NHERF family and NHE3 regulation
1 John Hopkins University School of Medicine, Departments of Medicine & Physiology, Baltimore, MD 21205, USA
Abstract
The intestinal and renal proximal tubule brush border (BB) Na+–H+ exchanger NHE3 binds to members of the NHERF (Na+–H+ exchanger regulatory factor) family. These are four proteins (current most used names include NHERF1, NHERF2, PDZK1 and IKEPP) which are related to each other, are present in locations in or close to the BB, and scaffold a variable series of proteins in NHE3-containing complexes in a dynamic manner that is altered by changes in signal transduction which affects NHE3 activity. The specific roles of these proteins in terms of NHE3 regulation as well as interactions with each other and with their many other substrates are only now being defined. Specificity for only one member of the NHERF family in one example of NHE3 regulation, inhibition by elevation in cGMP, is used to describe how NHERF family proteins are involved in NHE3 complex formation and its regulation. In this case, NHERF2 directly binds cGKII in the brush border to form an NHE3 complex, with cGKII also associating with the BB via its myristoylation.
, http://www.100md.com
The NHERF family
The NHERF family consists of four related PDZ domain-containing proteins that are present in the brush border (BB) of the mammalian small intestine, colon and renal proximal tubules (Weinman et al. 1995; Yun et al. 1997; Custer et al. 1997; Scott et al. 2002; Kocher et al. 2003), as well as in many other cells. These four proteins have two or four PDZ remains with many of their PDZ domains having a high identity with other PDZ domains of this protein family. This report begins to address why four highly homologous and related proteins might be necessary in the same localized domain in polarized epithelial cells. We focus on how each NHERF family protein is different from an evolutionary view and how each uniquely contributes to NHE3 regulation.
, 百拇医药
PDZ domains are one of the most commonly utilized human protein–protein interacting sequences. In the human genome, there are at least 500 proteins which contain at least 900 PDZ domains (Sheng & Sala, 2001; Hung & Sheng, 2002; Kim & Sheng, 2004; and SMART, a simple modular architecture research tool (http://SMART.embl-heidelberg.de)). There are at least three types of PDZ domains defined by the amino acid identity at the 0 and –2 positions in the C-terminal ligand sequence: Class I, X S/T X I/V/L/M; Class II, X X ( hydrophobic amino acid); and Class III, X D/E X V. PDZ domains can also bind to internal sequences, which resemble these C-terminal ligands, but have a B finger configuration (Cuppen et al. 1997). Binding of both C-terminal and internal PDZ domain ligands is by entry of the ligand into the linear groove of the PDZ domain (Cuppen et al. 1997; Sheng & Sala, 2001; Hung & Sheng, 2002).
, http://www.100md.com
Phylogenetics
Epithelial cells contain multiple PDZ proteins, which are localized in specific subcellular domains, including the BB, basolateral membranes, the tight junctions, as well as in the Golgi (CAL) and endosomes (sorting nexin 27) (Cheng et al. 2002, 2004; Brone & Eggermont, 2005) (Fig. 1A). The major identified class of PDZ domain-containing proteins in the BB is the NHERF family, although the unrelated Shank2 is also present. The NHERF protein family has four members, each of which have between two and four PDZ domains (Fig. 1B): NHERF1, also called NHERF or EBP50 (Reczek et al. 1997); NHERF2, also called E3KARP, SIP-1 or TKA-1; NHERF3, also called PDZK1, CLAMP, CAP70, DIPHOR-1 or NaPi-CaP1 (Wang et al. 2000); and NHERF4, also called IKEPP, DIPHOR-2 or NaPi-CaP2 (Custer et al. 1997). These proteins range in size between 337 and 519 amino acids. NHERF1 and NHERF2 also contain ERM (Ezrin, Radixin, Moesin) binding domains at their C-termini, while NHERF3 and NHERF4 do not.
, 百拇医药
A, localization of PDZ domain-containing proteins to epithelial cell brush border, basolateral membranes, tight junctions, Golgi or endosomes. B, NHERF gene family: shown are the suggested names (NHERF 1–4), multiple previous names of the proteins, protein–protein interacting domains, and number of amino acids. ERM, ezrin, radixin, moesin.
By performing phylogenetic analysis, we found that of all mammalian proteins, the NHERF family members are most highly related to each other, with the next most related protein being sorting nexin 27 (M. Donowitz, A. Sharma and C. Brett, unpublished results; Joubert et al. 2004). The 12 PDZ domains of the NHERF family were analysed for their relationships to each other. Based on identity ( 20%), they were divided into four groups (A–D) (Fig. 2). The evolution of each of these PDZ domains was traced from worm and fly. They did not originate in bacteria, plants or yeast. While bacteria, plants and yeast have distantly related proteins, these contain circular binding pockets, indicating that they are involved in different types of protein–protein interactions. The worm has two related PDZ domain-containing proteins, CO1F6.6 and 4J893. The PDZ domain of CO1F6.6 and the first PDZ domain of 4J893 are identical and are probably the predecessors of the human NHERF family group C PDZ domains (Fig. 2). The second PDZ domain of 4J893, which is only 5% identical to the first, is most similar to human group B PDZ domains and also to human group A. Group D human NHERF family PDZ domains are not related to PDZ domain proteins in C. elegans. D melanogaster also has two NHERF family-related protein orthologues which are only 10% identical to each other. These are CG10939-PA, which shows most homology to human group A PDZ domains, and CG32758-PA, which is most similar to human group D. Thus, group A PDZ domains appear to have separate genetic origins from both worm and fly, whereas the other three human NHERF family PDZ domain groups appear to have potentially originated from single worm or fly PDZ domain-containing genes. Of interest is the 22% identity in the amino acid composition of the second PDZ domain of worm 4J893 and the PDZ domain of fly CC610939.
, 百拇医药
PDZ domains of the NHERF family were defined using ScanSite MotifScan. The 12 human NHERF family PDZ domains were individually analysed for sequence relationship by the MegAlign program (DynaStar, Inc.). The domains were grouped by closest relationships. Relationship with C. elegans and D. melanogaster proteins were also analysed by MegAlign. All protein names and sequences were obtained from NCB1. C. elegans has two PDZ domain-containing proteins that are related to the NHERF family. CO1F6.6 contains one PDZ domain while 4J893 contains two PDZ domains, one identical to that in CO1F6.6 and the other 5% identical. This suggests that the second PDZ domain of 4J893 may have evolved by gene duplication. Each of the two worm PDZ domains is a precursor to certain groups of PDZ domains of the human NHERF family. The D. melanogaster proteins CG10939-PA and CG32758-PA each contain one PDZ domain (10% identical to each other) and are precursors to the human NHERF family.
, http://www.100md.com
Location and unique binding partners of NHERF proteins
We now return to the question of why there are four related and highly homologous PDZ proteins from the NHERF family in the BB of enterocytes or the renal proximal tubule in polarized Na+ absorptive epithelial cells. We suggest that they may have different locations and different binding partners both of which may relate to the fact that they evolved from different PDZ domains in worm or fly. Immunofluorescent localization studies were performed using confocal microscopy of the NHERF family members in polarized Caco-2 cells (a human colon cancer Na+ absorptive cell line), which contain NHE3, and in mouse ileum (N. C. Zachos, O. Kovbasnjuk & M. Donowitz, unpublished observation). In Caco-2 cells, NHERF1 and NHERF3 (PDZK1) are predominantly in the BB with marked colocalization with NHE3 under basal conditions (Table 1). NHERF2, as was initially reported by Wade et al. (2003), is present in the BB but predominantly localizes to the area just below the BB in the area of the intermicrovillar clefts. It has some colocalization with NHE3. NHERF4 (IKEPP) has minimal BB location and is primarily cytosolic. It overlaps with NHE3 in the area below the BB. Similar observations have been made in the mouse ileum. NHERF1 and NHERF3 (PDZK1) are predominantly in the ileal BB. NHERF2 localizes to the BB and just below the BB as well as more diffusely in the cytosol. NHERF4 (IKEPP) has minimal or no BB localization and is distributed throughout the cytosol. The localizations of NHERF1-3 appear similar to reports in the mouse proximal tubule (Wade et al. 2003; Madjdpour et al. 2004).
, http://www.100md.com
While NHERF1 and NHERF2 were initially recognized as NHE3 binding partners (they were named for that interaction), they have subsequently been identified as binding to many proteins, now approaching 60 (Shenolikar et al. 2004). While members of the NHERF family share protein binding partners, specific protein–protein interactions also have been discovered. As reviewed by Shenolikar et al. (2004), NHERF2 has N, Ne binding partners that do not bind to NHERF1, which include TAZ (transcriptional co-activator with PDZ-binding motif) (Kanai et al. 2000), -actinin-4 (Kim et al. 2002), phospholipase C B3 (Hwang et al. 2000), cGMP kinase I and II (Cha et al. 2005), DRA (down-regulated in adenoma) (Lamprecht et al. 2001), podocalyxin (Takeda et al. 2001), and SRY-1 (Poulat et al. 1997) and SGK1 (Yun et al. 2002). Thus given the scaffolding role recognized for PDZ domain proteins in general, this specificity of binding suggests that the organization of different proteins into unique complexes associating with the different NHERF family members may be one of the consequences of having multiple NHERF family members in a similar part of the cell. In this review, emphasis will be on how NHERF family members and related complex formation are involved in NHE3 regulation, as an example of exploration of the epithelial function of the NHERF family.
, http://www.100md.com
The NHE3 protein is estimated to have a size of 87 kDa. However, sucrose density gradient separation of multiple cell lines that express NHE3, as well as intact ileum, have demonstrated that NHE3 exists simultaneously in many large multiprotein complexes ranging in size from 200 to 1200 kDa under basal conditions (Li et al. 2004) (Fig. 3). These complexes probably indicate that NHE3 is binding to multiple proteins simultaneously, although multimerization of NHE3 may contribute. By comparing surface and total NHE3 size, it was observed that plasma membrane NHE3 is in larger complexes than intracellular NHE3. This indicates that not all complexes are formed in the ER/Golgi and it can be assumed that as NHE3 trafficks between the BB and recycling endosomes, it associates with some different proteins. Thus the NHE3-containing complexes are dynamic. One example of the dynamic nature of NHE3 complexes is illustrated in Fig. 4 and relates to acute inhibition of NHE3 activity by carbachol activation of M3 muscarinic receptors (Li et al. 2004). As part of this regulation, the amount of plasma membrane NHE3 is decreased in parallel with inhibition of NHE3 activity and as illustrated, NHE3 partially shifts into larger size complexes. -Actinin-4, which binds to NHE3 by scaffolding to NHERF2, also shifted into larger complexes in parallel with NHE3 after carbachol treatment (Kim et al. 2002; Li et al. 2004). A role for the NHERF family proteins in forming these large NHE3-containing complexes has been assumed but not established as yet.
, http://www.100md.com
NHE3 expressed in multiple cell types was solubilized in 1% Triton X-100 and placed on 5–35% discontinuous sucrose density gradients and separated by size, as described previously (Poulat et al. 1997). In cells expressing the largest amount of NHE3 on the plasma membrane (Caco-2 cell) and when the plasma membrane NHE3 was compared to total NHE3, there were generally larger NHE3 complexes on the plasma membrane (Li et al. 2004).
Sucrose density gradient separation similar to that in Fig. 3 (Poulat et al. 1997) was carried out with ileal Na+ absorptive cell BB prepared after 10 min exposure of ileum to carbachol in vitro. Western analysis demonstrated that the size of NHE3 complexes were larger after carbachol exposure. Similar to the increase in size of NHE3 after carbachol addition, -actinin-4 was distributed in multiple, large complexes which increased in size after carbachol, in parallel with the changes in size of NHE3 complexes (Li et al. 2004).
, http://www.100md.com
NHERF family members and NHE3 regulation
Experimental models used to understand the role of the NHERF family in NHE3 regulation have been developed using several cell and animal models. (1) The NHERF family members have been individually expressed in NHERF family null fibroblasts (PS120 cells), which also stably express exogenous NHE3. The cells contain small amounts of NHERF1 endogenously but do not express the other NHERF family members (Yun et al. 1997; Ahn et al. 2001). Studies of how regulation of NHE3 is affected by expressing single or multiple members of the NHERF family give insight into the role of NHERF family members in terms of scaffolding NHE3-containing complexes. (2) In epithelial cell models, including Caco-2 cells that endogenously express NHE3 and all four NHERF family members, RNAi has been successfully used to knock down members of the NHERF family individually and should be useful in reducing them in combinations. Thus eliminating single or multiple NHERF family members and then evaluating NHE3 activity should allow a better understanding of NHERF family effects on NHE3 regulation. However, there is the limitation that remaining NHERF family members might compensate for the knock down by changing expression levels or changing intracellular location. Thus, while knockout or knockdown models might reveal the importance of redundancy, this may not necessarily indicate the role of the NHERF family members under normal conditions in which all four members are present.
, 百拇医药
Another polarized Na+ absorptive cell that endogenously expresses NHE3 is the OK (opossum kidney) renal proximal tubule cell line. Most OK cell lines express NHERF1, NHERF3 and NHERF4 but no or minimal amounts of NHERF2 (Yun et al. 2002). Thus it is possible to ask what the effect of NHERF2 in a polarized epithelial cell is by studying OK cells ectopically expressing NHERF2. (3) Knockout mouse models have been developed for NHERF1 and NHERF2 and preliminary study of their effects on NHE3 regulation in proximal tubule and small intestine have begun, providing initial insights in the roles of these two proteins in NHE3 regulation in an intact epithelial tissue (Shenolikar et al. 2002; Weinman et al. 2003; Cunningham et al. 2004; Morales et al. 2004). The long-term goal of these studies is to understand the role of the NHERF family members in human ileum and renal proximal tubule. At this time no studies have been carried out to define the roles of NHERF family members in human epithelial tissue.
, 百拇医药
NHE3 in the small intestine is part of electroneutral NaCl absorption in which NHE3 is linked to a brush border Cl––HCO–3 exchanger thought to either be DRA (down-regulated in adenoma) or PAT-1 (putative anion transporter-1) (Petrovic et al. 2002; Jacob et al. 2002). NHE3 is up- and down-regulated as part of digestion, initially being inhibited and then stimulated later by changes in the intestinal neurohumoral milieu. In diarrheal diseases there is exaggeration of this inhibition of NHE3, which is more prolonged. Acute regulation of small intestinal neutral NaCl absorption and specifically of NHE3, occurs within minutes to hours, whereas in the kidney this occurs within many hours to a few days. In understanding acute NHE3 regulation in intact epithelial tissues, consideration of NHE3 complexes are required. When considering NHE3 complexes and the role of PDZ proteins, it is important to remember that NHERF family members hetero- and homodimerize or heteromultimerize using their PDZ domains (Lau & Hall, 2001). In addition, NHERF1, NHERF2 and NHERF3 have C-terminal PDZ binding ligands. Thus, BB PDZ proteins are likely to be in the same or related complexes binding to each other as well as assembling scaffolding proteins with other protein ligands, including NHE3.
, http://www.100md.com
cGMP inhibition of NHE3 requires NHERF2
Here we draw on the example of the recently characterized process of NHERF2-mediated cGMP inhibition of NHE3 to illustrate the role of NHERF proteins in NHE3 regulation involving BB NHE3-containing complexes (see Fig. 5). NHERF2 is needed as a scaffold to allow cyclic GMP to inhibit NHE3 by a cyclic GMP kinase II (cGKII)-dependent mechanism within a large multiprotein complex (Hwang et al. 2000). To understand the mechanism, it is important to realize that most cell culture models lack cGKII (Jarchau et al. 1994; Vaandrager et al. 1998), including both PS120 cells and OK cells, which were used to develop this model. Adding cGMP to PS120 cells containing NHERF2 and NHE3 in the presence of cGKII (but not in its absence) acutely inhibited NHE3 activity. In contrast, when NHERF2 was replaced with NHERF1, cGMP failed to alter NHE3 activity. Thus specificity for NHERF2 compared with NHERF1 was demonstrated. Moreover, in the absence of either cGKII or NHERF2, there was no change in NHE3 activity. The possibility that this cGMP effect was mediated by PKA instead of PKG was eliminated since a cGMP kinase-specific ligand similarly inhibited NHE3, and a specific cGMP kinase inhibitor blocked the effect. This mechanism was separable from cAMP-mediated inhibition of NHE3 in these cells, as demonstrated by showing that maximal concentrations of cGMP and cAMP caused an additive inhibition of NHE3 (Cha et al. 2005).
, 百拇医药
NHERF2 is required for the cGMP inhibition of NHE3. NHERF2 acts as a cGMP kinase anchoring protein (GKAP), forming a complex of NHE3, NHERF2 and cGKII. cGKII is required for cGMP to inhibit NHE3. To carry out this effect, cGKII must be fixed in two locations, by its N-terminus via myristoylation to the plasma membrane and to the second PDZ domain of NHERF2 (Cha et al. 2005). Given the BB localization of guanylate cyclase C, cGMP is generated at the BB where it probably acts on the cGKII, which is part of the BB NHE3 complex.
, 百拇医药
The specificity for NHERF2 in this process was conserved in OK cells (Cha et al. 2005). OK cells endogenously express NHERF1, NHERF3 and NHERF4 and no or small amounts of NHERF2 (Yun et al. 2002). In these cells, cGMP failed to inhibit NHE3 in the presence or absence of transiently expressed cGKII. In contrast, in cells stably transfected with NHERF2, cGMP inhibited NHE3, but only in cells expressing cGKII. This effect requires NHERF2 binding to NHE3 because when NHE3 is mutated not to bind NHERF2, cyclic GMP no longer inhibits NHE3. Thus cGMP inhibits NHE3 both in fibroblasts and in polarized epithelial cells expressing NHE3 on the apical surface by a process that requires both NHERF2 and cGKII.
, 百拇医药
Further studies examined the mechanisms by which NHERF2 allowed cGMP to inhibit NHE3. Based on overlay and pull-down assays of protein–protein interactions, NHERF2 bound cGKII, while NHERF1 failed to bind with similar affinity. In vivo studies used a previously described chimera of cGKI and the first N-terminal 29 amino acids from cGKII, as well as intact cGKI, and a cGKII mutant (G2A) that lacks its myristoylation site (which is required for plasma membrane association) (Vaandrager et al. 1998; Vaandrager et al. 1996). These cGMP kinase mutants were studied in PS120 cells containing NHERF2. Neither cyclic cGKI nor cGKII lacking the myristoylation site reconstituted cGMP inhibition of NHE3. In contrast, a chimera of wild-type cGKI and the first 29 amino acids of cGKII which was myristolyated and thus bound to the plasma membrane did reconstitute cGMP inhibition of NHE3. Thus cGKII must be myristoylated and attached to the plasma membrane and bound to NHERF2 (binds to PDZ domain 2) to allow cyclic GMP inhibition of NHE3, and this effect involves a complex of NHE3–NHERF2–cGKII. Why cGKI when myristoylated could also reconstitute cGMP inhibition of NHE3 is probably because NHERF2 is a broad specificity cGMP kinase anchoring protein (GKAP) and binds GKI as well as cGKII (Cha et al. 2005).
, http://www.100md.com
Current models describing NHERF family reconstitution of cAMP inhibition of NHE3 include the concept that NHERF1 or NHERF2 using their ERM binding domain, bind to ezrin, which serves as a low affinity cAMP kinase anchoring protein (AKAP) (Yun et al. 1998; Dransfield et al. 1997) to place PKAII (and consequently the locally liberated catalytic subunit of PKA) in a location to phosphorylate NHE3 on at least two Ser residues, which affects NHE3 activity. It was thus asked whether NHERF2 binding to ezrin was similarly required for cyclic GMP inhibition of NHE3. In these studies the NHERF2 C-terminal 30 amino acids (NHERF230) were truncated and this truncation mutant was expressed in PS120/NHE3 cells. NHERF230 failed to bind ezrin but continued to bind cGMP kinase II (Cha et al. 2005). Furthermore, NHERF230 reconstituted cGMP inhibition of NHE3 but failed to reconstitute cAMP inhibition of NHE3. Thus ezrin binding is not necessary for the cGMP inhibition of NHE3. This difference provides part of the explanation for the additivity of the cAMP and cGMP inhibition of NHE3.
, 百拇医药
Thus our current model for cGMP inhibition of NHE3 involves NHE3, NHERF2 and cGKII forming a complex at the plasma membrane, with cGKII having to bind to the plasma membrane by two mechanisms, one involving its myristoylation and the other binding to the PDZ domain 2 of NHERF2. Activation of guanylate cyclase-C, which is present in the BB, by guanylin or E. coli stable toxin A, increases guanylate cyclase activity and cGMP content near the BB to locally activate cGKII (Fig. 5). While in this model NHERF2 is attached to the cytoskeleton via ezrin binding to the NHERF2 ERM domain, this is not necessary for the cGMP inhibition of NHE3. Further questions that must be answered to understand cGMP inhibition of NHE3 include whether the substrate cGMP/cGKII directly phosphorylates is NHE3, whether cGMP inhibition of NHE3 is associated with increased trafficking of NHE3 off the plasma membrane, and what are all the mechanisms which explain the additively of cGMP plus cAMP inhibition of NHE3. Moreover, since guanylate cyclase C binds NHERF4, and even larger complex in the BB may be involved in this signaling complex related to cGMP inhibition of NHE3.
, 百拇医药
Not presented in this review in detail is that NHERF2 has also been demonstrated to be involved with NHE3 inhibition by elevated intracellular calcium ([Ca2+]i) in a mechanism that involves both complex formation and changes in trafficking (Kim et al. 2002; Lee-Kwon et al. 2003; Li et al. 2004). NHERF2 but not NHERF1 reconstitutes Ca2+ inhibition of NHE3 (Kim et al. 2002; Lee-Kwon et al. 2003; Li et al. 2004). In this effect, NHERF2 scaffolds a complex involving NHE3, -actinin-4 and PKC. The -actinin-4 comes from the focal adhesions in fibroblasts and probably from the tight junctions in epithelial cells (Li et al. 2004), while PKC joins the complexes from the cytosol (Lee-Kwon et al. 2003). The specificity of the NHERF2 effect comes from the fact that NHERF2 but not NHERF1 binds -actinin-4 (Kim et al. 2000). The mechanism involves initial forming of complexes which become larger over time and are followed by endocytosis at least of NHE3 (Kim et al. 2002; Li et al. 2004). Actinin-4 is involved in the complex formation as well as endocytosis since dominant-negative actinin-4 acts as an inhibitor of Ca2+-induced inhibition of NHE3 (Kim et al. 2000). PKC binds to NHERF2 but also binds to NHERF1 (Lee-Kwon et al. 2003). The role of PKC is not in forming the complexes but in allowing endocytosis, and it does not appear to be involved with NHE3 phosphorylation, probably instead phosphorylating components of the endocytic machinery (Lee-Kwon et al. 2003).
, http://www.100md.com
In summary, NHE3 exists simultaneously in multiple large multiprotein complexes. These are dynamic and change as part of acute NHE3 regulation. Proteins that are part of these complexes include the NHERF family of PDZ domain proteins. Certain NHERF family members are required for specific examples of acute NHE3 regulation. Identified mechanisms of specificity involve binding partners brought into the NHE3 complexes and different locations in or near the BB of each of the members of the NHERF family. Roles identified for the NHERF family members in acute NHE3 regulation include scaffolding to allow complex formation and also in changing rates of regulated endocytosis and/or exocytosis. While the amount of plasma membrane NHE3 does not appear to be dependent on the NHERF family (implying lack of involvement in either delivery from the Golgi or plasma membrane retention), other processes which are regulated by other PDZ domain proteins or the NHERF family with different ligands, such as mechanisms of recycling under basal or regulated conditions and phosphorylation-dependent complex formation, have yet to be evaluated for NHE3.
, 百拇医药
Footnotes
This report was presented at The Journal of Physiology Symposium on PDZ domain scaffolding proteins and their functions in polarized cells, San Diego, CA, USA, 4 April 2005. It was commissioned by the Editorial Board and reflects the views of the authors.
References
Ahn W, Kim KH, Lee JA, Kim JY, Choi JY, Moe OW, Milgram SL, Muallem S & Lee MG (2001). Regulatory interaction between the cystic fibrosis transmembrane conductance regulator and HCO3 salvage mechanisms in model systems and the mouse pancreatic duct. J Biol Chem 276, 17236–17243.
, http://www.100md.com
Brone B & Eggermont J (2005). PDZ proteins retain and regulate membrane transporters in polarized epithelial cell membranes. Am J Physiol Cell 288, C20–C29.
Cha B, Kim JH, Hut H, Hogema BM, Nadarja J, Zizak M, Cavet M, Lee-Kwon W, Lohmann SM, Smolenski A, Tse CM, Yun C, de Jonge HR & Donowitz M (2005). cGMP inhibition of Na+/H+ antioporter 3 (NHE3) requires PDZ domain adapter NHERF2, a broad specificity protein kinase G-anchoring protein. J Biol Chem 280, 16642–16650.
, http://www.100md.com
Cheng J, Moyer BD, Milewski M, Loffing J, Ikeda M, Micke JE, Cutting GR, Li M, Stanton BA & Guggino WB (2002). A Golgi-associated PDZ domain protein modulates cystic fibrosis transmembrane regulator plasma membrane expression. J Biol Chem 277, 3520–3529.
Cheng J, Wang H & Guggino WB (2004). Modulation of mature cystic fibrosis transmembrane regulator protein by the PDZ domain protein CAL. J Biol Chem 279, 1892–1898.
Cunningham R, Steplock D, Wang F, Huang H, Shenolikar S & Weinman EJ (2004). Defective parathyroid hormone regulation of NHE3 activity and phosphate adaptation in cultured NHERF-1–/– renal proximal tubule cells. J Biol Chem 279, 37815–37821.
, 百拇医药
Cuppen E, Gerrits H, Pepers B, Wieringa B & Hendriks W (1997). PDZ motifs in PTP-BL and RIL bind to internal protein segments in the LIM domain protein RIL. Mol Biol Cell 9, 671–683.
Custer M, Spindler B, Verrey F, Murer H & Biber J (1997). Identification of a new gene product (diphor-1) regulated by dietary phosphate. Am J Physiol 273, F801–F806.
Dransfield DT, Bradford AJ, Smith J, Martin M, Roy C, Mangeat PH & Goldenring JR (1997). Ezrin is a cyclic AMP-dependent protein kinase anchoring protein. EMBO J 16, 35–43.
, http://www.100md.com
Hung AY & Sheng M (2002). PDZ domains: structural modules for protein complex assembly. J Biol Chem 277, 5699–5702.
Hwang JI, Heo K, Shin KJ, Kim E, Yun CHC, Ryu SH, Shin HS & Suh PG (2000). Regulation of phospholipase C-3 activity by Na+/H+ exchanger regulatory factor 2. J Biol Chem 275, 16632–16637.
Jacob P, Rossmann H, Lamprecht G, Kretz A, Neff C, Lin-Wu E, Grego M, Groneberg DA, Kere J & Seidler U (2002). Down-regulated in adenoma mediates apical Cl–HCO3-exchange in rabbit, rat, and human duodenum. Gastroenterology 122, 709–724.
, 百拇医药
Jarchau T, Hausler C, Markert T, Pohler D, Vanderkerckhove J, De Jonge HR, Lohmann SM & Walter U (1994). Cloning, expression, and in situ localization of rat intestinal cGMP-dependent protein kinase II. Proc Nat Acad Sci U S A 91, 9426–9430.
Joubert L, Hanson B, Barthet G, Sebben M, Claeysen S, Hong W, Marin P, Dumuis A & Bockaert J (2004). New sorting nexin (SNX27) and NHERF specifically interact with the 5-HT receptor splice variant: roles in receptor targeting. J Cell Sci 117, 5367–5379.
, 百拇医药
Kanai F, Marignani PA, Sarbassova D, Yagi R, Hall RA, Donowitz M, Hisaminato A, Fujiwara T, Ito Y, Cantley LC & Yaffe MB (2000). TAZ: a novel transcriptional co-activator regulated by interactions with 14-3-3 and PDZ domain proteins. EMBO J 19, 6778–6791.
Kim JH, Lee-Kwon W, Park JB, Ryu SH, Yun CXC & Donowitz M (2002). Ca2+-dependent inhibition of Na+/H+ exchanger 3 (NHE3) requires an NHE3-E3KARP--actinin-4 complex for oligomerisation and endocytosis. J Biol Chem 277, 23714–23724.
, 百拇医药
Kim E & Sheng M (2004). PDZ domain proteins of synapses. Nat Rev Neurosci 5, 771–781.
Kocher O, Yesilaltay A, Cirovic C, Pal R, Rigotti A & Krieger M (2003). Targeted disruption of the PDZK1 gene in mice causes tissue-specific depletion of the high density lipoprotein receptor scavenger receptor class I type I and altered lipoprotein metabolism. J Biol Chem 278, 52820–52825.
Lamprecht G, Heil A, Baisch S, Lin-Wu E, Yun C, Kalbacher H, Gregor M & Seidler U (2002). The down regulated in adenoma (dra) gene product binds to the second PDZ domain of the NHE3 kinase A regulatory protein (E3KARP), potentially linking intestinal Cl–/HCO3-exchange to Na+/H+ exchange. Biochemistry 41, 12336–12342.
, http://www.100md.com
Lau AG & Hall RA (2001). Oligomerization of NHERF-1 and NHERF-2 PDZ domains: differential regulation by association with receptor carboxyl-termini and by phosphorylation. Biochemistry 40, 8572–8580.
Lee-Kwon W, Kim JH, Choi JW, Kawano K, Cha B, Dartt DA, Zoukhri D & Donowitz M (2003). Ca2+-dependent inhibition of NHE3 requires PKC which binds to E3KARP to decrease surface NHE3 containing plasma membrane complexes. Am J Physiol Cell 285, C1527–C1536.
, 百拇医药
Li X, Zhang H, Cheong A, Leu S, Chen Y, Elowsky CG & Donowitz M (2004). Carbachol regulation of rabbit ileal brush border Na+–H+ exchanger 3 (NHE3) occurs through changes in NHE3 trafficking and complex formation and is Src dependent. J Physiol 556, 791–804.
Madjdpour C, Bacic D, Kaissling B, Murer H & Biber J (2004). Segment-specific expression of sodium-phosphate cotransporter NaPi-IIa and -IIc and interacting proteins in mouse renal proximal tubules. Pflugers Arch 448, 402–410.
, 百拇医药
Morales FC, Takahashi Y, Kreimann EL & Georgescu MM (2004). Ezrin-radixin-moesin (ERM) -binding phosphoprotein 50 organizes ERM proteins at the apical membrane of polarized epithelia. Proc Natl Acad Sci U S A 101, 17705–17710.
Petrovic S, Wang Z, Ma L, Seidler U, Forte JG, Shull GE & Soleimani M (2002). Co-localization of the apical Cl–HCO3-exchanger PAT1 and gastric H-K-ATPase in stomach parietal cells. Am J Physiol 283, G1207–G1216.
, http://www.100md.com
Poulat F, de Santa Barbara P, Desclozeaux M, Soullier S, Moniot B, Bonneaud N, Boizet B & Philippe B (1997). The human testis determining factor SRY binds a nuclear factor containing PDZ protein interaction domains. J Biol Chem 272, 7167–7172.
Reczek D, Berryman M & Bretscher A (1997). Identification of EBP50: a PDZ containing phosphoprotein that associates with members of the ezrin-radixin-moesin family. J Cell Biol 139, 169–179.
, http://www.100md.com
Scott RO, Thelin WR & Milgram SL (2002). A novel PDZ protein regulates the activity of guanylyl cyclase C, the heat-stable enterotoxin receptor. J Biol Chem 277, 22934–22941.
Sheng M & Sala C (2001). PDZ domains and the organization of supramolecular complexes. Annu Rev Neurosci 24, 1–29.
Shenolikar S, Voltz JW, Cunningham R & Weinman EJ (2004). Regulation of ion transport by the NHERF family of PDZ proteins. Physiology 19, 362–369.
, 百拇医药
Shenolikar S, Voltz JW, Minkoff CM, Wade JB & Weinman EJ (2002). Targeted disruption of the mouse NHERF-1 gene promotes internalization of proximal tubule sodium-phosphate cotransporter type IIa and renal phosphate wasting. Proc Natl Acad Sci U S A 99, 11470–11475.
Takeda T, McQuistan T, Orlando RA & Farquhar MG (2001). Loss of glomerular foot processes is associated with uncoupling of podocalyxin from the actin cytoskeleton. J Clin Invest 108, 289–301.
, 百拇医药
Vaandrager AB, Ehlert EM, Jarchau T, Lohmann SM & de Jonge HR (1996). N-terminal myristoylation is required for membrane localization of cGMP-dependent protein kinase type II. J Biol Chem 271, 7025–7029.
Vaandrager AB, Smolenski A, Tilly BC, Houtsmuller AB, Ehlert EM, Bot AG, Edixhoven M, Boomaars WE, Lohmann SM & de Jonge HR (1998). Membrane targeting of cGMP-dependent protein kinase is required for cystic fibrosis transmembrane conductance regulator Cl– channel activation. Proc Natl Acad Sci U S A 95, 1466–1471.
, http://www.100md.com
Wade JB, Liu J, Coleman RA, Cunningham R, Steplock D, Lee-Kwon W, Pallone TL, Shenolikar S & Weinman EJ (2003). Localization and interaction of NHERF isoforms in the renal proximal tubule of the mouse. Am J Physiol Cell 285, C1494–C1504.
Wang S, Yue H, Derin RB, Guggino WB & Li M (2000). Accessory protein facilitated CFTR–CFTR interaction, a molecular mechanism to potentiate the chloride channel activity. Cell 103, 169–179.
, http://www.100md.com
Weinman EJ, Steplock D & Shenolikar S (2003). NHERF-1 uniquely transduces the cAMP signals that inhibit sodium-hydrogen exchange in mouse renal apical membranes. FEBS Lett 536, 141–144.
Weinman EJ, Steplock D, Wang Y & Shenolikar S (1995). Characterization of a protein co-factor that mediates protein kinase A regulation of the renal brush border membrane Na+/H+ exchange. J Clin Invest 95, 2143–2149.
Yun CC, Chen Y & Lang F (2002). Glucocorticid activation of Na+/H+ exchanger isoform 3 revisited. The roles of SGK1 and NHERF2. J Biol Chem 277, 7676–7683.
, http://www.100md.com
Yun CH, Lamprecht G, Forster DV & Sidor A (1998). NHE3 kinase A regulatory protein E3KARP binds the epithelial brush border Na+/H+ exchanger NHE3 and the cytoskeletal protein ezrin. J Biol Chem 273, 25856–25863.
Yun CH, Oh S, Zizak M, Steplock D, Tsao S, Tse CM, Weinman EJ & Donowitz M (1997). cAMP-mediated inhibition of the epithelial brush border Na+/H+ exchanger, NHE3, requires an associated regulatory protein. Proc Natl Acad Sci U S A 94, 3010–3015., http://www.100md.com(Mark Donowitz, Boyoung Ch)
Abstract
The intestinal and renal proximal tubule brush border (BB) Na+–H+ exchanger NHE3 binds to members of the NHERF (Na+–H+ exchanger regulatory factor) family. These are four proteins (current most used names include NHERF1, NHERF2, PDZK1 and IKEPP) which are related to each other, are present in locations in or close to the BB, and scaffold a variable series of proteins in NHE3-containing complexes in a dynamic manner that is altered by changes in signal transduction which affects NHE3 activity. The specific roles of these proteins in terms of NHE3 regulation as well as interactions with each other and with their many other substrates are only now being defined. Specificity for only one member of the NHERF family in one example of NHE3 regulation, inhibition by elevation in cGMP, is used to describe how NHERF family proteins are involved in NHE3 complex formation and its regulation. In this case, NHERF2 directly binds cGKII in the brush border to form an NHE3 complex, with cGKII also associating with the BB via its myristoylation.
, http://www.100md.com
The NHERF family
The NHERF family consists of four related PDZ domain-containing proteins that are present in the brush border (BB) of the mammalian small intestine, colon and renal proximal tubules (Weinman et al. 1995; Yun et al. 1997; Custer et al. 1997; Scott et al. 2002; Kocher et al. 2003), as well as in many other cells. These four proteins have two or four PDZ remains with many of their PDZ domains having a high identity with other PDZ domains of this protein family. This report begins to address why four highly homologous and related proteins might be necessary in the same localized domain in polarized epithelial cells. We focus on how each NHERF family protein is different from an evolutionary view and how each uniquely contributes to NHE3 regulation.
, 百拇医药
PDZ domains are one of the most commonly utilized human protein–protein interacting sequences. In the human genome, there are at least 500 proteins which contain at least 900 PDZ domains (Sheng & Sala, 2001; Hung & Sheng, 2002; Kim & Sheng, 2004; and SMART, a simple modular architecture research tool (http://SMART.embl-heidelberg.de)). There are at least three types of PDZ domains defined by the amino acid identity at the 0 and –2 positions in the C-terminal ligand sequence: Class I, X S/T X I/V/L/M; Class II, X X ( hydrophobic amino acid); and Class III, X D/E X V. PDZ domains can also bind to internal sequences, which resemble these C-terminal ligands, but have a B finger configuration (Cuppen et al. 1997). Binding of both C-terminal and internal PDZ domain ligands is by entry of the ligand into the linear groove of the PDZ domain (Cuppen et al. 1997; Sheng & Sala, 2001; Hung & Sheng, 2002).
, http://www.100md.com
Phylogenetics
Epithelial cells contain multiple PDZ proteins, which are localized in specific subcellular domains, including the BB, basolateral membranes, the tight junctions, as well as in the Golgi (CAL) and endosomes (sorting nexin 27) (Cheng et al. 2002, 2004; Brone & Eggermont, 2005) (Fig. 1A). The major identified class of PDZ domain-containing proteins in the BB is the NHERF family, although the unrelated Shank2 is also present. The NHERF protein family has four members, each of which have between two and four PDZ domains (Fig. 1B): NHERF1, also called NHERF or EBP50 (Reczek et al. 1997); NHERF2, also called E3KARP, SIP-1 or TKA-1; NHERF3, also called PDZK1, CLAMP, CAP70, DIPHOR-1 or NaPi-CaP1 (Wang et al. 2000); and NHERF4, also called IKEPP, DIPHOR-2 or NaPi-CaP2 (Custer et al. 1997). These proteins range in size between 337 and 519 amino acids. NHERF1 and NHERF2 also contain ERM (Ezrin, Radixin, Moesin) binding domains at their C-termini, while NHERF3 and NHERF4 do not.
, 百拇医药
A, localization of PDZ domain-containing proteins to epithelial cell brush border, basolateral membranes, tight junctions, Golgi or endosomes. B, NHERF gene family: shown are the suggested names (NHERF 1–4), multiple previous names of the proteins, protein–protein interacting domains, and number of amino acids. ERM, ezrin, radixin, moesin.
By performing phylogenetic analysis, we found that of all mammalian proteins, the NHERF family members are most highly related to each other, with the next most related protein being sorting nexin 27 (M. Donowitz, A. Sharma and C. Brett, unpublished results; Joubert et al. 2004). The 12 PDZ domains of the NHERF family were analysed for their relationships to each other. Based on identity ( 20%), they were divided into four groups (A–D) (Fig. 2). The evolution of each of these PDZ domains was traced from worm and fly. They did not originate in bacteria, plants or yeast. While bacteria, plants and yeast have distantly related proteins, these contain circular binding pockets, indicating that they are involved in different types of protein–protein interactions. The worm has two related PDZ domain-containing proteins, CO1F6.6 and 4J893. The PDZ domain of CO1F6.6 and the first PDZ domain of 4J893 are identical and are probably the predecessors of the human NHERF family group C PDZ domains (Fig. 2). The second PDZ domain of 4J893, which is only 5% identical to the first, is most similar to human group B PDZ domains and also to human group A. Group D human NHERF family PDZ domains are not related to PDZ domain proteins in C. elegans. D melanogaster also has two NHERF family-related protein orthologues which are only 10% identical to each other. These are CG10939-PA, which shows most homology to human group A PDZ domains, and CG32758-PA, which is most similar to human group D. Thus, group A PDZ domains appear to have separate genetic origins from both worm and fly, whereas the other three human NHERF family PDZ domain groups appear to have potentially originated from single worm or fly PDZ domain-containing genes. Of interest is the 22% identity in the amino acid composition of the second PDZ domain of worm 4J893 and the PDZ domain of fly CC610939.
, 百拇医药
PDZ domains of the NHERF family were defined using ScanSite MotifScan. The 12 human NHERF family PDZ domains were individually analysed for sequence relationship by the MegAlign program (DynaStar, Inc.). The domains were grouped by closest relationships. Relationship with C. elegans and D. melanogaster proteins were also analysed by MegAlign. All protein names and sequences were obtained from NCB1. C. elegans has two PDZ domain-containing proteins that are related to the NHERF family. CO1F6.6 contains one PDZ domain while 4J893 contains two PDZ domains, one identical to that in CO1F6.6 and the other 5% identical. This suggests that the second PDZ domain of 4J893 may have evolved by gene duplication. Each of the two worm PDZ domains is a precursor to certain groups of PDZ domains of the human NHERF family. The D. melanogaster proteins CG10939-PA and CG32758-PA each contain one PDZ domain (10% identical to each other) and are precursors to the human NHERF family.
, http://www.100md.com
Location and unique binding partners of NHERF proteins
We now return to the question of why there are four related and highly homologous PDZ proteins from the NHERF family in the BB of enterocytes or the renal proximal tubule in polarized Na+ absorptive epithelial cells. We suggest that they may have different locations and different binding partners both of which may relate to the fact that they evolved from different PDZ domains in worm or fly. Immunofluorescent localization studies were performed using confocal microscopy of the NHERF family members in polarized Caco-2 cells (a human colon cancer Na+ absorptive cell line), which contain NHE3, and in mouse ileum (N. C. Zachos, O. Kovbasnjuk & M. Donowitz, unpublished observation). In Caco-2 cells, NHERF1 and NHERF3 (PDZK1) are predominantly in the BB with marked colocalization with NHE3 under basal conditions (Table 1). NHERF2, as was initially reported by Wade et al. (2003), is present in the BB but predominantly localizes to the area just below the BB in the area of the intermicrovillar clefts. It has some colocalization with NHE3. NHERF4 (IKEPP) has minimal BB location and is primarily cytosolic. It overlaps with NHE3 in the area below the BB. Similar observations have been made in the mouse ileum. NHERF1 and NHERF3 (PDZK1) are predominantly in the ileal BB. NHERF2 localizes to the BB and just below the BB as well as more diffusely in the cytosol. NHERF4 (IKEPP) has minimal or no BB localization and is distributed throughout the cytosol. The localizations of NHERF1-3 appear similar to reports in the mouse proximal tubule (Wade et al. 2003; Madjdpour et al. 2004).
, http://www.100md.com
While NHERF1 and NHERF2 were initially recognized as NHE3 binding partners (they were named for that interaction), they have subsequently been identified as binding to many proteins, now approaching 60 (Shenolikar et al. 2004). While members of the NHERF family share protein binding partners, specific protein–protein interactions also have been discovered. As reviewed by Shenolikar et al. (2004), NHERF2 has N, Ne binding partners that do not bind to NHERF1, which include TAZ (transcriptional co-activator with PDZ-binding motif) (Kanai et al. 2000), -actinin-4 (Kim et al. 2002), phospholipase C B3 (Hwang et al. 2000), cGMP kinase I and II (Cha et al. 2005), DRA (down-regulated in adenoma) (Lamprecht et al. 2001), podocalyxin (Takeda et al. 2001), and SRY-1 (Poulat et al. 1997) and SGK1 (Yun et al. 2002). Thus given the scaffolding role recognized for PDZ domain proteins in general, this specificity of binding suggests that the organization of different proteins into unique complexes associating with the different NHERF family members may be one of the consequences of having multiple NHERF family members in a similar part of the cell. In this review, emphasis will be on how NHERF family members and related complex formation are involved in NHE3 regulation, as an example of exploration of the epithelial function of the NHERF family.
, http://www.100md.com
The NHE3 protein is estimated to have a size of 87 kDa. However, sucrose density gradient separation of multiple cell lines that express NHE3, as well as intact ileum, have demonstrated that NHE3 exists simultaneously in many large multiprotein complexes ranging in size from 200 to 1200 kDa under basal conditions (Li et al. 2004) (Fig. 3). These complexes probably indicate that NHE3 is binding to multiple proteins simultaneously, although multimerization of NHE3 may contribute. By comparing surface and total NHE3 size, it was observed that plasma membrane NHE3 is in larger complexes than intracellular NHE3. This indicates that not all complexes are formed in the ER/Golgi and it can be assumed that as NHE3 trafficks between the BB and recycling endosomes, it associates with some different proteins. Thus the NHE3-containing complexes are dynamic. One example of the dynamic nature of NHE3 complexes is illustrated in Fig. 4 and relates to acute inhibition of NHE3 activity by carbachol activation of M3 muscarinic receptors (Li et al. 2004). As part of this regulation, the amount of plasma membrane NHE3 is decreased in parallel with inhibition of NHE3 activity and as illustrated, NHE3 partially shifts into larger size complexes. -Actinin-4, which binds to NHE3 by scaffolding to NHERF2, also shifted into larger complexes in parallel with NHE3 after carbachol treatment (Kim et al. 2002; Li et al. 2004). A role for the NHERF family proteins in forming these large NHE3-containing complexes has been assumed but not established as yet.
, http://www.100md.com
NHE3 expressed in multiple cell types was solubilized in 1% Triton X-100 and placed on 5–35% discontinuous sucrose density gradients and separated by size, as described previously (Poulat et al. 1997). In cells expressing the largest amount of NHE3 on the plasma membrane (Caco-2 cell) and when the plasma membrane NHE3 was compared to total NHE3, there were generally larger NHE3 complexes on the plasma membrane (Li et al. 2004).
Sucrose density gradient separation similar to that in Fig. 3 (Poulat et al. 1997) was carried out with ileal Na+ absorptive cell BB prepared after 10 min exposure of ileum to carbachol in vitro. Western analysis demonstrated that the size of NHE3 complexes were larger after carbachol exposure. Similar to the increase in size of NHE3 after carbachol addition, -actinin-4 was distributed in multiple, large complexes which increased in size after carbachol, in parallel with the changes in size of NHE3 complexes (Li et al. 2004).
, http://www.100md.com
NHERF family members and NHE3 regulation
Experimental models used to understand the role of the NHERF family in NHE3 regulation have been developed using several cell and animal models. (1) The NHERF family members have been individually expressed in NHERF family null fibroblasts (PS120 cells), which also stably express exogenous NHE3. The cells contain small amounts of NHERF1 endogenously but do not express the other NHERF family members (Yun et al. 1997; Ahn et al. 2001). Studies of how regulation of NHE3 is affected by expressing single or multiple members of the NHERF family give insight into the role of NHERF family members in terms of scaffolding NHE3-containing complexes. (2) In epithelial cell models, including Caco-2 cells that endogenously express NHE3 and all four NHERF family members, RNAi has been successfully used to knock down members of the NHERF family individually and should be useful in reducing them in combinations. Thus eliminating single or multiple NHERF family members and then evaluating NHE3 activity should allow a better understanding of NHERF family effects on NHE3 regulation. However, there is the limitation that remaining NHERF family members might compensate for the knock down by changing expression levels or changing intracellular location. Thus, while knockout or knockdown models might reveal the importance of redundancy, this may not necessarily indicate the role of the NHERF family members under normal conditions in which all four members are present.
, 百拇医药
Another polarized Na+ absorptive cell that endogenously expresses NHE3 is the OK (opossum kidney) renal proximal tubule cell line. Most OK cell lines express NHERF1, NHERF3 and NHERF4 but no or minimal amounts of NHERF2 (Yun et al. 2002). Thus it is possible to ask what the effect of NHERF2 in a polarized epithelial cell is by studying OK cells ectopically expressing NHERF2. (3) Knockout mouse models have been developed for NHERF1 and NHERF2 and preliminary study of their effects on NHE3 regulation in proximal tubule and small intestine have begun, providing initial insights in the roles of these two proteins in NHE3 regulation in an intact epithelial tissue (Shenolikar et al. 2002; Weinman et al. 2003; Cunningham et al. 2004; Morales et al. 2004). The long-term goal of these studies is to understand the role of the NHERF family members in human ileum and renal proximal tubule. At this time no studies have been carried out to define the roles of NHERF family members in human epithelial tissue.
, 百拇医药
NHE3 in the small intestine is part of electroneutral NaCl absorption in which NHE3 is linked to a brush border Cl––HCO–3 exchanger thought to either be DRA (down-regulated in adenoma) or PAT-1 (putative anion transporter-1) (Petrovic et al. 2002; Jacob et al. 2002). NHE3 is up- and down-regulated as part of digestion, initially being inhibited and then stimulated later by changes in the intestinal neurohumoral milieu. In diarrheal diseases there is exaggeration of this inhibition of NHE3, which is more prolonged. Acute regulation of small intestinal neutral NaCl absorption and specifically of NHE3, occurs within minutes to hours, whereas in the kidney this occurs within many hours to a few days. In understanding acute NHE3 regulation in intact epithelial tissues, consideration of NHE3 complexes are required. When considering NHE3 complexes and the role of PDZ proteins, it is important to remember that NHERF family members hetero- and homodimerize or heteromultimerize using their PDZ domains (Lau & Hall, 2001). In addition, NHERF1, NHERF2 and NHERF3 have C-terminal PDZ binding ligands. Thus, BB PDZ proteins are likely to be in the same or related complexes binding to each other as well as assembling scaffolding proteins with other protein ligands, including NHE3.
, http://www.100md.com
cGMP inhibition of NHE3 requires NHERF2
Here we draw on the example of the recently characterized process of NHERF2-mediated cGMP inhibition of NHE3 to illustrate the role of NHERF proteins in NHE3 regulation involving BB NHE3-containing complexes (see Fig. 5). NHERF2 is needed as a scaffold to allow cyclic GMP to inhibit NHE3 by a cyclic GMP kinase II (cGKII)-dependent mechanism within a large multiprotein complex (Hwang et al. 2000). To understand the mechanism, it is important to realize that most cell culture models lack cGKII (Jarchau et al. 1994; Vaandrager et al. 1998), including both PS120 cells and OK cells, which were used to develop this model. Adding cGMP to PS120 cells containing NHERF2 and NHE3 in the presence of cGKII (but not in its absence) acutely inhibited NHE3 activity. In contrast, when NHERF2 was replaced with NHERF1, cGMP failed to alter NHE3 activity. Thus specificity for NHERF2 compared with NHERF1 was demonstrated. Moreover, in the absence of either cGKII or NHERF2, there was no change in NHE3 activity. The possibility that this cGMP effect was mediated by PKA instead of PKG was eliminated since a cGMP kinase-specific ligand similarly inhibited NHE3, and a specific cGMP kinase inhibitor blocked the effect. This mechanism was separable from cAMP-mediated inhibition of NHE3 in these cells, as demonstrated by showing that maximal concentrations of cGMP and cAMP caused an additive inhibition of NHE3 (Cha et al. 2005).
, 百拇医药
NHERF2 is required for the cGMP inhibition of NHE3. NHERF2 acts as a cGMP kinase anchoring protein (GKAP), forming a complex of NHE3, NHERF2 and cGKII. cGKII is required for cGMP to inhibit NHE3. To carry out this effect, cGKII must be fixed in two locations, by its N-terminus via myristoylation to the plasma membrane and to the second PDZ domain of NHERF2 (Cha et al. 2005). Given the BB localization of guanylate cyclase C, cGMP is generated at the BB where it probably acts on the cGKII, which is part of the BB NHE3 complex.
, 百拇医药
The specificity for NHERF2 in this process was conserved in OK cells (Cha et al. 2005). OK cells endogenously express NHERF1, NHERF3 and NHERF4 and no or small amounts of NHERF2 (Yun et al. 2002). In these cells, cGMP failed to inhibit NHE3 in the presence or absence of transiently expressed cGKII. In contrast, in cells stably transfected with NHERF2, cGMP inhibited NHE3, but only in cells expressing cGKII. This effect requires NHERF2 binding to NHE3 because when NHE3 is mutated not to bind NHERF2, cyclic GMP no longer inhibits NHE3. Thus cGMP inhibits NHE3 both in fibroblasts and in polarized epithelial cells expressing NHE3 on the apical surface by a process that requires both NHERF2 and cGKII.
, 百拇医药
Further studies examined the mechanisms by which NHERF2 allowed cGMP to inhibit NHE3. Based on overlay and pull-down assays of protein–protein interactions, NHERF2 bound cGKII, while NHERF1 failed to bind with similar affinity. In vivo studies used a previously described chimera of cGKI and the first N-terminal 29 amino acids from cGKII, as well as intact cGKI, and a cGKII mutant (G2A) that lacks its myristoylation site (which is required for plasma membrane association) (Vaandrager et al. 1998; Vaandrager et al. 1996). These cGMP kinase mutants were studied in PS120 cells containing NHERF2. Neither cyclic cGKI nor cGKII lacking the myristoylation site reconstituted cGMP inhibition of NHE3. In contrast, a chimera of wild-type cGKI and the first 29 amino acids of cGKII which was myristolyated and thus bound to the plasma membrane did reconstitute cGMP inhibition of NHE3. Thus cGKII must be myristoylated and attached to the plasma membrane and bound to NHERF2 (binds to PDZ domain 2) to allow cyclic GMP inhibition of NHE3, and this effect involves a complex of NHE3–NHERF2–cGKII. Why cGKI when myristoylated could also reconstitute cGMP inhibition of NHE3 is probably because NHERF2 is a broad specificity cGMP kinase anchoring protein (GKAP) and binds GKI as well as cGKII (Cha et al. 2005).
, http://www.100md.com
Current models describing NHERF family reconstitution of cAMP inhibition of NHE3 include the concept that NHERF1 or NHERF2 using their ERM binding domain, bind to ezrin, which serves as a low affinity cAMP kinase anchoring protein (AKAP) (Yun et al. 1998; Dransfield et al. 1997) to place PKAII (and consequently the locally liberated catalytic subunit of PKA) in a location to phosphorylate NHE3 on at least two Ser residues, which affects NHE3 activity. It was thus asked whether NHERF2 binding to ezrin was similarly required for cyclic GMP inhibition of NHE3. In these studies the NHERF2 C-terminal 30 amino acids (NHERF230) were truncated and this truncation mutant was expressed in PS120/NHE3 cells. NHERF230 failed to bind ezrin but continued to bind cGMP kinase II (Cha et al. 2005). Furthermore, NHERF230 reconstituted cGMP inhibition of NHE3 but failed to reconstitute cAMP inhibition of NHE3. Thus ezrin binding is not necessary for the cGMP inhibition of NHE3. This difference provides part of the explanation for the additivity of the cAMP and cGMP inhibition of NHE3.
, 百拇医药
Thus our current model for cGMP inhibition of NHE3 involves NHE3, NHERF2 and cGKII forming a complex at the plasma membrane, with cGKII having to bind to the plasma membrane by two mechanisms, one involving its myristoylation and the other binding to the PDZ domain 2 of NHERF2. Activation of guanylate cyclase-C, which is present in the BB, by guanylin or E. coli stable toxin A, increases guanylate cyclase activity and cGMP content near the BB to locally activate cGKII (Fig. 5). While in this model NHERF2 is attached to the cytoskeleton via ezrin binding to the NHERF2 ERM domain, this is not necessary for the cGMP inhibition of NHE3. Further questions that must be answered to understand cGMP inhibition of NHE3 include whether the substrate cGMP/cGKII directly phosphorylates is NHE3, whether cGMP inhibition of NHE3 is associated with increased trafficking of NHE3 off the plasma membrane, and what are all the mechanisms which explain the additively of cGMP plus cAMP inhibition of NHE3. Moreover, since guanylate cyclase C binds NHERF4, and even larger complex in the BB may be involved in this signaling complex related to cGMP inhibition of NHE3.
, 百拇医药
Not presented in this review in detail is that NHERF2 has also been demonstrated to be involved with NHE3 inhibition by elevated intracellular calcium ([Ca2+]i) in a mechanism that involves both complex formation and changes in trafficking (Kim et al. 2002; Lee-Kwon et al. 2003; Li et al. 2004). NHERF2 but not NHERF1 reconstitutes Ca2+ inhibition of NHE3 (Kim et al. 2002; Lee-Kwon et al. 2003; Li et al. 2004). In this effect, NHERF2 scaffolds a complex involving NHE3, -actinin-4 and PKC. The -actinin-4 comes from the focal adhesions in fibroblasts and probably from the tight junctions in epithelial cells (Li et al. 2004), while PKC joins the complexes from the cytosol (Lee-Kwon et al. 2003). The specificity of the NHERF2 effect comes from the fact that NHERF2 but not NHERF1 binds -actinin-4 (Kim et al. 2000). The mechanism involves initial forming of complexes which become larger over time and are followed by endocytosis at least of NHE3 (Kim et al. 2002; Li et al. 2004). Actinin-4 is involved in the complex formation as well as endocytosis since dominant-negative actinin-4 acts as an inhibitor of Ca2+-induced inhibition of NHE3 (Kim et al. 2000). PKC binds to NHERF2 but also binds to NHERF1 (Lee-Kwon et al. 2003). The role of PKC is not in forming the complexes but in allowing endocytosis, and it does not appear to be involved with NHE3 phosphorylation, probably instead phosphorylating components of the endocytic machinery (Lee-Kwon et al. 2003).
, http://www.100md.com
In summary, NHE3 exists simultaneously in multiple large multiprotein complexes. These are dynamic and change as part of acute NHE3 regulation. Proteins that are part of these complexes include the NHERF family of PDZ domain proteins. Certain NHERF family members are required for specific examples of acute NHE3 regulation. Identified mechanisms of specificity involve binding partners brought into the NHE3 complexes and different locations in or near the BB of each of the members of the NHERF family. Roles identified for the NHERF family members in acute NHE3 regulation include scaffolding to allow complex formation and also in changing rates of regulated endocytosis and/or exocytosis. While the amount of plasma membrane NHE3 does not appear to be dependent on the NHERF family (implying lack of involvement in either delivery from the Golgi or plasma membrane retention), other processes which are regulated by other PDZ domain proteins or the NHERF family with different ligands, such as mechanisms of recycling under basal or regulated conditions and phosphorylation-dependent complex formation, have yet to be evaluated for NHE3.
, 百拇医药
Footnotes
This report was presented at The Journal of Physiology Symposium on PDZ domain scaffolding proteins and their functions in polarized cells, San Diego, CA, USA, 4 April 2005. It was commissioned by the Editorial Board and reflects the views of the authors.
References
Ahn W, Kim KH, Lee JA, Kim JY, Choi JY, Moe OW, Milgram SL, Muallem S & Lee MG (2001). Regulatory interaction between the cystic fibrosis transmembrane conductance regulator and HCO3 salvage mechanisms in model systems and the mouse pancreatic duct. J Biol Chem 276, 17236–17243.
, http://www.100md.com
Brone B & Eggermont J (2005). PDZ proteins retain and regulate membrane transporters in polarized epithelial cell membranes. Am J Physiol Cell 288, C20–C29.
Cha B, Kim JH, Hut H, Hogema BM, Nadarja J, Zizak M, Cavet M, Lee-Kwon W, Lohmann SM, Smolenski A, Tse CM, Yun C, de Jonge HR & Donowitz M (2005). cGMP inhibition of Na+/H+ antioporter 3 (NHE3) requires PDZ domain adapter NHERF2, a broad specificity protein kinase G-anchoring protein. J Biol Chem 280, 16642–16650.
, http://www.100md.com
Cheng J, Moyer BD, Milewski M, Loffing J, Ikeda M, Micke JE, Cutting GR, Li M, Stanton BA & Guggino WB (2002). A Golgi-associated PDZ domain protein modulates cystic fibrosis transmembrane regulator plasma membrane expression. J Biol Chem 277, 3520–3529.
Cheng J, Wang H & Guggino WB (2004). Modulation of mature cystic fibrosis transmembrane regulator protein by the PDZ domain protein CAL. J Biol Chem 279, 1892–1898.
Cunningham R, Steplock D, Wang F, Huang H, Shenolikar S & Weinman EJ (2004). Defective parathyroid hormone regulation of NHE3 activity and phosphate adaptation in cultured NHERF-1–/– renal proximal tubule cells. J Biol Chem 279, 37815–37821.
, 百拇医药
Cuppen E, Gerrits H, Pepers B, Wieringa B & Hendriks W (1997). PDZ motifs in PTP-BL and RIL bind to internal protein segments in the LIM domain protein RIL. Mol Biol Cell 9, 671–683.
Custer M, Spindler B, Verrey F, Murer H & Biber J (1997). Identification of a new gene product (diphor-1) regulated by dietary phosphate. Am J Physiol 273, F801–F806.
Dransfield DT, Bradford AJ, Smith J, Martin M, Roy C, Mangeat PH & Goldenring JR (1997). Ezrin is a cyclic AMP-dependent protein kinase anchoring protein. EMBO J 16, 35–43.
, http://www.100md.com
Hung AY & Sheng M (2002). PDZ domains: structural modules for protein complex assembly. J Biol Chem 277, 5699–5702.
Hwang JI, Heo K, Shin KJ, Kim E, Yun CHC, Ryu SH, Shin HS & Suh PG (2000). Regulation of phospholipase C-3 activity by Na+/H+ exchanger regulatory factor 2. J Biol Chem 275, 16632–16637.
Jacob P, Rossmann H, Lamprecht G, Kretz A, Neff C, Lin-Wu E, Grego M, Groneberg DA, Kere J & Seidler U (2002). Down-regulated in adenoma mediates apical Cl–HCO3-exchange in rabbit, rat, and human duodenum. Gastroenterology 122, 709–724.
, 百拇医药
Jarchau T, Hausler C, Markert T, Pohler D, Vanderkerckhove J, De Jonge HR, Lohmann SM & Walter U (1994). Cloning, expression, and in situ localization of rat intestinal cGMP-dependent protein kinase II. Proc Nat Acad Sci U S A 91, 9426–9430.
Joubert L, Hanson B, Barthet G, Sebben M, Claeysen S, Hong W, Marin P, Dumuis A & Bockaert J (2004). New sorting nexin (SNX27) and NHERF specifically interact with the 5-HT receptor splice variant: roles in receptor targeting. J Cell Sci 117, 5367–5379.
, 百拇医药
Kanai F, Marignani PA, Sarbassova D, Yagi R, Hall RA, Donowitz M, Hisaminato A, Fujiwara T, Ito Y, Cantley LC & Yaffe MB (2000). TAZ: a novel transcriptional co-activator regulated by interactions with 14-3-3 and PDZ domain proteins. EMBO J 19, 6778–6791.
Kim JH, Lee-Kwon W, Park JB, Ryu SH, Yun CXC & Donowitz M (2002). Ca2+-dependent inhibition of Na+/H+ exchanger 3 (NHE3) requires an NHE3-E3KARP--actinin-4 complex for oligomerisation and endocytosis. J Biol Chem 277, 23714–23724.
, 百拇医药
Kim E & Sheng M (2004). PDZ domain proteins of synapses. Nat Rev Neurosci 5, 771–781.
Kocher O, Yesilaltay A, Cirovic C, Pal R, Rigotti A & Krieger M (2003). Targeted disruption of the PDZK1 gene in mice causes tissue-specific depletion of the high density lipoprotein receptor scavenger receptor class I type I and altered lipoprotein metabolism. J Biol Chem 278, 52820–52825.
Lamprecht G, Heil A, Baisch S, Lin-Wu E, Yun C, Kalbacher H, Gregor M & Seidler U (2002). The down regulated in adenoma (dra) gene product binds to the second PDZ domain of the NHE3 kinase A regulatory protein (E3KARP), potentially linking intestinal Cl–/HCO3-exchange to Na+/H+ exchange. Biochemistry 41, 12336–12342.
, http://www.100md.com
Lau AG & Hall RA (2001). Oligomerization of NHERF-1 and NHERF-2 PDZ domains: differential regulation by association with receptor carboxyl-termini and by phosphorylation. Biochemistry 40, 8572–8580.
Lee-Kwon W, Kim JH, Choi JW, Kawano K, Cha B, Dartt DA, Zoukhri D & Donowitz M (2003). Ca2+-dependent inhibition of NHE3 requires PKC which binds to E3KARP to decrease surface NHE3 containing plasma membrane complexes. Am J Physiol Cell 285, C1527–C1536.
, 百拇医药
Li X, Zhang H, Cheong A, Leu S, Chen Y, Elowsky CG & Donowitz M (2004). Carbachol regulation of rabbit ileal brush border Na+–H+ exchanger 3 (NHE3) occurs through changes in NHE3 trafficking and complex formation and is Src dependent. J Physiol 556, 791–804.
Madjdpour C, Bacic D, Kaissling B, Murer H & Biber J (2004). Segment-specific expression of sodium-phosphate cotransporter NaPi-IIa and -IIc and interacting proteins in mouse renal proximal tubules. Pflugers Arch 448, 402–410.
, 百拇医药
Morales FC, Takahashi Y, Kreimann EL & Georgescu MM (2004). Ezrin-radixin-moesin (ERM) -binding phosphoprotein 50 organizes ERM proteins at the apical membrane of polarized epithelia. Proc Natl Acad Sci U S A 101, 17705–17710.
Petrovic S, Wang Z, Ma L, Seidler U, Forte JG, Shull GE & Soleimani M (2002). Co-localization of the apical Cl–HCO3-exchanger PAT1 and gastric H-K-ATPase in stomach parietal cells. Am J Physiol 283, G1207–G1216.
, http://www.100md.com
Poulat F, de Santa Barbara P, Desclozeaux M, Soullier S, Moniot B, Bonneaud N, Boizet B & Philippe B (1997). The human testis determining factor SRY binds a nuclear factor containing PDZ protein interaction domains. J Biol Chem 272, 7167–7172.
Reczek D, Berryman M & Bretscher A (1997). Identification of EBP50: a PDZ containing phosphoprotein that associates with members of the ezrin-radixin-moesin family. J Cell Biol 139, 169–179.
, http://www.100md.com
Scott RO, Thelin WR & Milgram SL (2002). A novel PDZ protein regulates the activity of guanylyl cyclase C, the heat-stable enterotoxin receptor. J Biol Chem 277, 22934–22941.
Sheng M & Sala C (2001). PDZ domains and the organization of supramolecular complexes. Annu Rev Neurosci 24, 1–29.
Shenolikar S, Voltz JW, Cunningham R & Weinman EJ (2004). Regulation of ion transport by the NHERF family of PDZ proteins. Physiology 19, 362–369.
, 百拇医药
Shenolikar S, Voltz JW, Minkoff CM, Wade JB & Weinman EJ (2002). Targeted disruption of the mouse NHERF-1 gene promotes internalization of proximal tubule sodium-phosphate cotransporter type IIa and renal phosphate wasting. Proc Natl Acad Sci U S A 99, 11470–11475.
Takeda T, McQuistan T, Orlando RA & Farquhar MG (2001). Loss of glomerular foot processes is associated with uncoupling of podocalyxin from the actin cytoskeleton. J Clin Invest 108, 289–301.
, 百拇医药
Vaandrager AB, Ehlert EM, Jarchau T, Lohmann SM & de Jonge HR (1996). N-terminal myristoylation is required for membrane localization of cGMP-dependent protein kinase type II. J Biol Chem 271, 7025–7029.
Vaandrager AB, Smolenski A, Tilly BC, Houtsmuller AB, Ehlert EM, Bot AG, Edixhoven M, Boomaars WE, Lohmann SM & de Jonge HR (1998). Membrane targeting of cGMP-dependent protein kinase is required for cystic fibrosis transmembrane conductance regulator Cl– channel activation. Proc Natl Acad Sci U S A 95, 1466–1471.
, http://www.100md.com
Wade JB, Liu J, Coleman RA, Cunningham R, Steplock D, Lee-Kwon W, Pallone TL, Shenolikar S & Weinman EJ (2003). Localization and interaction of NHERF isoforms in the renal proximal tubule of the mouse. Am J Physiol Cell 285, C1494–C1504.
Wang S, Yue H, Derin RB, Guggino WB & Li M (2000). Accessory protein facilitated CFTR–CFTR interaction, a molecular mechanism to potentiate the chloride channel activity. Cell 103, 169–179.
, http://www.100md.com
Weinman EJ, Steplock D & Shenolikar S (2003). NHERF-1 uniquely transduces the cAMP signals that inhibit sodium-hydrogen exchange in mouse renal apical membranes. FEBS Lett 536, 141–144.
Weinman EJ, Steplock D, Wang Y & Shenolikar S (1995). Characterization of a protein co-factor that mediates protein kinase A regulation of the renal brush border membrane Na+/H+ exchange. J Clin Invest 95, 2143–2149.
Yun CC, Chen Y & Lang F (2002). Glucocorticid activation of Na+/H+ exchanger isoform 3 revisited. The roles of SGK1 and NHERF2. J Biol Chem 277, 7676–7683.
, http://www.100md.com
Yun CH, Lamprecht G, Forster DV & Sidor A (1998). NHE3 kinase A regulatory protein E3KARP binds the epithelial brush border Na+/H+ exchanger NHE3 and the cytoskeletal protein ezrin. J Biol Chem 273, 25856–25863.
Yun CH, Oh S, Zizak M, Steplock D, Tsao S, Tse CM, Weinman EJ & Donowitz M (1997). cAMP-mediated inhibition of the epithelial brush border Na+/H+ exchanger, NHE3, requires an associated regulatory protein. Proc Natl Acad Sci U S A 94, 3010–3015., http://www.100md.com(Mark Donowitz, Boyoung Ch)