THEMES
G Protein-Coupled Receptors in Gastrointestinal Physiology
III. Asymmetry in plasma membrane signal transduction: lessons from brush-border Na+/H+ exchangers*

M. Donowitz, S. Khurana, C. M. Tse, and C. H. C. Yun

Departments of Medicine and Physiology, Gastrointestinal Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

    ABSTRACT
Top
Abstract
Introduction
References

Signal transduction in epithelial cells adds another level of complexity to the signaling that occurs in symmetrical cells, in the form of the need to coordinate and keep separate signals at the apical and basolateral membranes. Regulation by protein kinases of ileal NaCl absorption and its component brush-border Na+/H+ exchanger are used as an example of how signaling in epithelial cells must deal with spatial localization of signals, protein-protein interactions, signaling molecules, and the involvement of the transport protein being regulated in collecting and focusing the signals generated at the receptor and beyond.

intestinal epithelial cells; epidermal growth factor; carbachol; NHE3; signalosomes

    INTRODUCTION
Top
Abstract
Introduction
References

WE RELATE SEVERAL RECENT ADVANCES in the understanding of signal transduction to the specific challenges of signaling in epithelial cells. The advances reviewed here are related to 1) spatial localization of signaling proteins, 2) protein-protein interactions, 3) involvement of signalosomes (23) or signaling complexes, and 4) cross talk between G protein-linked and tyrosine kinase signaling pathways. Although the same ligands, receptors, and signaling proteins appear to be involved in signaling in epithelial and nonepithelial cells, an added complexity in epithelia is the separation of the epithelial cell plasma membrane into two domains, the brush-border and basolateral membranes. For those conditions in which the receptors, effectors, and targets are not located in the same membrane domain, there are the added complexities of 1) maintaining the domains separate, 2) ways for the domains to communicate, and 3) ways for the regulated process to focus the various components of the signals generated from the receptors.

    LOCATION OF TARGET: REGULATION OF NEUTRAL NACL ABSORPTION

The process used for illustration is the neutral NaCl-linked absorptive process in the mammalian ileum and ascending colon (7). This process is made up of a brush-border Na+/H+ exchanger linked to an anion exchanger (functional Cl-/HCO-3 exchanger). The latter was previously thought to be a member of the AE gene family and now may be the gene product of the DRA (downregulated in adenoma) gene (8a). The brush-border Na+/H+ exchangers are members of the mammalian Na+/H+ exchanger gene family, of which at least six isoforms have now been identified (6, 18, 19). Two of these, NHE2 and NHE3, are present in the brush border of ileum and ascending colon of all species studied, including the human, dog, rabbit, rat, mouse, and chicken (9). NHE3 appears to be the major functional brush-border isoform in mammalian intestine. However, although only partially defined, the role of NHE2 seems to vary among species. For instance, in rabbit ileum, 50% of basal Na+/H+ exchange is due to NHE2 and 50% to NHE3 (24), whereas in the dog all basal Na+/H+ exchange is due to NHE3. In contrast, in the chicken, NHE2 makes up 85% of basal colonic Na+/H+ exchange and all of the increase induced by elevating aldosterone (4). Neutral NaCl absorption is important because it is 1) the major mechanism for ileal Na+ absorption during the period between meals, 2) accounts for the entire increase in canine ileal Na+ absorption occuring in the postprandial state, and 3) is the single transport process that becomes abnormally inhibited in most diarrheal diseases. Short-term regulation of NaCl absorption by growth factors and protein kinases, the topic of this review, appears to be exerted by an effect on the Na+/H+ exchanger component of neutral NaCl absorption. However, there has been no detailed study of direct effects on the Cl-/HCO-3 exchanger. Both neutral NaCl absorption and brush-border Na+/H+ exchange are unusual for transport processes in that they are not turned off under basal conditions. Rather, both neutral NaCl absorption and brush-border Na+/H+ exchange are set in a partially activated state. This allows rapid stimulation and inhibition similar to that occurring in the postprandial state (7). Although this was initially thought to be due to the presence of a myriad of neurohumoral-paracrine mediators in the normal intestine with different mixes of these mediators released in the basal and postprandial state, it now appears that this partial stimulation also occurs when NHE3 is expressed in the PS120 fibroblast and the human colon cancer cell line Caco-2. For instance, in ileal brush border, Caco-2 cells, and PS120 cells, 1) Ca2+-calmodulin and Ca2+-calmodulin kinase II inhibit NaCl absorption and/or NHE3 on the apical membrane under basal conditions, 2) tyrosine kinases stimulate NaCl absorption and NHE3, and 3) serine/threonine phosphatases 1 and 2A (okadaic acid sensitive) inhibit NaCl absorption and NHE3 (3, 16). Whether these effects are exerted directly on NHE3 or on NHE3 regulatory proteins is not known. Relevant to the kinase regulation of NHE3 expressed in PS120 cells and on the apical surface of Caco-2 cells and rabbit ileum, protein kinase C (PKC) inhibits, whereas fibroblast growth factor (FGF) or epidermal growth factor (EGF) stimulates, NHE3 (7, 15). This regulation involves changes in the maximal velocity (Vmax) of NHE3 and, at least for EGF, FGF, and PKC, does not appear to involve changes in the K'(H+i) (6, 15, 16). In contrast, the housekeeping isoform NHE1 is regulated by kinases through changes in K'(H+)i (15). Changes in Vmax could be due to changes either in the amount of NHE3 on the plasma membrane, in the turnover number of each NHE3 on the plasma membrane, or both. On the basis of cell surface biotinylation studies of NHE3 expressed in PS120 fibroblasts and confocal microscopy studies of NHE3 in Caco-2 cells, most regulation appears to be due to changes in both the amount of plasma membrane NHE3 and in the NHE3 turnover number (10).

    REGULATORY MECHANISMS

In this review, two examples are used in which activation of basolateral membrane receptors, one G protein-coupled receptor and one tyrosine kinase receptor, alters apical membrane NaCl absorption and brush-border Na+/H+ exchange. 1) The cholinergic agonist carbachol binds to M3 muscarinic cholinergic receptors to inhibit NaCl absorption. 2) EGF binds to the EGF receptor (EGFR) in the basolateral membrane to stimulate NaCl absorption and increase brush-border Na+/H+ exchange. These examples were chosen to illustrate that separate signal transduction steps occur in the basolateral and apical membranes and therefore both membranes must be studied to define the signaling events. We refer to this as the asymmetrical aspect of signal transduction. Furthermore, these models were chosen because one stimulates (EGF) and one inhibits (carbachol) ileal NaCl absorption and brush-border Na+/H+ exchange, yet both involve similar classes of intermediate and cross talk of tyrosine kinase and G protein-coupled signaling.

    LOCALIZATION OF THE SIGNAL

Carbachol/Ca2+ signal in ileum (Fig. 1). As in most epithelial cells, carbachol binds to muscarinic cholinergic receptors, which are localized to the basolateral membrane. Although only partially defined, these appear to be M3 receptors on the basis of pharmacological and molecular classification. When activated, these receptors lead to a biphasic increase in intracellular free Ca2+ ([Ca2+]i). In spite of the lack of apical membrane carbachol receptors, the elevation of [Ca2+]i occurs first at the apical membrane, within 1 s of addition of ligand, and sweeps as waves from the apical to the basolateral surface. These waves occur with a near-maximal agonist concentration, but at lower concentrations the [Ca2+]i elevation remains restricted to the apical pole. This phenomenon is best characterized in pancreatic acinar cells and hepatocytes but has also been shown to occur in lacrimal gland epithelial cells, Madin-Darby canine kidney (MDCK) cells, and in a human colon cancer cell line, HT-29 (11). It appears that [Ca2+]i is elevated close to the apical membrane, where it acts to regulate NaCl absorption and brush-border Na+/H+ exchange; the additional increase in [Ca2+]i near the basolateral membrane probably regulates K+ channels. Ca2+ is known to be a poorly diffusible messenger (1). Baker showed in the 1980s that injection of boluses of Ca2+ into squid axon diffused little, and Allbritton et al. (1) demonstrated that the free Ca2+ diffusion length was less than 0.1 µm. This distance is too small to transmit a signal from the basolateral membrane to the apical membrane, given that intestinal cells are 20-35 µm in length.

Several adaptations have been identified in ileal cells that focus the cholinergic elevation of [Ca2+]i at the apical membrane. The cholinergic receptor-linked increase in [Ca2+]i involves a phosphatidylinositol (PI)-specific phospholipase C (PLC). There is an unusual asymmetrical aspect of carbachol signaling through PLC. In most cells there appears to be direct coupling of the M3 receptor and PI-specific PLC-beta (PI-PLC-beta ). In the ileum there is asymmetrical distribution of the PI phospholipases in the apical vs. basolateral membranes. Under basal conditions, the basolateral membrane has PI-PLC-beta 2 and -delta 1 but not -beta 1 and -gamma 1, whereas the apical membrane has PI-PLC-beta 2, -delta 1, and -gamma 1 (12). Carbachol exposure does not alter basolateral membrane PI-PLC activity or the amount of PLC-beta 2 and PLC-delta 1 and does not cause PLC-gamma 1 to appear in the basolateral membrane, but rapidly increases PI-PLC activity and the amount of PLC-gamma 1 in the apical membrane. It is surprising that M3 receptors in the basolateral membrane do not appear to be coupled to PLC-beta 2. This effect is short-lived, occurring within 1 min of carbachol exposure, and PI-PLC activity and the amount of PLC-gamma 1 return to baseline by 21/2 min after carbachol addition. However, there is activation of another phospholipase that mimics the more prolonged time course of carbachol inhibition of NaCl absorption. Phospholipase D (PLD) activity is present in both the ileal apical and basolateral membranes under basal conditions, with basolateral activity exceeding apical activity. Carbachol increases apical but not basolateral membrane PLD activity, an effect that occurs within 1 min of carbachol exposure and lasts for at least 40 min. The increase in PLD appears to be initiated by PI-specific PLC activation. However, the prolonged activation of PLD is as yet unexplained. The initial elevation in [Ca2+]i is almost certainly due to the brush-border activation of PLC-gamma 1 to generate inositol 1,4,5-trisphosphate (IP3) (12). An epithelium-specific type of IP3 receptor (the high-sensitivity type 3 IP3 receptor) is localized to the subapical domain in intestinal cells such as HT-29 cells, although some other IP3 receptors are in this area as well (17). There is a further asymmetrical aspect of signaling related to apical membrane PI-PLC activation. Profilin is a phosphatidylinositol bisphosphate (PIP2) binding protein, with availability of the substrate for PI-PLC (PIP2) potentially limited by binding to profilin. Profilin is present in the apical but not the basolateral membrane of ileal epithelial cells (M. Donowitz and P. Goldschmidt-Clermont, unpublished observations). Moreover, within 1 min of carbachol exposure, profilin is released from the brush border. This would tend to increase the availability of PIP2 at the apical membrane, increasing the substrate for the activated PI-PLC. Thus activation of phospholipases at the apical membrane and local generation of IP3 to act on apically localized intracellular Ca2+ stores probably focuses elevation of [Ca2+]i in the apical membrane of intestinal cells. The Ca2+ waves follow from Ca2+-induced Ca2+ release acting at other nonapically localized intracellular Ca2+ stores, which are distributed throughout the cytosol. IP3 is able to diffuse across much longer distances than Ca2+ (1); however, the apical IP3 generation seems to give the cell much more precise control of the site at which the elevation in [Ca2+]i is focused.

The activation of PLC and PLD generates diacylglycerol directly (PLC) or indirectly (PLD), which potentially could activate PKC. Carbachol increases PKC activity and translocation to the ileal brush border, increasing PKC activity and amount within 1 min of carbachol exposure, an effect that continues for at least 40 min. PKC activation and translocation occur at the apical but not the basolateral membrane; in fact, over time basolateral PKC activity and the amount of PKC decrease. Brush-border PKC is involved in the inhibition of NaCl absorption and brush-border Na+/H+ exchange, because addition of PKC inhibitors either to the ileal apical membrane or to brush-border vesicles reverses the inhibition caused by carbachol. It is not known which isoform of PKC translocates to the brush border to regulate Na+/H+ exchange. Also still unknown is the signal generated by carbachol at the basolateral membrane that initiates the apical membrane signaling events to inhibit NaCl absorption.

EGF/tyrosine kinase receptor (Fig. 1). In the adult intestine the EGFR is localized to the basolateral membrane (22). Caco-2 cells grown as polarized cells have only a small fraction of EGFR on the apical membrane. In Caco-2 cells, the normal ligand for the EGFR is unknown; some Caco-2 cells make amphiregulin and others make transforming growth factor-alpha . It has not yet been established whether EGF is physiologically involved in short-term regulation of NaCl absorption. However, EGF at doses present in serum acutely stimulates NaCl absorption. This effect occurs quickly, reaching a maximum in 1-15 min, and is associated with an increase in NHE3 Vmax. EGF stimulation of NaCl absorption and brush-border Na+/H+ exchange can be demonstrated in intact ileal tissue, brush-border vesicles made from ileal villus cells exposed in vitro to EGF, and NHE3 expressed on the apical surface of Caco-2 cells. This short-term EGF stimulation of brush-border Na+/H+ exchange occurs specifically, with no accompanying increase in Na+-dependent D-glucose absorption over ~30 min in ileum or Caco-2 cells. However, longer exposure to EGF is associated with an increase in D-glucose uptake and in the size of the ileal brush border. The EGF effects on brush-border NHE3 asymmetrically involve at least two signaling molecules at the brush border, PI 3-kinase and c-Src. The EGF stimulation of brush-border Na+/H+ exchange can be prevented by apical membrane exposure of ileum or Caco-2 cells to the PI 3-kinase inhibitor wortmannin (13). In addition, brush-border PI 3-kinase activity and amount and tyrosine phosphorylation of the p85 subunit increase within 1 min of exposure to EGF. These changes parallel the EGF stimulation of brush-border Na+/H+ exchange activity, and wortmannin added to the apical membrane prevents the EGF stimulation of Na+/H+ exchange as well as the changes in the brush-border PI 3-kinase. In ileal cells, although there is basolateral PI 3-kinase activity under basal conditions, no change occurs with EGF exposure (13). Apical membrane tyrosine kinases are involved in the increase in PI 3-kinase at the apical membrane. Addition of the tyrosine kinase inhibitors tyrphostin-23 and genistein to the apical membrane of ileal cells and Caco-2 cells prevented the EGF-induced increase in PI 3-kinase activity in Caco-2 cells and the increase in activity of PI 3-kinase and tyrosine phosphorylation in ileal brush border. This effect occurred when genistein was added to ileal brush-border vesicles that had been exposed in vitro to EGF and had increased brush-border Na+/H+ exchange. However, addition of wortmannin to these vesicles did not reverse the EGF stimulation of brush-border Na+/H+ exchange, suggesting that the PI 3-kinase effect had already occurred, for instance, in delivering the NHE3 to the apical membrane.

A role for c-Src in the EGF stimulation of brush-border NHE3 has been partially defined (M. Donowitz, R. Patterson, and S. Khurana, unpublished observations). c-Src appears to be involved in the EGF activation and tyrosine phosphorylation of PI 3-kinase in the brush border. An Src kinase inhibitor, PP1, added to the ileal apical surface prevented EGF stimulation of NaCl absorption. Src is asymmetrically distributed in ileal villus cells, being present in the brush border but not the cytosol or basolateral membrane. EGF increases brush-border Src activity, an effect that occurs within 1 min of EGF addition to the basolateral membrane. PP1 inhibits basal and EGF-activated Src activity and also prevents EGF activation and tyrosine phosphorylation of brush-border PI 3-kinase. The increase in the amount of brush-border PI 3-kinase caused by EGF was not inhibited by PP1. This demonstrates that the EGF-induced movement of PI 3-kinase to the brush border was a separate step, independent of Src kinase, whereas the tyrosine phosphorylation and activation of PI 3-kinase were dependent on Src kinase. The signal(s) generated at the EGFR in the basolateral membrane, which moves PI 3-kinase from the cytosol to the brush border and which activates Src at the apical surface (which then activates PI 3-kinase and stimulates Na+/H+ exchange), has not been identified. Thus there are at least three asymmetrical aspects of EGFR signaling in the apical membrane involved in stimulation of brush-border Na+/H+ exchange: 1) activation of c-Src, 2) movement of PI 3-kinase to the apical membrane, and 3) Src activation and tyrosine phosphorylation of PI 3-kinase.

Thus we have identified asymmetrical aspects of signal transduction involving multiple proteins. Under basal conditions, the following are found only or primarily in the ileal brush-border membrane and not in the basolateral membrane: PI 3-kinase, PLC-gamma 1, profilin, c-Src, ezrin, and villin (this separation does not differentiate the brush border and the junctional complex, which is partially purified with the apical and partially purified with the basolateral membrane). Of the signaling proteins studied, only PLD is present at higher activity levels in the basolateral membrane than in the brush-border membrane under basal conditions. The identified changes in signaling proteins with EGF or carbachol involve changes in the activities and/or amounts of the molecules present under basal conditions in the brush border and/or cytosol but not in the basolateral membrane. There is parallelism in the signaling molecules activated by EGF and carbachol. Both activate lipid kinases in the cytosol (PI 3-kinase and PLC-gamma 1, respectively), which translocate to the brush border, where they have increased activity. In the brush border, both cause an increase in tyrosine kinase and PKC activity. We have not yet identified translocation of signaling proteins from the apical to the basolateral membrane as a part of signaling, and only PKC has been shown to move from the basolateral to the apical membrane. Conversely, the basolateral membrane has not been studied in sufficient detail to identify the signaling events initiated there.

    CROSSOVER BETWEEN SIGNALING BY G PROTEIN-COUPLED RECEPTORS AND TYROSINE KINASE RECEPTORS

Although initially thought to be entirely separate signaling pathways, crossover of tyrosine kinase and G protein-related signaling is now known to be a common event in signaling pathways. This adds a great deal of specificity to signaling based on integration of multiple components in an individual signaling pathway. Such crossover is recognized to occur in epithelial cells (2).

In the EGF signaling described above, there is stimulation of both serine/threonine kinases and tyrosine kinases in the brush border. In the brush border, c-Src is activated by EGF binding to the basolateral membrane, but there is also stimulation of the serine/threonine kinases Akt and PKC-zeta at the brush border (M. Donowitz, C. Shih, and S. Khurana, unpublished observations). Thus crossover of EGFR to serine/threonine kinases is not strictly a switch to serine/threonine signaling at the apical membrane.

Carbachol signaling involves both serine/threonine phosphorylation and tyrosine kinase activation in the apical membrane, as occurs with EGFR signaling. Carbachol binding to basolateral membrane receptors is associated with tyrosine kinase-dependent activation of PI-PLC and PLD in the brush border and activation of PKC as well. Which isoform of PKC is involved and whether its activation is dependent on tyrosine kinase have not been established, but the latter would be likely, since PKC activation is dependent on phospholipase activation (Fig. 1). PKC activation in the brush border is required for the inhibition of NHE3 by carbachol, and this effect occurs at the level of the brush border.


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Fig. 1.   Summary of asymmetrical aspects of signaling induced by basolateral membrane activation of epidermal growth factor receptor (EGFR) and M3 cholinergic receptors. Shaded area includes signaling events shown to occur in brush border. PI 3-K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PLC, phospholipase C; PA, phosphatidic acid; DAG, diacylglycerol.

    PROTEIN-PROTEIN INTERACTIONS

A major advance in understanding signal transduction is the recognition of the common involvement of physical attachment of proteins that occurs as a part of signaling, which to a significant extent occurs by means of interaction of specific domains (20). These include SH2, SH3, WW, PDZ, PTB, 14---3---3, and PH domains. Interacting proteins form collections of signaling molecules called signalosomes (23), which accumulate at both tyrosine kinase receptors and at seven-membrane-spanning domain/G protein-coupled receptors as part of signaling initiation. Various approaches have proven sensitive enough to at least suggest which molecules interact. These include yeast two-hybrid screening and other interactive cloning approaches, coprecipitations, and overlay techniques. In epithelial cells, these approaches have demonstrated that signaling complexes also exist at the brush border.

The presence of protein-protein interactions in signaling has provided some insight into a confusing aspect of studies of cloned Na+/H+ exchangers in model cell systems. Many aspects of NHE3 regulation in ileal brush border were similar in PS120 cells and Caco-2 cells. One exception is cAMP inhibition of ileal Na+/H+ exchange, because in PS120 cells and Caco-2 cells NHE3 is not regulated by cAMP. In another type of fibroblast, AP1 cells, cAMP does inhibit NHE3. At least part of this cell-specific response of NHE3 to cAMP appears to be due to two related proteins, E3KARP (NHE3 kinase A regulatory protein) and NHERF (Na+/H+ exchanger regulatory factor). These related proteins each have two PDZ domains, which are highly homologous to each other and are also highly conserved between E3KARP and NHERF. When transfected into PS120 cells, E3KARP and NHERF reconstitute cAMP inhibition of NHE3, establishing a role for these proteins in cAMP regulation (26). NHERF is a phosphoprotein under basal conditions, whereas E3KARP is not phosphorylated under basal conditions (G. Lamprecht and C. Yun, unpublished data), suggesting that their phosphorylation is not required for cAMP inhibition of NHE3. The human analog of NHERF, EBP50, binds ezrin, a protein that links the brush border to the actin cytoskeletal core, suggesting that a potential role of these proteins is to link transport proteins to cytoskeleton (21). This may serve to allow formation of a signaling complex that involves the transport protein being regulated. The role of these molecules in signaling has just begun to be understood, and our suggestion that they have a role in the formation of signalosomes is a hypothesis. In recognition of the fact that these proteins are involved in regulation of far more than Na+/H+ exchanges, we refer to them as the Regulatory Factor gene family.

The role of protein-protein interactions in signaling in general and in different locations in epithelial cells has just begun to be understood. For instance, there is very little information regarding the signaling complexes that involve the junctional complex, although these signaling complexes are known to be rich in tyrosine kinases.

    INVOLVEMENT OF THE TRANSPORT PROTEIN BEING REGULATED IN SIGNALING COMPLEXES

Signals initiated at plasma membrane receptors are focused at the receptors by protein-protein interactions that are initiated by parts of the receptor (20). Examples include the tyrosine kinase receptors, in which a phosphorylated tyrosine in the cytoplasmic domain of the receptor plus the three to five 3' amino acids selectively bind SH2 domains, and perhaps a comparable sequence of events in G protein-coupled receptors, in which phosphoserines of the receptor selectively bind 14---3---3 proteins. What serves to focus the signal at the apical membrane in an epithelial cell? The evidence we have generated suggests the presence of apical membrane signaling complexes, but it seems very likely that, as occurs in association with receptors, the signals are focused. We hypothesize that one way that the signals are accumulated is that the proteins being regulated focus the signals and signaling complexes. We use NHE3 as an example of a transport protein that is regulated in the brush border and appears to focus the signal on itself. The COOH-terminal ~300 amino acids of NHE3 are cytoplasmic and are required for NHE3 regulation by growth factors, protein kinases, and changes in osmolarity (6, 16). The COOH terminus of NHE3 is organized into a series of domains, with part of the COOH terminus required for stimulation and part for inhibition (Fig. 2). NHE1 and NHE3 are regulated partially by changes in their phosphorylation, but although they are phosphoproteins in the basal state, some kinase/growth factor regulation is not associated with changes in NHE phosphorylation (6, 18, 25). It appears that some of the regulatory subdomains in the NHE COOH termini act as hooks or scaffolds to interact with associated regulatory proteins. Some of these associated regulatory proteins help change the NHE phosphorylation, whereas others appear to regulate the NHEs in different ways, including the formation of signaling complexes. An example includes the regulatory factors E3KARP and NHERF, in their reconstitution of cAMP regulation of NHE3. E3KARP binds to NHE3 close to the domain necessary for cAMP inhibition (Fig. 2; C. Yun and G. Lamprecht, unpublished observations). NHE3 phosphorylation is increased by cAMP on amino acid Ser605 (14). NHE3 is not phosphorylated by the regulatory factors, since they are not protein kinases. As a hypothesis, we suggest that the regulatory factors either change the conformation of the COOH terminus of NHE3 or change the location of NHE3, perhaps clustering the molecule in the plasma membrane, to make it accessible to protein kinase A. One likely protein involved is the cytoskeletal linking protein ezrin, which has recently been shown to bind NHERF and is known to bind protein kinase A (21). We suggest the involvement of a complex, possibly formed between ezrin with the attached protein kinase A (20), E3KARP or NHERF, and NHE3 (Fig. 2). A second regulatory protein that binds to the COOH terminus of NHE3 is calmodulin (3). Calmodulin appears to be involved in the regulation of NHE1, NHE2, and NHE3 (5, 6, 8, 14). Calmodulin binds to the COOH-terminal 85 amino acids of NHE3, and inhibitors of calmodulin (W13) and calmodulin kinase II (KN-62) stimulate NHE3. The effects of W13 and KN-62 require the COOH-terminal 76 amino acids of NHE3 (5, 16). How calmodulin regulates NHE3 and whether a signaling complex forms around the calmodulin is not known. However, the fact that two signaling molecules have been shown to bind to the COOH terminus of NHE3 (Fig. 2) suggests that signaling complexes may form around specific domains of NHE3 and that NHE3 might focus regulation by acting as a scaffold for signaling proteins.


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Fig. 2.   Correlation of domains in the NHE3 COOH terminus identified as involved in functional regulation by protein kinases and growth factors and which bind associated regulatory proteins. Inset: ileal brush border and a hypothesis of the scaffolding role of the NHE3 COOH terminus and its relationship to binding of associated regulatory proteins, including RF (E3KARP), and calmodulin (CaM). RF, regulatory factors (NHERF, E3KARP); OA, okadaic acid; FBS, fetal bovine serum; Osm, hyperosmolarity.

    ACKNOWLEDGEMENTS

This study was supported in part by National Institutes of Health Grants RO1-DK-26523, PO1-DK-44484, RO1-DK-51116, American Digestive Health Foundation/American Gastroenterological Association/Hoechst Marion Rousel Research Scholar Award, and the Meyerhoff Digestive Diseases Center.

    FOOTNOTES

* Third in a series of invited articles on G Protein-Coupled Receptors in Gastrointestinal Physiology.

Of the many studies quoted, only selected references were listed due to space limitations.

Address for reprint requests: M. Donowitz, Johns Hopkins Univ. School of Medicine, 925 Ross Research Bldg., 720 Rutland Ave., Baltimore, MD 21205-2195.

    REFERENCES
Top
Abstract
Introduction
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Am J Physiol Gastroint Liver Physiol 274(6):G971-G977
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