HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Donowitz, M. Articles by Li, X. Search for Related Content PubMed PubMed Citation Articles by Donowitz, M. Articles by Li, X.

Regulatory Binding Partners and Complexes of NHE3

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

ABSTRACT

I. OVERVIEW

II. MECHANISMS OF ACUTE REGULATION OF NHE3

A. NHE3 Phosphorylation

1. Evidence that NHE3 is phosphorylated as part of its acute regulation

A) CAMP.

B) CGMP.

C) ELEVATED CALCIUM.

D) OTHER KINASES.

E) SER/THR PHOSPHATASES.

B. Acute Regulation of NHE3 Can Occur by Changes in Trafficking or Changes in Turnover Number

III. INTRACELLULAR NHE3 ACTIVITY

IV. NHE3 COOH-TERMINAL REGULATORY DOMAIN AND NHE3 COMPLEXES

A. Regulatory Domain

B. NHE3 Complexes

1. How many proteins are there in the NHE3 complexes?

C. Intramolecular Interactions

D. Dynamic Aspects With Signaling

V. NHE3 COOH-TERMINAL BINDING PARTNERS

A. COOH-Terminal Domain 1, Amino Acids 455–585: CHP, Ezrin, and Phosphoinositol Phosphates

1. CHP

2. Direct binding of ERM proteins

3. Phosphoinositol phosphates and ATP

B. COOH-Terminal Domain 2, Amino Acids 586–660: Calmodulin, Calmodulin Kinase II, and NHERF Family

1. CaM and CaM kinase II

2. NHERF family of multiple PDZ domain proteins

3. Overview of PDZ domain proteins (49, 63, 113, 156, 196, 215, 248, 352, 411, 455, 538)

4. Epithelial apical membrane PDZ domain proteins relevant to NHE3 regulation

A) APICAL SURFACE DISTRIBUTION OF NHERF FAMILY IN ILEUM AND RENAL PROXIMAL TUBULE.

B) NHERF FAMILY BINDING PARTNERS.

5. ''Hand-off'' hypothesis

6. Model systems used to understand the cell specific role for NHERF proteins in NHE3 regulation

A) NHE NULL FIBROBLASTS: EXPRESSION IN PS120 FIBROBLASTS INDIVIDUALLY OF NHERF1, NHERF2, NHERF3/PDZK1, AND NHERF4/IKEPP.

B) EPITHELIAL CELL CULTURE MODELS: EXPRESSION OF NHERF FAMILY MEMBERS IN EPITHELIAL CELLS, EITHER ENDOGENOUSLY OR EXOGENOUSLY.

C) NHERF1 FAMILY KNOCKOUT MICE.

7. NHERF-interacting domains of NHE3

8. Role of NHERF family members in NHE3 regulation (compared with functions of other PDZ domain containing proteins in polarized epithelial cells)

A) MAINTENANCE OF POLARITY OF EPITHELIAL CELLS.

B) DELIVER AND MAINTAIN NHE3 IN THE MEMBRANE UNDER BASAL CONDITIONS.

C) RECONSTITUTION OF REGULATED RAPID CHANGES IN NHE3 ACTIVITY BY ACTING AS SCAFFOLDS FOR NHE3 COMPLEX FORMATION.

9. Other BB PDZ proteins in NHE3 regulation

10. Regulation of NHERF proteins

11. Homo- and heteromultimerization of NHERF family

C. COOH-Terminal Domain 3, Amino Acids 661–756

D. COOH-Terminal Domain 4, Amino Acids 757–832: DPPIV(?), PP2A, and Megalin

1. DPPIV

2. PP2A

3. Megalin

VI. FUNCTIONAL INTERACTIONS OF NHE3 WITH PROTEINS NOT KNOWN TO DIRECTLY BIND NHE3

A. PEPT1/PEPT2

B. NHE1

C. SLC26A3 (DRA) and SLC26A6 (PAT1)

D. CFTR

GRANTS

ACKNOWLEDGMENTS

REFERENCES

	ABSTRACT

Top Next References

	I. OVERVIEW

Top Previous Next References

 
NHE3 (SLC26A3) is one of nine isoforms of the mammalian Na⁺/H⁺ exchanger (NHE) gene family (60, 62, 121, 357, 358, 476, 537). The mammalian NHEs consist of isoforms that occur primarily in the plasma membrane and those that appear to primarily reside in intracellular organelles (60). However, it may be that all NHEs have at least transient plasma membrane localization (62). The evolutionary history of the NHEs showed that the intracellular NHEs arose in a setting in which their transport was driven by H⁺ gradients generated by V-ATPases (62, 182, 342, 353, 354). The earliest evolving plasma membrane NHEs exist both in the plasma membrane and on the membranes of intracellular organelles (22, 33, 48, 255, 359, 448). These NHEs traffick continually between the plasma membrane and intracellular organelles (62, 226, 255, 434). They arose at a time of evolutionary appearance of the Na⁺-K⁺-ATPase, which creates a Na⁺ gradient and provides the energy for their exchange of extracellular Na⁺ for intracellular H⁺. The most recent NHEs to evolve appear to be the NHEs that are present predominantly on the plasma membrane (359, 401, 450, 451). The plasma membrane and intracellular NHEs can be separated by differences in the amino acid residues in their membrane-spanning domains, especially the putative P-loop (244, 335, 339, 417, 475). NHE1-5 are the plasma membrane NHEs. Of these, NHE3 and NHE5 traffic between the recycling endosomes and the plasma membrane under basal conditions, while NHEs 1, 2, 4 appear to be statically present in the plasma membrane. The intracellular NHEs include NHE6, -7, and -9. It is not known whether the intracellular NHE isoforms exist statically in specific organelles or whether individual intracellular NHEs exist in multiple organelles (328, 342, 505). NHE8 resembles the intracellular NHEs based on amino acid composition but appears to be present primarily on the plasma membrane, being found in the brush border of the renal proximal tubule and small intestine (20, 181, 182, 513). The driving force for the NHEs on the plasma membrane is the Na⁺ gradient generated by Na⁺-K⁺-ATPase (123). For resident organellar NHEs, which appear to generally exchange cytosolic K⁺ (perhaps also Na⁺) for intraorganellar H⁺, the driving force is primarily the H⁺ gradient generated by the organellar V-ATPase (61, 465). These NHEs transport H⁺ out of the compartment in exchange for cytosolic K⁺ or Na⁺ and thus help set the organellar pH (61, 465). NHE3 and NHE5 also appear to function on intracellular organelles (8, 134, 163, 434). In a subset of early endosomes, extracellular Na⁺ appears to provide the driving force for acidification (162–165). All plasma membrane members of the NHE gene family transport Na⁺ and H⁺ with a stoichiometry of 1:1 (20, 21, 476). The relative affinity for transport of Na⁺ versus K⁺ by the organellar NHEs in exchange for intraorganellar H⁺ is not known (60, 61). Please note, a distantly related sperm transporter is not considered a member of the SLC26A family. Also, a proposed colonic Cl^–-dependent NHE isoform (384, 385, 400) is believed to be a cloning artifact (403), and rather, the suggestive physiological studies are suspected of representing the now recognized Cl^– dependence of multiple NHE isoforms (4).

NHE3 is present in the brush border (BB) of small intestine (duodenum, jejunum, ileum), colon, gallbladder, parietal cells of some species but not others, cholangiocytes, some pancreatic duct cells, proximal tubule of the kidney, thick ascending limb of Henle and proximal portion of the long descending thin limb of Henle, a few neurons in the cerebellum and respiratory neurons of the ventrolateral medulla (where it is suggested as being involved in respiratory rhythm generation, neuronal chemosensitivity, and the central CO₂ respiratory response), in perineuronal cells (not nerves) of the laryngeal nerve, chondrocytes, parotid duct, and epidydimus duct (1–3, 20, 24, 25, 30, 47, 48, 56, 58, 60, 62, 64, 98, 99, 101, 138, 154, 159, 200, 207, 210, 226, 236, 254, 255, 259, 282, 291, 295, 313, 326, 345, 365, 375, 378, 383, 389, 394, 395, 414, 424, 438, 508, 517). In addition, NHE3 is present in gills of fish (86, 141, 509) and is involved in mouse blastocyst formation (237).

The major recognized functions of NHE3 are in the apical membrane of the proximal tubule and small intestine and colon (20, 121, 160, 403, 452, 511, 537). NHE3 is responsible for the majority of NaCl absorption in the intestine and kidney, acting predominantly in their proximal portions (explaining proximal tubule and small intestinal linked Na⁺ and Cl^– absorption). In addition, it accounts for the majority of HCO₃^– absorption in the kidney since the amount of NHE3 appears to exceed the amount of apical anion exchanger, with the residual Na⁺/H⁺ exchange activity being equivalent to HCO₃^– absorption (89, 121, 123, 184, 313, 314, 330, 512, 537). At least in the gastrointestinal (GI) tract and kidney, NHE3 is functionally linked to a BB Cl^–/HCO₃^– exchanger and takes part in neutral NaCl absorption (123, 259, 260, 275). In this process one molecule of Na⁺ and one molecule of Cl^– are absorbed together with no net movement of charge. This process depends on intracellular carbonic anhydrase to generate the H⁺ and HCO₃^– (123). The Cl^–/HCO₃^– exchanger involved probably varies among intestinal segments and appears to include PAT1 (putative anion transporter or SLC26A6) or DRA (downregulated in adenoma, SLC26A3) (20, 35, 159, 223, 268, 275, 406, 482, 483). The current suggestion is that PAT1 may dominate in Na⁺ absorptive cells in the duodenum and in jejunum, while DRA is involved in ileum and colon, although this appears to be species dependent. For instance, in some species, DRA is the duodenal villus apical anion exchanger. In addition, some tissues and even some cells may have both DRA and PAT1; for instance, in mouse duodenum, DRA is in crypt cells and PAT1 is in the villus cells (415). In the kidney, the involved anion exchanger also appears to be PAT1, although SLC26A7, another anion exchanger, is also reported to be in the proximal tubule BB apical membrane.

The neutral NaCl absorptive process is highly regulated (121, 123, 537). It is both rapidly (over minutes) stimulated and/or inhibited as part of digestion in the GI tract and in response to neurohormal stimulation in both GI tract and kidney proximal tubule. NHE3 is the part of neutral NaCl absorption that has been shown to be directly regulated, although similar detailed studies have not been reported for the BB Cl^–/HCO₃^– exchanger. In fact, in preliminary studies, the apical membrane anion exchange may be inhibited by protein kinase C (397, 398). In colon, in addition to the NHE3/PAT1 or DRA-linked NaCl absorption, there is an additional neutral Na⁺-linked anion absorptive process in which NHE3 or NHE2 is linked to a short-chain fatty acid/anion exchanger (Fig. 1). (176, 267, 466, 467). A congenital diarrheal disease due to lack of BB NHE activity (almost certainly NHE3) occurs (55, 238, 336). It is not due to a structural or localization abnormality in NHE3.

View larger version (21K): [in this window] [in a new window]   FIG. 1. Two forms of neutral Na+ anion absorption in mammalian colon. The neutral NaCl absorptive processes involve NHE3 linked to either SLC26A3 or -6 based on cells involved (A) and the short-chain fatty acid (SCFA)/anion exchanger linked to either NHE2 or NHE3, with NHE2-SCFA uptake active even in the presence of cAMP, which inhibits NHE3 (B).

Short-term regulation of NHE3 mimics digestive and renal physiology or regulation by changes in the dietary or neurohumoral environment of the intestine and kidney and also models pathological states, including diarrhea (13, 121, 123, 330, 537). The mechanisms involved in short-term NHE regulation include changes in plasma membrane turnover number, changes in amount of NHE3 expressed on the plasma membrane by altering rates of endocytosis and/or exocytosis, and changes in NHE3 half-life (50, 70, 97, 121, 130, 212, 226, 330, 358, 537). We have found that NHE3 simultaneously exists in multiple large multiprotein complexes particularly on the plasma membrane under basal conditions and that the NHE3 complexes change as part of some NHE3 regulation in terms of complex size and associating proteins (8, 71, 296, 298, 340). There appears to be coordinated regulation of NHE3 and anion secretion in murine jejunum, which would allow coordinated movement of intestinal Na⁺, Cl^–, HCO₃^–, and water transport (158, 159).

This review examines the current understanding of the mechanisms of NHE3 regulation, the identified NHE3 complexes, and the proteins which directly and/or indirectly interact with NHE3, with an emphasis on the NHE3 binding partners and their role in acute NHE3 regulation.

	II. MECHANISMS OF ACUTE REGULATION OF NHE3

Top Previous Next References

 
NHE3 is one of the most regulated transport proteins. It is both rapidly stimulated and inhibited as part of normal digestive physiology, and it contributes to multiple pathophysiological states when it is downregulated for a prolonged period (121, 130, 330, 537). Rapid regulation of NHE3 occurs over minutes in the intestine as part of digestion either in response to change in luminal end products of digestion (at least, generation of D-glucose and short-chain fatty acids) or to changes in the neurohumoral mileu of the intestine. Changes in NHE3 activity in the kidney occur with a somewhat slower time frame (many minutes to a few hours) in response to drugs, dietary changes (especially change in Na or K intake), or changes in blood pressure/volume (13, 321). While the agonists that alter NHE3 in the GI tract and kidney are somewhat different, the response of NHE3 to changes in second messengers is the same, i.e., NHE3 inhibition induced by parathyroid hormone (PTH) may occur over many minutes in the kidney proximal tubule via activation of the adenylate cyclase/cAMP/protein kinase A (PKA) II plus protein kinase C (PKC) systems, while similar effects on NHE3 activity occur in the small intestine over a few minutes in response to elevation of vasoactive intestinal polypeptide (VIP), secretin, or cholingergic agonists and accompanying changes in cAMP or Ca²⁺.

Transport kinetics for NHEs, including NHE3, include Michaelis-Menton kinetics for extracellular Na⁺ uptake (kinetics of K⁺ transport by intracellular organellar NHEs has not been defined as yet), with 1:1 exchange of extracellular Na⁺ for H⁺ (20, 21, 252, 358, 449, 452). However, intracellular H⁺ export is more complex, with H⁺ both being transported and activating the exchanger allosterically through the so-called H⁺ modifier site (21, 198, 422, 471, 472, 476). This leads to the K'(H⁺)_i being a complex term, with a Hill coefficient of 2 (21). Regulation of NHE3 generally involves changes in NHE3 V_max and often, but not always, changes in K'(H⁺)_i (121, 357, 537). However, usually this is not associated with changes in the NHE3 extracellular. Also, occasionally there are changes in the NHE3 set point (73). Two basic regulatory mechanisms are involved in short-term NHE3 regulation: 1) changes in turnover number with no changes in trafficking; and 2) changes in trafficking (121, 330, 537). The latter involves changes in regulated exocytosis and/or endocytosis, which can include effects in both by the same agonist.

Some of the agonists that rapidly regulate NHE3 in the intestine or renal proximal tubule are listed in Table 1. Categories of ligands include neurohumoral substances produced in the GI tract or systemically, neural mediators most made in the GI tract, kinase regulators, phosphatase inhibitors, changes in osmolarity, and end products of digestion.

View larger version (28K): [in this window] [in a new window]   FIG. 2. Putative signaling pathway by which luminal D-glucose acts via SGLT1 to stimulate NHE3 by increasing its exocytosis. RE, recycling endosomes. [Adapted from work of Turner and co-workers (213, 410, 458–460, 542).]

As examples of the rapid neurohumoral ligand regulation of NHE3 (Table 1), epidermal growth factor and 2-adrenergic agonists stimulate ileal Na⁺ absorption, and lysophosphatidic acid increases NHE3 in OK renal proximal tubule cell line. Stimulation of renal proximal tubule BB NHE3 also occurs with endothelin-1, angiotensin, and exposure to acidosis. Most of these agonists stimulate NHE3 exocytosis without changing the rate of endocytosis. These agonists act to increase BB NHE3 amount, with comparable increases in NHE3 activity and percent of NHE3 on the apical membrane (285, 295).

cAMP, cGMP, and elevated intracellular Ca²⁺ and the neurohumoral substances and bacterial toxins that cause these second messengers to increase all inhibit neutral NaCl absorption and its component BB NHE3 (121, 197, 330, 537). This occurs in both the GI tract and renal proximal tubule. The regulated mechanisms vary widely. At one extreme, cAMP inhibits NHE3 in PS120 fibroblasts by changing the NHE3 turnover number without affecting the amount of plasma membrane NHE3 (130, 277). In contrast, in OK cells, cAMP initially decreases the turnover number, but in 30 min, it is associated with an increase in rates of endocytosis, followed by inhibition of exocytosis (60 min) and a decrease in total NHE3 half-life (506, 543). This demonstrates highly coordinated NHE3 regulation in response to a single second messenger.

A. NHE3 Phosphorylation

NHE3 is phosphorylated under basal conditions (525), and that phosphorylation is often affected as part of acute NHE3 regulation (97, 115, 212, 271, 330, 331, 372, 453, 507, 547). Changes in NHE3 phosphorylation are necessary for some acute regulation of NHE3 but apparently not for others. There are examples of NHE3 regulation by both changes in trafficking and not involving trafficking that are dependent on changes in NHE3 phosphorylation. This basic regulatory mechanism is partially understood with identification of some phosphorylation sites of NHE3, some roles of phosphatases in NHE3 regulation, the role of kinase localization to specific subcellular domains, and involvement of NHE3 complexes all being partially defined. In contrast, further NHE3 phosphorylation sites, additional roles of phosphatases, and restricted spatial and temporal phosphorylation of NHE3 as part of regulation are all predicted, and cAMP has even been shown to act by a non-PKA-dependent mechanism that inhibits NHE3 in some cells without causing a change in NHE3 phosphorylation (EPAC, exchange protein directly activated by cAMP) (209). This area was reviewed with great insight by Moe (330), whose laboratory has provided many of the studies in this area.

NHE3 is phosphorylated under basal conditions in several cell culture models as well as in intact tissue. These include fibroblasts, OK proximal tubule, and Caco-2 cells as well as in renal proximal tubule and ileal BB. The great majority of basal and stimulated phosphorylation is on Ser with minor phosphorylation of Thr (212, 232, 330, 331, 372, 507, 525, 547). Tyr phosphorylation of NHE3 has not been identified. Of interest is that at least one Tyr residue (Y323 of rabbit NHE3) is predicted to be phosphorylated with a 95% probability by Netphos2.0. The phosphorylated sites have only been partially defined, and use of mass spectroscopic approaches has revealed previously unidentified phosphorylated residues of NHE3 (X. Li, A. Pandey, and M. Donowitz, unpublished data). The role of basal phosphorylation is unclear since mutagenesis which eliminated up to six putative PKC phosphorylation sites and sites phosphorylated by cAMP and other putative Ser phosphorylation sites did not alter basal NHE3 activity (507). This led to the conclusion that some basal phosphorylation does not have a major role in basal NHE3 activty. In contrast, the protein phosphatase inhibitor okadaic acid acutely stimulated NHE3 activity, demonstrating that at least some kinase(s) stimulate NHE3 activity under what is considered to be basal conditions (292).

In analyzing the role of stimulated phosphorylation of NHE3, Moe (330) appropriately pointed out that most rapid stimulation and inhibition of NHE3 are coupled to protein kinases, and pharmacological inhibitors of kinases prevent many examples of NHE3 regulation. He categorized the questions related to the role of phosphorylation of NHE3 in its regulation into 1) Is the site phosphorylated in vivo? 2) Is phosphorylation of a site of NHE3 necessary for a regulatory function? 3) Is phosphorylation of a site of NHE3 sufficient for a regulatory function? The overview, not surprisingly, given that complexes and associated regulatory proteins are known to be involved in NHE3 regulation and that the endocytic machinery itself is known to be regulated by phosphorylation, is that while phosphorylation of NHE3 appears to be necessary for some but not all its acute regulation, NHE3 phosphorylation may not be sufficient for either stimulation or inhibition (330, 507, 525). Interestingly, Netphos 2.0 software predicted 19 COOH-terminal Ser/Thr phosphorylation sites of rabbit NHE3 with probabilities of phosphorylation of >92%. All currently known phosphorylated residues of NHE3 are among the predicted list. This suggests that the role of Ser/Thr phosphorylation of NHE3 is only partially defined.

1. Evidence that NHE3 is phosphorylated as part of its acute regulation

A) CAMP.  In vitro, purified cAMP-dependent protein kinases phosphorylate NHE3 in the COOH terminus. More importantly, in intact cells exposed to elevated cAMP, immunoprecipitated NHE3 showed increased phosphorylation by specific phosphopeptide mapping, direct tandem mass spectroscopic analysis, and anti-phospho antibodies to specific NHE3 amino acids (97, 212, 264, 271, 330, 506, 525, 547). Moreover, a back-phosphorylation approach showed that PKA elevation in vivo led to occupation of PKA sites in NHE3 and inhibited subsequent in vitro phosphorylation (497, 500). The exact amino acid residues that are phosphorylated by cAMP elevation remain somewhat controversial. The most definitive studies showed that cAMP caused concentration-dependent in vivo phosphorylation of rat NHE3 Ser⁶⁰⁵ and Ser⁵⁵², which are in PKA consensus sequences (330, 543). Both were necessary for cAMP to maximally inhibit NHE3, since mutation of these Ser individually decreasing and both together preventing NHE3 inhibition by cAMP. In contrast, Kurashima et al. (271) showed that NHE3 truncated to amino acid (aa) 585 lost all cAMP inhibition and that Ser⁶⁰⁵ and Ser⁶³⁴ of rat NHE3 were necessary for NHE3 inhibition by cAMP (271). They showed that the Ser⁶⁰⁵ was phosphorylated in the presence of PKA. However, although Ser⁶³⁴ was not phosphorylated (either basal or with cAMP), it was also necessary for cAMP to fully inhibit NHE3. Each of these Ser contributed 50% to cAMP inhibition of NHE3, with mutation of both totally preventing cAMP-induced changes in NHE3 activity in fibroblasts. In these studies, Ser⁵⁵² was not phosphorylated. The reason for the discrepancy is unclear, although phosphopeptide mapping demonstrates that NHE3 that is immunoprecipitated from cells with elevated cAMP has changes in multiple phosphopeptides, suggesting changes in phosphorylation at multiple sites (272, 525, 547). It is of note that mutation of Ser from other putative PKA phosphorylation sites in the NHE3 COOH terminus did not alter cAMP effects on NHE3 activity (Ser-330, -514, -576, -662, -691, -692, -805). It remains to be determined whether phosphorylation of the Ser residues occurs as part of acute NHE3 regulation by mechanisms that do not involve changes in cAMP. As described in more detail below, cAMP-induced phosphorylation of NHE3 is necessary for all functional effects of cAMP on NHE3 described. Moreover, in OK renal proximal tubule cells, elevating cAMP with dopamine inhibited NHE3 over the same time and concentration range that increased NHE3 phosphorylation (212).

B) CGMP.  cGMP kinase II (cGKII) is a BB protein that is involved in acute NHE3 inhibition in response to the luminally released gut humoral substances guanylin and uroguanlyin. Guanylin is released as part of digestion. Heat-stable Escherichia coli enterotoxin is similar structurally to guanylin/uroguanylin and binds to the same BB receptor, guanylate cyclase C. It increases the cGMP content in the cell at the apical domain and activates cGKII. This activation leads to acute inhibition of NHE3 by a process in which NHE3 is phosphorylated (B. Y. Cha, H. Kochinsky, P. Aronson, H. de Jonge, and M. Donowitz, unpublished data).

C) ELEVATED CALCIUM.  Elevated Ca²⁺ inhibits NHE3 by a PKC--dependent process (250, 286). PKC- becomes part of the NHE3 complexes with elevated Ca²⁺ in both PS120 cells and ileal Na⁺ absorptive cell BB (286, 298). However, PKC phosphorylation of NHE3 could not be demonstrated in PS120 cells or in ileal BB, and mutagenesis studies of NHE3 suggest that PKC phosphorylation of NHE3 does not correlate with NHE3 regulation (507, 525). Wiederkehr et al. (507) showed that purified PKC phosphorylated similar phosphopeptides in the NHE3 COOH terminus in vitro that occurred with phorbol ester exposure to intact cells in vivo. However, Yip et al. (526) did not see consistent changes caused by phorbol ester exposure on NHE3 phosphopeptides from NHE3 transfected PS120 cells [although in retrospect this may not have occurred due to lack of expression of sufficient Na⁺/H⁺ exchanger regulatory factor (NHERF) proteins in these cells; see below]. In detailed studies using AP-1 cells, mutation of six Ser in the NHE3 COOH terminus which were in putative PKC consenses sequences prevented phorbol ester regulation of NHE3 in vivo (Ser-13, -552, -575, -661, -690, -804) (507). However, PKC has been associated with NHE3 stimulation, inhibition, or no effect depending on the cell model studied. Also, Wiederkehr and Moe and co-workers (507) importantly showed that when NHE3 was expressed in AP-1 cells and individual clones selected, phorbol esters stimulated, inhibited, or had no effect on NHE3 activity depending on the specific clone studied. In spite of this, phorbol esters stimulated NHE3 phosphorylation with identical phosphopeptide maps, and this was regardless of whether phorbol esters stimulated, inhibited, or had no effect on NHE3 activity (507). Thus identical changes in NHE3 phosphorylation were associated with the full spectrum of changes in NHE3 activity. Moreover, in preliminary studies of effects of elevated Ca²⁺ on NHE3 regulation in PS120 cells and OK cells in which NHERF2 complexes are required for inhibition, we could not demonstrate changes in phosphorylation of IP NHE3. Thus the conclusion for Ca²⁺/PKC inhibition of NHE3 is that while PKC (PKC-) is involved and joins the NHE3 complexes, the PKC phosphorylated substrate does not appear to be NHE3, although at least one of the Ser that can be phosphorylated by PKC is necessary for the inhibition of NHE3.

That NHE3 phosphorylation is necessary for some acute regulation but not others eliminates the possibility that phosphorylation is sufficient for all NHE3 regulation. Even for cAMP inhibition, phosphorylation is not sufficient. For instance, in OK cells, dopamine elevated cAMP content acting both by DA-1 and DA-2 receptors, with the effects on NHE3 being synergistic. DA-1 inhibited NHE3 activity, while DA-2 itself had no effect on NHE3 activity but DA-1 plus DA-2 caused a greater effect than DA-1 alone. In spite of this, DA-1 and DA-2 individually increased phosphorylation of NHE3 on the same sites as when they were studied together (212, 330). These findings, plus the failure of PKC phosphorylation to consistently affect NHE3 activity (507), demonstrate that phosphorylation of NHE3 is not sufficient to explain NHE3 regulation. In addition, the quantitative role of NHE3 phosphorylation in NHE3 regulation needs further examination.

In spite of the correlation of phosphorylation of specific amino acids of NHE3 as part of its regulation with changes in turnover number, for instance, with cAMP inhibition, the series of molecular events that lead to the regulation downstream from the changes in phosphorylation are not understood. The changes in NHE3 trafficking are partially understood at the level of changes in associating proteins and changes in associating with some parts of the trafficking machinery, although again molecular details are lacking (see below).

D) OTHER KINASES.  Several other kinases also either associate with or regulate NHE3 activity. PI 3-K, a major NHE3 regulating enzyme, is discussed below. A kinase which binds directly to NHE3 is calmodulin (CaM) kinase II (545), while other kinases which join NHE3 are part of complexes involved in regulation. These include PKA, PKG, and PKC, all of which associate with NHE3 via BB PDZ proteins of the NHERF family. Understanding how kinases regulate proteins now includes understanding how kinases are activated locally in cells with the consequence that specific signaling pathways appear to involve a few substrates, rather than all putative cell substrates. This specificity is attributed, at least partially, to activation of kinases at specific locations in the cells as part of specific receptor/signaling pathways. The location of NHE3 complexes with BB PDZ proteins and their association with kinases may partially explain this phenomenon for NHE3 at the BB. In addition, multiple other kinases are involved in acute NHE3 regulation, although apparently several steps away from NHE3. For instance, already mentioned is that D-glucose activates Caco-2 BB NHE3 by a process that involves the p38 MAP kinase, MAPAKP2, PI 3-K, and Akt2. Also, endothelin both stimulates and inhibits NHE3 in a dose-dependent manner by a process that involves the Ca²⁺-sensitive kinase PYK2 (274). Thus phosphorylation of NHE3 by kinases that associate with it via its signaling complexes or serve as intermediates in the signaling networks are necessary for some but not all aspects of acute NHE3 regulation both via changes in turnover number and by trafficking. This incompletely defined area requires much further study to define other associating kinases and phosphatases and how changes in phosphorylation leads to changes in NHE3 complex formation, trafficking, turnover number, as well as how the NHE3 signal complexes are involved in controlling NHE3 phosphorylation as well as in phosphorylation of the NHE3 regulatory machinery. NHE3 stimulation by SGK1 is described in section VB8CVI.

E) SER/THR PHOSPHATASES.  Another understudied area is the role of regulated phosphatase activity in NHE3 regulation. Protein phosphatase (PP) 2A was reported as associating with NHE3 when cAMP was elevated acutely (380). The role of this association has not been defined. However, the PP1 and PP2A inhibitor okadaic acid stimulates NHE3 activity (292). This suggests that basal phosphatase activity inhibits NHE3 activity, although it was not established whether the phosphatase involved physically associated with NHE3 or whether it was changes in NHE3 phosphorylation or in NHE3 regulatory proteins that were responsible for changes in NHE3 activity. Given the large number of kinases shown to associate with NHE1, at least calls for further examination of kinase and phosphatase binding partners of NHE3, both directly and as part of its signaling complexes.

In addition, not yet explored is the role of localization of NHE3 into lipid rafts (LR) in the BB in determining the state of NHE3 phosphorylation under basal or stimulated conditions. Some NHE3 is in LR and is involved in basal cycling and endocytosis and growth factor stimulation of NHE3 (295, 339). While NHERF1 and NHERF2 in the BB do not appear to be LR associated (295), the state of the other NHERF family members is not known, nor is it known whether the kinases involved with NHE3 regulation are in apical membrane LR.

B. Acute Regulation of NHE3 Can Occur by Changes in Trafficking or Changes in Turnover Number

In all cells and tissues in which NHE3 has been studied, NHE3 stimulation and inhibition are at least partially due to changes in trafficking, with separate endocytic and exocytic processes that can be regulated independently (88, 97, 121, 226, 270, 537). In many cases there is also regulation by changes in NHE3 turnover number, which in some cases occurs faster than changes in rates of trafficking (121, 537).

In evaluating mechanisms of regulation of NHE3, the model used determines some of the results. Of the models used, changes in turnover number as well as regulated endocytosis and exocytosis have been demonstrated in the renal proximal tubule cell line, OK cells, the intestinal Na⁺ absorptive cell line, Caco-2 cells, intact small intestine, and NHE3 null fibroblasts transfected with NHE3. In all cells/tissues, NHE3 exists both on the plasma membrane and in intracellular vesicles, which are probably endosomes, under basal conditions.

The tissue which NHE3 seems to be differently regulated from others is intact renal proximal tubule (45, 288–290, 321, 322, 519, 521, 522, 526, 539–541). This in spite of the fact there is an intracellular pool of NHE3, similar to other cells, which is probably endosomal, as has been demonstrated by electron microscopy (47, 48). In intact proximal tubule, NHE3 seems to cycle predominantly between microvilli and intervillus areas. In the apical membrane of proximal tubule cells, Biemesderfer et al. (45) showed that NHE3 exists in two pools, thought to be in the microvilli and in the intervillus spaces (45). In the former, NHE3 is in lighter complexes (9. 2s), does not associate with megalin, and appears to be active. In contrast, in the latter, NHE3 is in heavier complexes (18s), associates with megalin, and appears to be less active or inactive. As part of signaling, NHE3 in proximal tubule partially moves from a lighter distribution on sucrose density gradients that includes apical membrane markers, to heavier fractions which have more markers of the intervillus compartment but have some overlap with both BB and intracellular endosomal markers. In these density gradient studies initiated by Mircheff and pursued by McDonough, NHE3 was shown to be present in three fractions. The debate is what they represent and how distinct they are: alkaline phosphatase is enriched in the microvillus fraction, galactosyltransferases are enriched in microvillus clefts, and acid phosphatase is enriched in endosomes. As an example, with PTH treatment, 20% of total NHE3 shifts from low-density membranes enriched in microvillar markers (541) to mid-density membranes enriched in intermicrovillar cleft markers and later to heavier density membrane with some endosomal markers. Models used to evaluate changes in NHE3 trafficking in proximal tubule have included acute hypertension-induced natriuresis, PTH, and an angiotensin-converting enzyme inhibitor, all of which caused rapid and reversible inhibition of NHE3 activity and redistribution of NHE3 in the sucrose gradients from lighter to heavier density fractions interpreted as away from BB to intervillus clefts. McDonough and co-workers (519, 526, 539, 540) showed that 20 min of acute hypertension led to redistribution of NHE3 from BB to the base of the BB, while a control protein, NaPi2a, internalized into endosomes. However, how much of each fraction is BB versus microvillus clefts versus endosomes was not resolved.

Of importance, the Biemesderfer studies characterized the Triton X-100 soluble pools of NHE3 (45, 46). Only 50% of total proximal tubule NHE3 was Triton X-100 soluble. Thus this model does not consider 50% of BB NHE3. They also demonstrated that anti-NHE3 antibodies did not recognize the entire NHE3 pool and showed that at least part of what was not recognized was bound to megalin (46). The role of megalin specifically in NHE3 regulation has not been resolved (see below).

In ileal Na⁺ absorptive cells, epidermal growth factor (EGF) and clonidine stimulated active NaCl absorption and BB NHE3 activity, which correlated with the increase in BB NHE3 amount (130, 246, 295). This stimulation was LR dependent, being inhibited by disrupting LR with methyl--cyclodextrin (MCD) (295, 340). Basal endocytosis was also LR dependent with the amount of NHE3 coimmunopurified with EEA1 containing vesicles decreasing with MCD and with cytoskeleton disruption with cytochalasin D (295, 340). Thus, in ileum, unless antibodies are blocked from recognizing large populations of NHE3, the addition of NHE3 to the BB as part of rapid stimulation is not associated with shifts within the BB of NHE3 pools characterized by differences in detergent solubility. Whether Biemesderfer's and McDonough's careful studies in proximal tubule and our ileal studies of the EGF and clonidine induced increases in BB NHE3 are describing comparable pools of NHE3 is not known. However, the current view is that ileal NHE3 but not renal proximal tubule NHE3 traffics between endosomes and BB. It is also not known whether ileal NHE3 exists in a large intermicrovillar membrane pool, as occurs in proximal tubule. In addition, since a significant portion of renal NHE3 is Triton X-100 insoluble, the role of LR in renal NHE3 trafficking should be determined.

Oppositely, carbachol rapidly decreased the amount of NHE3 in the BB and increased the amount coimmunopurified with EEA1, suggesting regulation by trafficking (298). However, whether the regulated process is endocytosis, exocytosis, or both in intact tissue is still not established. Also, in rabbit ileum, the amount of NHE3 in BB increases over minutes with EGF and clonidine and decreases with carbachol (295, 298). This supports trafficking of NHE3 as a mechanism involved with acute NHE3 regulation. This is specific for ileum, and mechanisms in the ileum and proximal tubule could be separate. A portion of ileal Na⁺ absorptive cell NHE3 is in the same fractions of sucrose density or OptiPrep gradients as EEA1 (30%), and some NHE3 can be coimmunopurified with endosomal vesicles by using EEA1 antibody coupled to beads (295). The amount of NHE3 copurified with EEA1 vesicles based on immunopurification and colocalized with EEA1 based on sucrose density gradient was reduced by cytochalasin D exposure. These results strongly indicate that there is an early endosomal pool of NHE3.

In epithelial cell culture models, basal NHE3 activity is regulated by a balance of basal endocytosis and exocytosis. A major difference that has been recognized among cell and tissue models of NHE3 is the percent of NHE3 on the plasma membrane under basal conditions. This includes 15% in fibroblasts (PS120 and AP-1 cells), 15–50% in OK cells based on the application of both cell surface biotinylation and confocal microscopy, 80% in Caco-2 cells measured both by cell surface biotinylation and confocal microscopy, 80% in renal proximal tubule, and 80% in rabbit ileal BB (7, 8, 116, 197, 371, 523). In OK cells, NHE3 is in BB and an intracellular pool that colocalizes with Rab 11 (8). In BB, the amount of NHE3 in the central portion of BB over the recycling compartment may be increased compared with in other parts of the apical membrane (8). Based on fluorescence recovery after photobleaching (FRAP) and use of cross-linkers, it appears that basal trafficking is preferentially to this area of the BB where NHE3 density is increased (71). This suggests the presence of a targeting signal for NHE3 in this localized domain in the BB.

Acute regulation of NHE3 in OK cells initially involves changes in turnover number or amount based on the stimulus followed by changes in trafficking. Endothelin and metabolic acidosis increased the surface amount of NHE3 and in parallel increased NHE3 activity (371, 523). PTH inhibited NHE3 acting by cAMP. At 30 min NHE3 activity was decreased, but surface amount of NHE3 was not affected. Amount of BB NHE3 was reduced 4–12 h after PTH by a mechanism that involves increased endocytosis and no change in exocytosis (97). This process was dynamin dependent, being inhibited by a dominant negative dynamin mutant. Increased endocytosis of NHE3 was associated with increased association of NHE3 with clathrin and AP-2. How this time-dependent regulation of NHE3 occurs is postulated to be either initial inhibition of all the affected NHE3 molecules followed by their removal from the BB versus initial inactivation of NHE3 with subsequent removal of only some of the BB NHE3 and reactivation of the remaining. Unknown also is whether NHE3 is active or not at the site of removal and intracellularly. There is evidence that some of the recycling NHE3 is functionally active (8, 134, 481), but it is not clear if this represents the pool removed from the BB. Thus, in cell culture models, both changes in turnover number and trafficking on and off the BB has been shown to contribute to NHE3 regulation in multiple cell types from different tissues, using different ligands, and by different techniques.

A myriad of signaling molecules are linked to plasma membrane receptors, the activation of which affect NHE3 activity. In addition, however, there are signaling molecules that are involved with NHE3 regulation at or close to the apical membrane. Most clearly identified as having a role in basal NHE3 activity is PI 3-K (78, 134, 136, 178, 224, 246, 270, 284, 295, 296). In all cells and most tissue models of NHE3, basal NHE3 activity is under control of PI 3-K, with PI 3-K inhibitors decreasing basal transport rate and percent of NHE3 on the plasma membrane. In rabbit ileum, wortmannin did not alter basal NaCl absorption, which must be confirmed as this is the only example of PI 3-K not being involved in basal regulation of NHE3 (246). In contrast, in NHE null fibroblasts in which NHE3 is expressed (PS120 and AP-1), Caco-2 cells, and OK cells, wortmannin inhibits basal NHE3 activity by 50% (8, 130, 224, 270). PI 3-K is also involved in stimulated NHE3 activity. Constitutive activation of PI 3-K or Akt stimulates NHE3 activity and increases plasma membrane amount in fibroblasts and OK cells, while growth factor stimulation of NHE3 in fibroblasts is blocked by 50% by PI 3-K inhibitors (224, 284), and lysophosphatidic acid (LPA) stimulation of NHE3 in OK cells is 100% blocked by wortmannin (285).

Comparison of NHE3 trafficking mechanisms should be made with GLUT4, the best studied transporter that trafficks to the plasma membrane, which occurs through insulin-stimulated exocytosis (80, 222, 233, 420, 461, 486, 487, 544). The GLUT4 studies are reviewed to serve as background to consider the various pathways and regulatory proteins known to be involved in regulation of trafficking of a transport protein, although it must be pointed out that NHE3 trafficking between the plasma membrane and intracellular pools is much less dramatic than regulated GLUT4 trafficking. A minimal amount of GLUT-4 is on the plasma membrane under basal conditions, with minimal basal exocytosis from the recycling compartment. In contrast, after insulin stimulation, GLUT-4 traffics via both a storage vesicular pool and increased exocytosis from the recycling compartment. In NHE3, Alexander and Grinstein (11) performed a detailed kinetic analysis of NHE3 expressed exogenously in the distal renal tubule cell line MDCK (11). They identified four pools of NHE3: 1) a mobile apical membrane population, the trafficking of which could be rapidly stimulated; 2) an immobile apical membrane pool that binds the cytoskeleton by interaction with Rho GTPases; and 3 and 4) two intracellular pools with a fast and less rapid exchange rate with the apical membrane. The relevance of this distal tubule model to normal physiology of the proximal tubule is unknown. Also unknown is how similar is the mechanism of regulation of exocytosis of NHE3 to the GLUT-4 model, which predicts a storage pool that as yet is not well characterized biochemically and the recycling endosomes as distinct, although perhaps interacting pools.

	III. INTRACELLULAR NHE3 ACTIVITY

Top Previous Next References

 
Does intracellular NHE3 transport Na⁺ and H⁺? Our interpretation is that the answer is yes, although intracellular NHE3 function and its regulation have not been studied in detail. Some of the reasons to suspect that this pool of NHE3 is functional include the following. 1) In several cell culture models, inhibition of NHE3 alkalinizes an intracellular compartment thought to be early endosomes (8, 134, 163, 164). This indicates NHE3 normally acidifies this compartment. 2) In proximal tubule of wild-type mice, NHE3 inhibition by cAMP increased early endosomal pH within 1 min, as marked by FITC-BSA, implying cAMP inhibits both BB and intracellular NHE3 activity and demonstrates that intracellular NHE3 is active. Note this effect is too fast to be explained by changes in trafficking (164). 3) In NHE3 knock-out mouse, there is reduced renal proximal tubule albumin uptake (165). Whether this involves intracellular NHE3 is not known. 4) In proximal tubule of ClC-5 knock-out mouse, NHE3 has primarily an intracellular location (373), which has been assumed to be in some pool of endosomes and does not resemble the distribution of Golgi. This intracellular pool is still able to allow albumin endocytosis, which is strong evidence that intracellular NHE3 functions.

	IV. NHE3 COOH-TERMINAL REGULATORY DOMAIN AND NHE3 COMPLEXES

Top Previous Next References

 
The NHE3 COOH terminus is necessary for all identified examples of acute NHE3 regulation (293, 534). This generally has been examined by truncation or point mutants of the NHE3 COOH terminus. Truncation of the NHE3 COOH terminus alters basal as well as regulated NHE activity. Truncation to aa 756 increases NHE3 activity twofold, while truncation to aa 690 increases it to sixfold of basal (7). This is accompanied by similar increases in the amount of NHE3 on the plasma membrane in PS120 fibroblasts (2 x for 756, 6 x for 690) with the transport per plasma membrane exchanger remaining constant. Further truncating NHE3 to aa 585 did not further alter the percent of NHE3 on the plasma membrane or the activity per plasma membrane exchanger, although total expression was diminished. This suggested that there are two endocytosis signals in the NHE3 COOH terminus, one between aa 756–832 and the other dominant signal between aa 690 and 756. Additional truncation of NHE3 to aa 509, 475, and 455 decreased NHE3 activity, with NHE3 445 activity minimally detectable in the absence of stimulation with serum. As shown in Figure 3, study of truncation mutants demonstrated that the multiple acute regulators of NHE3 activity require specific domains of the COOH terminus to act. The sole exception identified in any mammalian NHE is the domain required for NHE response to hyperosmolarity. While not studied in NHE3, the effect of hyperosmolarity to stimulate NHE1 and inhibit NHE2 was shown to involve the first extracellular loop between aa 41–53 of NHE1 (aa 31–43 of NHE2) (422). This is an area of great divergence among the NHEs. This domain is believed to represent the first extracellular loop for NHE1, which is not believed to contain a cleavable signal peptide, while we found that NHE3 did have a cleavable signal peptide, thus making the initial expressed part of NHE3 extracellular (475, 546). Similar results were found for Nhx1, the yeast intracellular NHE (505). Wakabayashi and co-workers also reported similar suggestive evidence for a cleaved signal peptide in NHE6, although his other evidence does not support this view (328, 475). This hyperosmolar response of NHE1/NHE2 is the only NHE regulation that does not depend entirely on the intracellular COOH-terminal domain (422). Not only is the NHE3 COOH terminus necessary for rapid regulation of NHE3, but it defines the nature of the regulation. Swapping the COOH terminus of NHE3 with that of NHE1 or NHE2 led to regulation consistent with the NHE providing the COOH terminus (534).

View larger version (35K): [in this window] [in a new window]   FIG. 3. A: NHE3 organization including COOH-terminal domains involved in acute regulation and binding partners. The NHE3 NH2 and COOH termini are illustrated. Shown above the COOH terminus in blue are sites of action of regulatory stimuli defined by the truncations which abolish the specific regulatory effects. The direction of the arrow indicates acute stimulation () or inhibition () of NHE3 activity. Below the COOH terminus in yellow are listed associating proteins and the areas of their binding sites on NHE3. B: classification of NHE3 COOH terminus into 4 domains for discussion of binding partners.

The NHE3 (rabbit) COOH terminus is predicted to begin at aa 455 based on the assumption of similarity in structure to the crystallized bacterial Na⁺/H⁺ exchanger, NhaA, which lacks any significant intracellular COOH-terminal domain (216). The NHE3 COOH terminus from aa 455–832 (378 aa) almost certainly has tertiary structure, although this has not been determined using physical techniques. Programs that predict secondary structure indicate that the first 130 aa after the transport domain is predominantly -helical (74). In contrast, the NHE1 COOH terminus between aa 503–545 was crystallized (cocrystallized with its binding partner CHP2) and shown to be -helical as was predicted from secondary structure predicting programs (18, 40).

The NHE COOH terminus has multiple functional subdomains. Initial hints of the existence of functional subdomains in the COOH terminus come from COOH-terminal truncation studies of NHE1. Based on the intracellular pH (pH_i) sensitivity and changes in acute NHE1 regulation, Ideka et al. (219) divided the NHE1 COOH terminus into four functional domains, and these are discussed on the assumption that all NHEs probably have some similarities in the organization of their intrinsic regulatory mechanisms (219). These four domains of NHE1 are as follows: 1) aa 515–595, 2) aa 596–635, 3) aa 636–659, and 4) aa 660–815 (219). Subdomain 1 had a pH_i maintenance function, preserving pH_i sensitivity in the physiological range, whereas subdomain 2, overlapped with the high-affinity CaM-binding site in subdomain 3, and exhibited an autoinhibitory function. Subdomains 2 and 4 had no function for pH_i sensitivity. Deletion of subdomain 1 abolished the decrease of pH_i sensitivity induced by cellular ATP depletion, suggesting that this subdomain is involved in ATP regulation of NHE1. Deletion of subdomain 3 rendered the inhibition by ATP depletion less efficiently, suggesting a possible functional interaction between subdomains 1 and 3. However, deletion of subdomain 2 partially abolished the inhibitory effect of subdomain 3. Therefore, subdomain 2 was proposed to function as a mobile "flexible loop," permitting the CaM-binding subdomain in subdomain 3 to exert its normal function. These findings further showed that the COOH-terminal domain of the NHEs plays a key role in regulating NHE activity. Fliegel and associates (294, 297) examined the structure of NHE1 aa 516–815 by circular dichroism spectroscopy and concluded that it was 35% -helix, 17% -turn, and 48% random coil. They concluded that NHE1 was largely -helical near the NH₂-terminal transport domain (aa 516–632), while aa 633–815 was largely -sheet. Moreover, elevating Ca²⁺ decreased the amount of -helix and increased the amount of -sheets in the COOH terminus.

The COOH terminal 377 aa of NHE3 are intracellular, based on accessibility to antibodies in the unpermeabilized compared with permeabilized state. A single study, using monoclonal antibodies, suggested that some COOH-terminal epitopes were accessible to extracellular antibodies, and by implication that this part of NHE3 contained extracellular epitopes (48). There has been no explanation provided for this finding. The cytosolic NHE3 COOH terminus interacts with proteins, which play essential roles in regulating NHE3 activity, trafficking, and phosphorylation. For NHE1, at least for CHP, this association is complicated and involves a structural contribution that is necessary for NHE1 to have any transport activity (see below). Several NHE3 COOH-terminal interacting proteins have been reported, and domains required for specific regulatory effects are beginning to be defined (Fig. 3B). Four subdomains of the NHE3 COOH terminus are arbitrarily defined in Figure 3. Detailed description of NHE3 associating proteins that interact with each subdomain are described later.

B. NHE3 Complexes

What are the mechanisms by which the NHE3 COOH terminus takes part in the acute regulation of NHE3? The identified mechanisms include 1) acting as a kinase substrate and undergoing changes in Ser/The phosphorylation, 2) interacting with the NH₂ terminus, and 3) interacting with other proteins or lipids to change the location or signaling molecules associating with NHE3. This section deals with NHE3 interacting proteins and putative lipid interactions. Consistent with its association with multiple binding partners, the estimated size of NHE3 on density gradient centrifugation (OptiPrep or sucrose density gradients) indicates that NHE3 rarely exists as a monomer or dimer (90 or 180 kDa), but after solubilization in 1% Triton X-100 exists simultaneously in complexes between 350 and at least 1,000 kDa (Fig. 4A) (8, 295, 296, 298, 340). This observation was made in multiple intact tissue types and cell culture models, including mouse and rabbit small intestine, mouse proximal tubule, the intestinal epithelial cell line Caco-2, the proximal tubule cell line OK, and PS120 fibroblasts. Several characteristics of these complexes have been established. 1) In multiple cell types, the size of NHE3 complexes has been compared on the apical surface, identified by cell surface biotinylation versus the total cell pool (8, 298; Li and Donowitz, unpublished data). The plasma membrane NHE3 complexes were larger than the total pool in OK cells (the majority of the total NHE3 in OK cells is intracellular with estimates from 50 to 85% of NHE3 being intracellular) (8, 196) and PS120 cells (85% of total NHE3 is intracellular). 2) These complexes are dynamic with acute regulation of NHE3 (298). Evidence that the NHE3 complexes were dynamic comes from studies in which ileum was exposed in vitro to carbachol, which inhibited NHE3 by elevating intracellular Ca²⁺ and stimulated endocytosis, and in which OK cells were exposed to LPA, which stimulated NHE3 by increasing exocytosis. BB membranes were prepared from ileal Na⁺ absorptive cells, and total membrane was prepared from the OK cells; in both cases, NHE3 shifted into larger complexes as part of acute regulation of NHE3 (see Fig. 4B for carbachol).

View larger version (24K): [in this window] [in a new window]   FIG. 4. A: NHE3 is present simultaneously in large, multiprotein complexes. Triton X-100 solubilized cells were separated on discontinuous sucrase gradients with molecular weight standards run on parallel gradients, as shown below. B: carbachol increases the size of NHE3 complexes in the ileal brush border. Ileum was exposed to carbachol (1 µM) for 10 min. Brush border was prepared and studied as in A. [From Li et al. (298), with permission from Blackwell Publishing.]

1. How many proteins are there in the NHE3 complexes?

With the assumption that the average size of an NHE3-interacting protein is 70 kDa, the number of NHE3 proteins associating with a dimer of NHE3 (NHE3 like all mammalian NHEs characterized exists as a dimer under basal conditions) at any point in time is estimated to be 3 to 10 (based on complex sizes ranging from 350 to 1,000 kDa minus the size of a dimer of NHE3 divided by 70 kDa). It is still to be determined why NHE3 complexes exhibit such a large range in size, the meaning of simultaneous isolation of many sized complexes containing NHE3, and how these complexes are regulated. Our current view is that different combinations of known and yet to be identified NHE3-interacting proteins exist simultaneously in NHE3 complexes producing different sizes of complexes. In fact, given the possibility of some complex degeneration during processing, we hypothesize that even more proteins associate with NHE3.

C. Intramolecular Interactions

Does dimerization or oligomerization of NHE3 occur in vivo and contribute to the formation of the large complexes of NHE3? Disuccinimidyl suberate (DSS) cross-linking showed that homodimerization exists for NHE1 and NHE3 (146). The NH₂-terminal transmembrane domain is sufficient for this dimerization since deletion of the 300 aa COOH terminus of NHE1 did not alter dimerization. However, the COOH terminus appears to take part in dimerization. Domain 1 of the NHE1 COOH terminus, aa 562–579, was identified as the "dimerization domain" (208). Deletion of this region disrupted disulfide cross-linking between the COOH termini and functionally markedly reduced the intracellular pH sensitivity of the exchanger, suggesting that this aspect of the dimer might be necessary for the pH-dependent regulation of NHE1 (208). Multimerization of NHE3 was also reported when NHE3 was analyzed by SDS-PAGE and Western blotting under denaturing conditions (250); however, whether this represents a true picture of in vivo complex formation is not known.

Using cross-linking reagents such as the Ca²⁺ phenanthroline or the bifunctional sulfhydryl reagent methanethiosulfonate, Wakabayashi et al. (208) suggested that NHE1 forms a dimer between the COOH-terminal domain intrinsic Cys⁷⁹⁴ and Cys⁵⁶¹. This was the first evidence of the existence of intermolecular interactions between the COOH terminus of NHE1. However, because NHE3 does not contain Cys residues corresponding to Cys⁷⁹⁴ and Cys⁵⁶¹ of NHE1, similar intramolecular interactions are not expected to occur in NHE3. If there were a Cys-mediated COOH-terminal dimerization in NHE3, the only potential residue would be the sole Cys⁵⁹⁵ in the COOH terminus of NHE3. It is conserved in NHE3 and NHE5 across mammalian species including human, rat, and rabbit. Whe