaling pathways. COOH-terminal serine residues of NHE1 are phosphorylated by the ERK-regulated kinase p90RSK (55) and the Ste20-like Nck-interacting kinase (NIK) (73) in response to activation of growth factor receptors and by Rho kinase 1 (ROCK1) in response to activation of integrin receptors (59) and G protein-coupled receptors for thrombin and lysophosphatidic acid (49, 60). Phosphorylation of COOH-terminal serine residues increases ion translocation by the transmembrane domain, and phosphorylation of Ser703 by p90RSK promotes direct binding of the adaptor protein 14-3-3β (28). Although NIK phosphorylates distal COOH-terminal serine residues, phosphorylation of NHE1 in vivo by NIK is dependent on direct binding of the regulatory domain of NIK to a central domain of the NHE1 tail (73). Distal to the NIK-binding domain are adjacent high- and low-affinity binding sites for calmodulin (CaM) (62, 63). Additional proteins binding directly to the COOH terminus of NHE1 include heat shock protein (HSP)70 (53) and carbonic anhydrase II (31), although the binding sites for these proteins have not been determined.
Scaffolding by transmembrane proteins often occurs through modular protein-protein interaction domains. A common theme for the targeting and assembly of signaling proteins by transmembrane scaffolds is through the binding of proteins containing PDZ domains (46, 76). PDZ domain interactions promote the assembly of protein modules by N-methyl-d-aspartate (NMDA) NR2 receptors (52), nicotinic receptors (10), K+ channels (25), the cystic fibrosis transmembrane receptor (54, 65), and 5-HT and β-adrenergic G protein-coupled receptors (20, 26). Scaffolding by NHE1, however, occurs independently of PDZ domain interactions. The COOH-terminal cytoplasmic domain of NHE1 lacks canonical binding sites for PDZ domains, and none of the identified NHE1-binding proteins contains PDZ domains. In contrast, the NHE isoform NHE3, which is localized at the apical domain of epithelia in kidney and the gastrointestinal tract (6), binds the PDZ domain-containing adaptor proteins NHERF (67) and E3KARP (75). An additional mechanism by which transmembrane scaffolds assemble signaling complexes is through the association of adaptor proteins containing SH2 domains, which bind to phosphorylated tyrosine residues, and SH3 domains, which bind to proline-rich sequences (44). Scaffolding and signal relay by receptor tyrosine kinases require binding of SH2/SH3 adaptor proteins, including Grb2 (57) and Nck/Dock (30). Scaffolding by NHE1 is not mediated through interactions with SH2 domains, because the COOH terminus of NHE1 is phosphorylated on serine, but not tyrosine, residues. The distal COOH terminus of NHE1 contains three PxxP consensus sites (amino acids 721–724, 724–727, 809–812 in human NHE1) for binding SH3 domains; however, no SH3 domain-containing proteins have been reported to bind at these sites.
NHE1 AS SPATIALLY RESTRICTED SCAFFOLDING PLATFORM
In several cell types NHE1 is not uniformly distributed along the plasma membrane but is predominantly localized at distinct plasma membrane domains. NHE1 is localized at the basolateral membrane of polarized epithelial cells (11, 40), at intercalated disks and transverse tubules in cardiac myocytes (45), and at membrane protrusions or lamellipodia in fibroblasts (Fig. 2A; Refs. 16, 19, 51). In Dictyostelium discoideum, a recently identified NHE (DdNHE1, GenBank Accession No. AAM33760), with sequence identity most closely related to mammalian NHE8, is localized at the anterior pole of chemotaxing cells (Fig. 2A; H. Patel and D. L. Barber, unpublished observations). Hence, a restricted localization of NHE1 at specialized membrane domains may be conserved across tissues and species.
In most cell types, mechanisms restricting the localization of NHE1 have not been determined. In fibroblasts, however, NHE1 is retained at the distal margin of lamellipodia by binding ERM proteins (16). The NH2-terminal FERM domain of ERM proteins binds to two motifs of clustered lysines and arginines in the juxtamembrane domain of NHE1 (16), and the COOH terminus of ERM proteins binds to F-actin (8). Hence, through binding ERM proteins, NHE1 anchors actin filaments to the leading-edge membrane of lamellipodia. ERM binding to NHE1 is impaired by alanine substitutions in the distal cluster of charged lysines and arginines but not in the proximal cluster. This mutant, NHE1-KR/A, is not retained predominantly at lamellipodia but is more uniformly distributed along the plasma membrane (Fig. 2B) (16). Mislocalization of NHE1-KR/A also impairs the localization of ERM proteins and results in the loss of actin filaments tethered to the distal edge of lamellipodia (Fig. 2B). Ion translocation by the transmembrane domain is not necessary to maintain NHE1 in lamellipodia, because a mutant NHE1-E266I that lacks transport activity localizes in lamellipodia and anchors actin filaments, like wild-type NHE1. Hence, ERM binding is necessary to retain the localization of NHE1 in fibroblasts. Whether ERM binding is sufficient, however, has not been determined. PIP2 and ERM proteins bind to the same two clusters of basic amino acids within the juxtamembrane domain of NHE1 (1). PIP2 binding to the proximal cluster, however, is retained with mutations at the distal site in NHE1-KR/A, indicating that PIP2 binding is not sufficient to retain the localization of NHE1. PIP2, which resides at the inner leaflet of the plasma membrane and is enriched in lamellipodia (66) and lipid rafts (34), could facilitate the localization of NHE1 by inducing a conformation of the COOH terminus that allows binding of ERMs and other proteins. The juxtamembrane region of the structurally analogous anion exchanger AE1 is a flexible hinge region (64), suggesting that this region in NHE1 may undergo conformational changes to allow the binding of multiple proteins. Additionally, through its binding to proteins other than NHE1, PIP2 could facilitate the assembly of signaling complexes at NHE1.
What is the functional significance of restricting NHE1 to specialized membrane domains? The probable answer relates to the coordinate functions of NHE1 in ion transport, actin anchoring, and scaffolding. As an ion transport protein, localized H+ efflux by NHE1 likely promotes signaling or regulatory events. An increase in intracellular pH (pHi) has long been speculated to be necessary for de novo assembly of cytoskeletal filaments, as determined in earlier work on the fertilization of sea urchin eggs (3), the acrosomal reaction in ecinoderm sperm cells (58), and the motility of Ascaris sperm cells (23). At the leading edge of migrating mammalian fibroblasts (14) and chemotaxing Dictyostelium cells (H. Patel and D. L. Barber, unpublished observations) NHE1 is necessary for maintaining polarity and directed movement by promoting local assembly and remodeling of the actin cytoskeleton. Localized H+ efflux at intercalated disks and transverse tubules in cardiac myocytes promotes gap junction conductance (69) and Ca2+ release pathways (70, 71) in regulating impulse conduction and excitation-contraction coupling. Some actions of NHE1, including increased cell proliferation, require global, but not localized, increases in pHi. The proliferative rate of fibroblasts expressing a mutant NHE1-KR/A that lacks ERM binding and is not localized in lamellipodia is similar to that of fibroblasts expressing wild-type NHE1 but approximately fourfold faster than fibroblasts expressing NHE1-E266I, which lacks ion translocation (16, 48). Mice with homologous inactivation of nhe1, however, are viable and lack morphogenic defects (4, 12), suggesting that in the absence of NHE1 compensatory or redundant pHi-regulatory mechanisms occur during development. Recent findings (72) indicate that in mice null for NHE1 the expression of other membrane transporters is altered, including an increase in the acid extruder NHE3 and a decrease in the acid loader AE3.
As an actin anchoring protein, NHE1 localized in specialized membrane domains acts to tether actin filaments to the plasma membrane and maintain cell shape (16). Actin anchoring and H+ efflux by NHE1 coordinately function in remodeling of the actin cytoskeleton and cell-substrate adhesions (14, 16). As a scaffolding protein, NHE1 clustered in distinct membrane domains likely facilitates a focal recruitment site for NHE1-interacting proteins, analogous to ionotrophic neurotransmitter receptors at postsynaptic sites (52) and T cell receptors at the immunologic synapse (68), which facilitate localized signal relay through forced proximity. Scaffolding by NHE1 could promote the localized assembly of signaling complexes and, coordinately with H+ efflux, facilitate signal relay.
SCAFFOLDING BY NHE1 IN ASSEMBLY OF SIGNALING COMPLEXES
Many of the signaling molecules interacting with NHE1 regulate NHE1 activity (kinases, CHP, CaM, 14-3-3, PIP2) and NHE1 localization (ERM proteins, PIP2) (15, 41, 49). Although only binary interactions of NHE1 and associated signaling molecules have been reported, scaffolding by NHE1 likely assembles complexes of signaling molecules that interact structurally or functionally. Several NHE1-interacting proteins function in maintaining ERM proteins in an activated conformation at the plasma membrane (Fig. 3). Inactive ERM proteins are retained in the cytosol in a closed conformation that masks binding sites in their NH2-terminal FERM domain for transmembrane proteins and binding sites in their COOH terminus for F-actin (8). ERM proteins function as coincidence detectors and require two inputs for an activated open conformation. Binding of PIP2 by the NH2 terminus and phosphorylation of a conserved threonine residue in the COOH terminus (Thr558 in human moesin) (8) disrupt the “head-to-tail” conformation and allow F-actin binding (21, 39). The proximity of PIP2 and ERM binding on NHE1 likely maintains ERM proteins in an open, active conformation, which facilitates F-actin binding and maintains the localization of NHE1. Moreover, a conserved threonine residue in the COOH terminus of ERM proteins is a substrate for two kinases that also phosphorylate NHE1: ROCK1 (38) and NIK (M. Baumgartner et al., unpublished observations). In addition to phosphorylating an ERM COOH-terminal regulatory threonine residue NIK binds directly to the NH2 terminus of ERM proteins, which likely further promotes an active, open conformation. Thus the forced proximity of ERM proteins, PIP2, NIK, and ROCK1 by associating with NHE1 could facilitate the activation of ERM proteins, stabilize F-actin binding to ERM proteins, and, in fibroblasts, regulate cytoskeletal dynamics in lamellipodia.
PIP2, NIK, and ROCK1 also function in cytoskeletal remodeling independently of their actions on ERM proteins (Fig. 3). PIP2 plays a critical role in actin polymerization by coordinating with Cdc42-GTP the activation of Wiskott-Aldrich syndrome protein (WASP), which promotes the actin-nucleating and actin filament-branching activity of Arp2/3 (22). In a possibly related signaling pathway, NIK binds to the adaptor protein Nck, which also activates WASP (50), and we recently determined that NIK binds and phosphorylates Arp2/3 (M. Baumgartner and D. L. Barber, unpublished observation). ROCK regulates cytoskeletal remodeling by promoting the assembly of actin filaments and focal adhesions, two actions that require localized NHE1 (14, 59, 61).
Phosphorylation of Ser703 on NHE1 by p90RSK induces activation of NHE1 by growth factors (55), and phospho-Ser703 binds 14-3-3β with high affinity (28). The 14-3-3 proteins have multiple functions, including shielding phosphorylated residues from dephosphorylation (2), and binding of 14-3-3β to a phosphorylated glutathione S-transferase fusion protein of the COOH terminus of NHE1 attenuates dephosphorylation by protein phosphatase 1 (28); 14-3-3 also binds to p90RSK and inhibits p90RSK catalytic activity (9). Hence, 14-3-3 binding to NHE1 likely sustains NHE1 activity by acting in a positive feedback loop that includes maintaining phosphorylation of Ser703 and competing for binding to p90RSK, which would increase p90RSK activity. Additionally, 14-3-3 proteins act as adaptors to sequester signaling complexes at the plasma membrane (2), and they may facilitate signal relay at an NHE1 scaffold by promoting the assembly of multiprotein complexes.
Several Ca2+-binding proteins bind directly to the COOH terminus of NHE1 to regulate ion exchange activity. The binding site for the EF-hand Ca2+-binding protein CHP1 was initially reported to include amino acids 520–535 (33) but subsequently confirmed to require amino acids 567–637 (42). Homologous proteins CHP2 (43) and tescalin (32, 36) also bind directly to NHE1, although binding sites have not been identified. Transient overexpression of CHP1 (33) and tescalin (32) attenuates NHE1 activity; however, an inhibitory action of CHP on NHE1 may reflect an artifact of overexpression because recent findings indicate that CHP may be an essential cofactor for promoting NHE1 activity. Preventing the binding of endogenous CHP to NHE1 by expressing a mutant NHE1 containing glutamine substitutions (526–531Q) or by injecting a competing peptide of the CHP-binding domain of NHE1 decreases NHE1 activity (42). CaM binds to NHE1 at two sites, a high-affinity site (∼20 nM) at amino acids 637–656 and a low-affinity site (∼350 nM) at amino acids 657–700 (62). Both CaM-binding sites reside within an autoinhibitory domain that suppresses NHE1 activity in quiescent cells by reducing the affinity for intracellular H+ sensing (63). Ca2+-dependent CaM binding is predicted to increase NHE1 activity by inducing a conformational change that relieves suppression by the autoinhibitory domain. CHP, like calcineurin B, can bind CaM, which suggests the possibility of cooperative or facilitated binding of CaM to NHE1. CHP, but not CaM, is an essential cofactor for maintaining NHE1 activity (42), and CHP binding to NHE1 could facilitate binding of CaM, particularly at the low-affinity CaM-binding site. CaM, like PIP2 and 14-3-3 proteins, facilitates the assembly of multiprotein complexes, thereby possibly enhancing a scaffolding function of NHE1.
SCAFFOLDING BY NHE1 AND SIGNAL RELAY
If scaffolding by NHE1 assembles signaling complexes, does it also facilitate signal relay? If scaffolding by NHE1 does facilitate signal relay, the number of functionally distinct proteins binding to NHE1 indicates that it does not promote signal relay through a single linear signaling pathway, analogous to soluble scaffolds such as Ste5p and Jun NH2-terminal kinase-interacting protein (JIP)1. Ste5p assembles a mitogen-activated protein kinase module in the mating pheromone pathway of the budding yeast (17), and JIP1 facilitates signaling through the Jun NH2-terminal kinase pathway in mammalian cells (74). Instead, NHE1 likely supports expansion of molecular complexity through multiple divergent signaling cascades, analogous to signal amplification through scaffolding by the T cell receptor, which promotes signal relay through coreceptors, kinases, phosphatases, and adaptors (68). In fibroblasts, a scaffolding platform of NHE1 promotes signal relay from growth factor and integrin receptors to cytoskeleton-dependent pathways regulating cell shape determination (16), cell-matrix adhesion (16, 59), and directed cell migration (14). The structurally and functionally analogous ion exchanger AE1 assembles signaling units at the erythrocyte membrane in response to environmental changes to regulate the metabolic, transport, and mechanical properties of red blood cells (56). Hence, scaffolding by ion exchangers, including NHE1, is likely promoted by extracellular cues to coordinate divergent signaling pathways regulating distinct cellular responses.
If an NHE1 scaffolding platform facilitates signal relay, does NHE1 activity augment the output signal of assembled protein complexes? For a scaffold to increase signal output, computational modeling indicates that the abundance of the scaffold protein should not exceed that of the assembled signaling proteins, so as to prevent combinatorial inhibition (7, 29), analogous to the optimal balance of antigen and antibody in the precipitin reaction. This criterion is met by the lower abundance of NHE1 relative to a number of its associated proteins, including ERM proteins, CaM, and CHP. If NHE1 does increase the output signal of assembled protein complexes, an intriguing possibility is that localized H+ efflux alters protein affinities and catalytic activities to promote stronger or faster signals, or to regulate responses between switchlike or graded and processive. The binding affinities and activities of a number of proteins acting in signaling pathways regulated by NHE1 are pH sensitive, including cofilin (5, 47), gelsolin (27), and talin (18). The pH sensitivity of proteins is mediated by protonation of histidine residues. With an acidic dissociation constant (pKa) of 6–7, histidine is the only amino acid that undergoes strongly altered protonation within the physiological pH range of the cytosol. Site-directed substitution of histidine residues abolishes the pH sensitivity of a number of proteins, including the anion exchanger (24), the elongation factor Tu (13), the inwardly rectifying K+ channel GIRK4 (37), and an H+-sensing G protein-coupled receptor (35). More than 50% of enzymes have histidine residues in their active sites, including NIK, which has five histidines clustered at the ATP-binding pocket (142HLHIHHVIHRD152). Hence, H+ efflux by NHE1 might promote signal output by facilitating the catalytic activity of its associated kinases. This possibility can now be tested by using a mutant mammalian NHE1 that lacks H+ efflux but retains ERM binding, cytoskeletal anchoring, and localization in lamellipodia (14, 16).
The action of NHE1 as a membrane scaffold is a relatively new perspective that warrants further investigation. Current evidence favors a cooperative action of NHE1 as an ion transport protein and as a membrane scaffold in promoting the assembly of signaling complexes and signal relay in specialized membrane domains. As we learn more about the complexity of signaling units assembled at NHE1 and the role of NHE1 activity in signal output, we will better understand the pivotal role NHE1 plays in diverse cellular processes.
Unpublished work in this review was supported by National Institute of General Medical Sciences Grants GM-47413 and GM-58642 (to D. L. Barber). M. Baumgarter is supported by the Swiss National Science Foundation.
We are grateful to John Orlowski for providing an image of NHE1 localization in rat heart. We thank members of the Barber lab for helpful comments and Mary McKenney for editorial assistance.
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