Page 1Articles
of 51 in PresS. Am J Physiol Gastrointest Liver Physiol (June 22, 2006). doi:10.1152/ajpgi.00135.2006
1
The emerging role of PDZ adapter proteins for regulation of intestinal ion
transport
G. Lamprecht1 and U. Seidler2*
(1) 1st Medical Department, University of Tuebingen, Germany
and
(2) Department of Gastroenterology, Hepatology and Endocrinology, Medical School of
Hannover, Germany
Running title:
Regulation of intestinal ion transport by PDZ adapter proteins
Corresponding author:
Ursula Seidler
Department of Gastroenterology, Hepatology and Endocrinology
Medical School of Hannover, Germany
Carl-Neuberg-Str. 1
30625 Hannover
[email protected]
Phone: +49 (0) 511 532 94 27
Copyright © 2006 by the American Physiological Society.
Page 2 of 51
2
Abstract
In the gastrointestinal tract CFTR, in conjunction with one or several members of the SLC26 anion exchanger family, mediates electrogenic Cl- and HCO3- secretion. NHE3, on the other hand,
coupled to one or several of the SLC26 isoforms, mediates electroneutral NaCl absorption. The
agonist-induced activation of anion secretion and inhibition of salt absorption causes secretory
diarrhea. Current dogma sees the formation of a multiprotein complex of transport proteins, PDZ
adapter proteins, anchoring proteins, the cytoskeleton, and the involved protein kinases as one
crucial step in the regulation of these transport processes. Data obtained in heterologous expression studies suggest an important role of these PDZ adapter proteins in trafficking, endocytic recycling, and membrane retention of the respective transmembrane proteins. This article
reviews recent advances in our understanding of the role of the PDZ adapter proteins NHERF,
E3KARP, PDZK1, IKEPP (NHERF-1 to NHERF-4), CAL and Shank-2 that bind to CFTR, NHE3
and the intestinal SLC26 members, in the regulation of intestinal fluid transport. Current concepts are mostly derived from heterologous expression studies, and studies on their role in organ physiology are still in infancy. Recently, however, PDZ adapter protein deficient mice and
organ-specific cell lines have become available, and the first results suggest a more cell-type
and possibly signal-specific role of these adapter proteins. This opens the potential for drug development targeted to PDZ domain interactions, which is in theory one of the most efficient antidiarrheal strategies.
Keywords
CFTR, NHE3, DRA, PAT1, SLC26A3, SLC26A6, NBC3, PDZ, NHERF, E3KARP, PDZK1, CAL,
Shank2, IKEPP, NHERF-1, NHERF-2, NHERF-3, NHERF-4, gastrointestinal ion transport, Cl/HCO3- exchange, Na+/H+ exchange, Na+/HCO3- cotransport, chloride secretion, bicarbonate secretion, NaCl absorption, PKC, Calcium, PKA, SGK1, Guanylate cyclase C, knockout mice.
Page 3 of 51
3
Abbreviations
CFTR, cystic fibrosis transmembrane regulator; NHE3, Na/H exchanger isoform 3; DRA, down
regulated in adenoma (SLC26A3); PAT1, putative anion transporter 1 (SLC26A6); NHERF,
NHE3 regulatory factor; E3KARP, NHE3 kinase A regulatory protein; PDZK1 PDZ domain protein kidney 1; IKEPP, intestinal and kidney enriched PDZ protein; GCC, guanylate cyclase C;
CAL, CFTR associated ligand; Shank2, SH3 and Ankyrin repeats containing protein 2; CortBP1,
Cortactin binding protein 1; SGK1, serum glucocorticoid inducible kinase, isoform 1; PKA, protein kinase A; PKC, protein kinase C.
Page 4 of 51
4
Introduction
During the normal digestive process, the gastrointestinal tract secretes and absorbs large quantities of electrolytes and water. Both processes involve the concerted action of a number of ion
transport proteins, and derangement of either function causes severe disease.
It is current dogma that fluid secretion in the intestine is secondary to anion secretion and fluid
absorption secondary to sodium absorption (100). The best known and most studied of the ion
transport proteins involved in anion secretion is the CFTR Cl- channel. This channel mediates
electrogenic Cl-, as well as HCO3- secretion throughout the gut (2;37), and, although able to mediate the secretion of both ions by itself (87;91), appears to work optimally in conjunction with
apical Cl-/HCO3- exchangers (58;59;71).
Sodium absorption is mediated by several brush border ion transport proteins, which are operative in different segments and during different phases of the digestive process. Postprandially,
sugar- and amino acid-coupled sodium absorption prevails in the upper small intestine (100).
Electroneutral sodium absorption is mediated predominantly by the Na+/H+ exchanger isoform
NHE3, with some contribution of other ill-defined sodium uptake mechanisms. It has the highest
net sodium absorptive capacity and is present along the total length of the gut, with predominance in the ileum and proximal colon (100). Electrogenic sodium absorption is present in the
distal colon and can reduce the sodium concentration in the stool to a few millimolar (64). Electroneutral Cl- uptake parallels Na+ uptake in a one to one ratio, and is believed to be mediated to
a large part by the operation of apical anion exchangers of the SLC26 gene family (79). However, the contribution of trans- versus paracellular Cl- uptake is ill-defined at the present moment.
To provide driving force, ensure electroneutrality, preserve cellular ion homeostasis and maintain cellular volume, each transport process involves a coordinated action of a number of ion
transporters in the apical as well as basolateral membrane. Originally thought to be regulated by
Page 5 of 51
5
changes in membrane potential and transepithelial ion concentration gradients, it has become
clear that changes in ion flux involve extremely complicated signal transduction networks regulating the trafficking and interaction of multiple proteins involved in a regulatory process with
more diversity than we ever thought of. In this review, we will focus on the recent progress that
has been made in the understanding of the role of PDZ domain proteins in regulating the position, the interaction with other proteins, and the activity of the major players in intestinal anion
secretion and salt absorption. After briefly reviewing the basic properties of this subset of PDZ
adapter proteins, we will discuss the role of PDZ adapter proteins in regulating trafficking, control
of transport activity, and interaction of different transporters. Finally, we will summarize recent
findings on anion secretory and salt absorptive regulatory dysfunctions seen in knockout mice
for several of these PDZ adapter proteins.
PDZ domain proteins that bind to CFTR, NHE3, SLC26A3 and
SLC26A6
PDZ domains are the most abundant protein-protein interaction modules in the human genome.
Their name is derived from the first three proteins in which such domains were detected, – the
postsynaptic density protein PSD95, the Drosophila homologue Disc-large, and the tight junction
protein ZO-1. PDZ domains are 90 - 100 amino acid long globular structures of six -sheets and
two -helices (41). They bind the PDZ motif of their ligands in a hydrophobic binding pocket between the second -chain and the second -helix. The affinity of the ligand-PDZ domain binding
can be regulated by phosphorylation and other conformational changes both in the PDZ binding
motif of the ligand (7;15;17;72) as well as the PDZ domain protein (74;89;90).
PDZ adapter proteins usually possess several PDZ domains, and often contain other non-PDZ
protein interaction domains. The multidomain structure of the PDZ adapter proteins allows them
to interact simultaneously with multiple binding partners and theoretically to mediate the forma-
Page 6 of 51
6
tion of large protein networks, although up to this point, such large networks have not been isolated or visualized directly.
As mentioned above, a large number of proteins containing PDZ domains are found in the human genome. Each of these is assumed to bind to a limited set of ligands. Based on our understanding of the requirements for PDZ binding motif – PDZ domain interaction, databases can be
searched for potential PDZ binding motifs in the C-terminus of a protein, corresponding to class
1-3 binding patterns (41). Concurrently, crystallographic studies have revealed a more complex
nature of the PDZ binding motif – PDZ domain interaction, with more amino acids involved than
just the last four (102). Furthermore, certain PDZ-domains can also interact with internal protein
sequences that adopt a -hairpin structure (43). This, for example, is the case for the NHERFNHE3 interaction (123). Therefore, a new classification is being awaited in the near future.
In the following section, we will briefly discuss the properties of PDZ proteins that are expressed
in the intestinal tract and have been found to bind to the PDZ motif of CFTR (table 1). Most of
these have been found to also bind to NHE3, and to the anion exchangers SLC26A3 (DRA) and
SLC26A6 (PAT1), the likely interaction partners of CFTR in bicarbonate secretion and of NHE3
in salt absorption (1;4;33;58;59). From this binding pattern alone, it is tempting to speculate that
this subset of PDZ proteins may play a dominant role in regulating intestinal anion secretion and
salt absorption.
NHERF, E3KARP, PDZK1 and IKEPP (NHERF-1 to NHERF-4): NHERF (NHE3 regulatory factor), also called NHERF-1 or EBP50, and E3KARP (NHE3 kinase A regulatory protein), also
called NHERF-2, as well as PDZK1 (PDZ domain protein kidney 1) also called CAP70, PDZ-dc1
or NHERF-3, and IKEPP (intestinal and kidney enriched PDZ protein) also called NHERF-4, re-
Page 7 of 51
7
spectively, are highly homologous (35;123;124)1. Both NHERF (NHERF-1) and E3KARP
(NHERF-2) consist of two PDZ domains and a C-terminal Ezrin/Radixin/Moesin (ERM) binding
domain, which links these proteins via Ezrin to the cytoskeleton (110;123). Both proteins bind to
CFTR, NHE3 and SLC26A3 (DRA) in heterologous expression systems (66;110;123). NHERF is
a phosphoprotein while E3KARP is not (39;67)2. Constitutive phosphorylation of NHERF at S289
by G protein coupled receptor kinase 6A (GRK6A) enhances oligomerization of NHERF (68).
Regulated phosphorylation of S289 in response to PKA or PKC has been demonstrated in kidney slices, resulting in decreased association of NHERF with NaPi-IIa (23). Furthermore NHERF
is phosphorylated by cyclin dependent kinase 2 (cdc2) and dephosphorylated by protein phosphatase 1 and/or 2A at Serine-279 and Serine-301 (42). Both NHERF and E3KARP have been
shown to dimerize both homotypically and heterotypically (31;68;103). Dimerization or oligomerization may allow the formation of larger clusters of adapter proteins below the apical plasma
membrane.
PDZK1 (NHERF-3) contains four PDZ domains, but no other protein interaction domains have
been identified (60;114). Phosphorylation of PDZK1 at serine 509 by PKA has recently been
demonstrated (83). With regard to gastrointestinal ion transport it is most relevant that PDZK1
binds CFTR (114), SLC26A3 (DRA) (97) and NHE3 (34;125;126). PDZK1 forms heterooligomers
1
The PDZ adapter proteins dealt with in this report have been identified in different settings and
different species and are thus known under various names. In order to minimize the number of
acronyms, we use the most common name irrespective of the original reference. Synonyms and
the names of orthologs from other species are summarized in table 1. Recently it has been suggested to rename these proteins as NHERF-11 to NHERF-4 (28a) and these new designations
are given in brackets.
2
There is some indirect evidence though that SGK1 may phosphorylate E3KARP (16).
Page 8 of 51
8
with NHERF in vitro (34), and as mentioned for NHERF and E3KARP, this may allow the formation of an entire network of PDZ adapter proteins underneath the plasma membrane.
IKEPP (NHERF-4) was identified as an interactor of guanylate cyclase C (GCC) (99). IKEPP
contains four PDZ domains and is expressed in the kidney, in the small and in mouse large intestine and in Caco-2 and in T84 cells (35;99). In the intestine and in the proximal tubule IKEPP
is localized to subapical vesicular structures (28;35), while PDZK1 resides in or immediately under the plasma membrane (28;35). In COS7 cells IKEPP inhibits the activity of GCC (99). Controversial results concerning the binding of IKEPP to NHE3 have been published (35). One recent report demonstrates the binding of IKEPP to NHE3 and Ca2+-dependent stimulation of
NHE3 transport activity (125).
CAL (CFTR associated ligand) has two N-terminal coiled-coil domains that facilitate homomultimerization and probably also membrane association. Based on colocalization studies CAL is
mostly expressed in the Golgi (12). The interaction of the second coiled-coil domain with TC10
(85), a small GTPase, and Syntaxin 6 (11), a SNARE protein, suggests that CAL is involved in
vesicle trafficking. Indeed Cheng et al have shown that CAL influences the distribution of CFTR
between intracellular membrane compartments and the plasma membrane (12-14).
Shank2/CortBP1 (SH3 and Ankyrin repeats containing protein 2 / Cortactin binding protein 1)
belongs to the family of Shank adapter proteins, which includes Shank1-3 (101). Shank2 binds
to CFTR, NHE3 and NBC3 in yeast-two-hybrid assays (54). Among eight different PDZ adapter
proteins known to be expressed in brain, Shank2/CortBP1 was shown to be specifically expressed in isolated rat pancreatic ducts (96), where CFTR, NHE3 and NBC3 are involved in apical Cl- and HCO3- transport (54). Shank2/CortBP1 is also present along the entire crypt-villus
axis of rat colon. In addition a novel splice variant, Shank2E, has recently been described in
hepatocytes and cholangiocytes with an apical expression (77). Shank2 counteracts the cAMPmediated stimulation of CFTR (see further below) (54) as well as the cAMP-mediated inhibition
of NHE3 (40), but its role in NBC3 regulation has not been clarified yet.
Page 9 of 51
9
The role of PDZ adapter proteins in intestinal anion secretion
As mentioned in the introduction, intestinal anion secretion is predominantly mediated by the
CFTR channel, possibly in conjunction with one of the SLC26 Cl/HCO3 exchangers in those
epithelial cells where both transporters are significantly coexpressed in the same membrane
(107). Under those circumstances, the recycling of chloride may increase the transport rates of
both CFTR and the anion exchangers (58;59;107). PDZ protein – anion transport protein interactions have been implicated in the agonist-mediated regulation of the individual transport proteins,
as well as in the cooperation of CFTR with the SLC26 anion exchangers (58;59). In addition,
they have been implicated in trafficking, phosphorylation, homo- and heterodimerization, and
membrane retention of CFTR (45;93;94;106;109-111;114). IKEPP has also been implicated in
the regulation of anion secretion by binding to the guanylate cyclase C (99).
In the following sections, we will review current knowledge on the interaction of these PDZ proteins with the various components necessary for regulated intestinal anion secretion.
CFTR trafficking
Recent studies suggest that CFTR activity is to a large extent regulated by membrane insertion
and removal (6). In this context, PDZ interactions have been demonstrated at several points
along the biosynthetic and protein secretory pathway of CFTR (figure 1). CFTR was among the
first proteins shown to require a C-terminal PDZ interaction motif for apical expression (80;81). A
more recent study by the same group demonstrated that the C-terminus of CFTR contains additional apical sorting signals, but that the PDZ-binding motif is required for prolonging the CFTR
plasma membrane half-life (111). The same study also showed that the PDZ interaction motif of
CFTR was required for reinsertion of CFTR from the recycling endosomes into the plasma
membrane (111). The endocytosis of CFTR, on the other hand, has been shown to be clathrinmediated and to involve dynamin (reviewed in (6)). Since the above-mentioned experiments
Page 10 of 51
10
were performed by expressing CFTR truncated for its PDZ-motif (111), it is presently unknown
which of the above-named PDZ proteins, or whether all of them, may serve that function in certain cell types.
Recently, Benharouga et al studied various truncation mutants of CFTR in epithelial and nonepithelial cells (5). The authors demonstrated that CFTR sorts to the apical membrane with or
without a PDZ-binding motif, but that its presence increased the effect of cAMP on CFTR transport activity. Ostedgaard et al have also studied several mutations of the CFTR C-terminus by
overexpressing them in CF airway epithelial cells (88). Again, CFTR was sorted to the apical
membrane also in the absence of the PDZ interaction motif, but its biochemical stability was reduced.
The validity of these in vitro studies are underlined by the finding that a patient with the clinical
features of cystic fibrosis was identified, whose CFTR lacks only the C-terminal four last amino
acids [http://genet.sickkids.on.ca]. Obviously, the lack of the PDZ-binding motif alone causes a
serious overall cellular functional defect of CFTR, even if the exact biochemical nature of this
defect awaits further clarification.
The Golgi-associated PDZ-protein CAL, also known as PIST (85) or FIG (11), retains CFTR
within the cell and enhances its lysosomal degradation (13). TC10, a small rho-GTPase that interacts with CAL and likely inhibitis CFTR-CAL binding, resulting in an increased CFTR expression in the plasma membrane (14). When NHERF was coexpressed with CAL, the inhibitory effect of CAL on CFTR surface expression was also overcome (12). This suggests that inhibitors
of CFTR-CAL binding, or agonists acting through NHERF and enhancing CFTR-NHERF binding,
may promote enhanced apical CFTR expression. Structural studies of the C-terminal domain
bound to the PDZ-domains of NHERF and other CFTR-associated proteins such as CAL may
provide a framework for developing novel drug therapies to enhance CFTR membrane expression and activity. Proof of concept for such an approach has recently been published by Guerra
et al, who demonstrated than overexpression of NHERF in
508CFTR expressing airway cells
Page 11 of 51
11
results in more mutant CFTR molecules to reach the plasma membrane as well as more functional activity upon PKA stimulation (38).
Regulation of CFTR channel gating
CFTR channel gating is thought to involve three distinct processes: 1) phosphorylation of the
regulatory domain, 2) ATP binding and hydrolysis by the nucleotide binding domains and 3) interactions of CFTR molecules amongst themselves and with other proteins (reviewed in
(32;55;105)). PDZ-domain interactions are important for the first and third process. A model has
been developed for PKA activation of membrane resident CFTR (45;106;109;110). It suggests
that PKA is linked to CFTR via Ezrin and NHERF or E3KARP, and that this close proximity allows PKA to phosphorylate and stimulate CFTR (figure 2B). By binding to NHERF, CFTR may
also associate with the
2-adrenergic
receptor in the apical plasma membrane of bronchial
epithelial cells, and such a macromolecular complex may mediate local receptor initiated PKAactivation and signaling to CFTR (84).
The effect of NHERF and E3KARP in facilitating the phosphorylation of CFTR is counteracted by
Shank2 (54). It diminishes basal as well as cAMP-induced phosphorylation of CFTR, resulting in
a reduction of the open-probability of the channel (54). Conversely, knock down of Shank2 in
T84 cells by antisense treatment augmented CFTR currents (54). It has been speculated that
Shank2 inhibits CFTR by association with a phosphatase, or by competition with a PDZ adapter
protein that mediates CFTR activation (54).
There is growing evidence that CFTR forms dimers in the plasma membrane (114), and this dimerization also influences channel gating (figure 2A). This dimerization appears to be mediated
by an interaction with NHERF (94) or PDZK1 (114), and results in an increased open-probability
of CFTR. Raghuram et al have further shown that dimerization is negatively regulated by phosphorylation of the second PDZ domain of NHERF (93). A recent study has further characterized
the steps leading to CFTR dimerization by NHERF (74). In the absence of other influences, the
Page 12 of 51
12
C- and N-termini of Ezrin have a tendency to auto-aggregate. The interaction with Rho, phosphorylation by PKC, and other influences, lead to an “open” configuration, in which the ERM
domain of NHERF can interact with Ezrin. In the absence of an “open” Ezrin, the C-terminus of
CFTR only binds to the first PDZ domain of NHERF with high affinity. Binding of this complex to
the conformational “open” Ezrin molecule increases the affinity of the second PDZ domain for
the C-teriminus of CFTR and a second CFTR molecule is bound to one NHERF molecule (figure
2A). Another group showed that only about 2% of CFTR were associated with NHERF, suggesting that CFTR dimerization may occur by other PDZ-proteins or PDZ-domain-unrelated events
as well (73).
Coupling of CFTR to SLC26A3 (DRA) and SLC26A6 (PAT1)
Coexpression of CFTR with SLC26A3 (DRA) and SLC26A6 (PAT1) in HEK cells has been
shown to result in activation of Cl-/HCO3- exchange (59;70;71) even when chloride transport by
CFTR was inhibited using glibenclamide3. The activation of SLC26A3 or SLC26A6 activity was
further stimulated by cAMP-dependent activation of CFTR. Likewise, cAMP-stimulated CFTR
transport rate was higher in CFTR and SLC26A3 (DRA) or SLC26A6 (PAT1) coexpressing cells.
A structural interaction of CFTR and the anion exchangers is believed to be the underlying
mechanism for this reciprocal activation of transport function. Phosphorylation of the regulatory
domain of CFTR facilitates its binding to the so-called STAS domain of the SLC26 anion exchangers (58) (figure 2C). The interaction necessitates the binding of CFTR and SLC26A3
(DRA) or SLC26A6 (PAT1) to a common PDZ adapter protein (58). These functional studies are
supported by the fact, that CFTR and members of the SLC26 family were coprecipitated from
3
The expression of CFTR also induces the expression of SLC26A3 (DRA) and SLC26A6
(PAT1) on the transcriptional level in tracheal epithelia cells and in cultured pancreatic duct cells
(36;118).
Page 13 of 51
13
heterologous expression systems as well as from mouse pancreatic tissue (59) and are partially
colocalized in mouse pancreas and mouse small intestine (58).
In native duodenum or pancreatic ducts, cAMP-mediated activation of CFTR-dependent bicarbonate secretion does not require the activity of an anion exchanger (48;108). In murine duodenum, the absence of SLC26A6 does not inhibit cAMP-stimulated Cl- or HCO3- secretion, although it significantly reduced basal secretory rate (115). The absence of CFTR, however, has
been found to reduce the maximal anion exchange rate in murine duodenum (107). Obviously,
further studies in native tissues are needed to resolve these questions.
In the pancreas, models are required to explain the exceptionally high HCO3- concentration in
the distal pancreatic ducts. In this organ, the model of CFTR-anion exchanger coupling was first
developed, because early data suggested a low HCO3- permeability of epithelial Cl- channels
(86). It is now clear that the expression and activity of basolateral anion uptake mechanisms
may also dramatically change the anion secretion through an anion conductance (25) and that
they are likely to play a major role in the anion composition of the pancreatic fluid (30;47). Methods have been developed to measure secretory activity in murine pancreatic ducts (30;70).
Thus, the regulation of pancreatic anion secretion and the role of PDZ proteins will likely be further elucidated in the near future.
The role of PDZ adapter proteins in intestinal electroneutral NaCl absorption
Electroneutral NaCl absorption in the ileum and proximal colon is largely mediated by a coupled
action of the Na+/H+ exchanger isoform NHE3 and the anion exchanger SLC26A3 (DRA). Both
the absence of NHE3 in a knockout model (98) or that of SLC26A3 (DRA) in the human autosomal recessive disease congenital chloride diarrhea (51) causes severe diarrhea and changes
in plasma electrolyte composition. Both NHE3 and SLC26A3 (DRA) have PDZ binding motifs
Page 14 of 51
14
and bind to the same set of PDZ proteins (66;123). However, to date most studies on the functional relevance of PDZ protein interaction have only been performed for NHE3. Similar to
CFTR, NHE3 is regulated by changes in membrane insertion, and probably by changes in the
transport activity of membrane resident NHE3 molecules (26). PDZ adapter-NHE3 interactions
are likely involved in both modes of regulation. A large number of endogenous substances, as
well as bacterial and viral enterotoxins or drugs have been shown to affect electroneutral salt
absorption in the intestine (65). For some of these, a direct effect on NHE3 has been established
(21;27;52;121;122). PDZ adapter proteins are likely involved both in the agonist-mediated stimulation as well as inhibition of NHE3.
cAMP- and cGMP-mediated inhibition of NHE3
In PS120/NHE3 cells, a fibroblast cell line that lacks endogenous NHEs and has been stably
transfected with NHE3, either E3KARP or NHERF is required for cAMP-mediated inhibition of
NHE3 (124) (figure 3, right panel). Coprecipitation studies revealed that not only NHE3, but also
the PKA-anchoring protein ezrin, binds to NHERF and E3KARP (67;116). This suggested that
E3KARP or NHERF serves as an adapter between NHE3 and ezrin. Ezrin is a known PKA anchoring (AKAP) protein. In analogy to CFTR phosphorylation, the close proximity of PKA to
NHE3, brought about by the PDZ adapter and ezrin, is currently thought to be a necessary prerequisite for PKA-mediated phosphorylation of NHE3 (67;123;127).
The kinetic analysis of cAMP-mediated NHE3 inhibition in OK cells suggested that the amount of
transporter in the plasma membrane remains unchanged during the early phase of cAMPelevation (67). At later time points a decrease in cell surface NHE3 was also observed (44).
Thus, phosphorylation of NHE3 both directly inhibits the transport rate, and may serve as a signal allowing endocytosis of NHE3 to occur.
Guanylin and its bacterial analogue STa (heat stable enterotoxin) inhibit salt absorption in the
small intestine through intracellular cGMP elevation (112). In PS120/NHE3 fibroblasts, E3KARP
Page 15 of 51
15
has been shown to be required for cGMP-mediated inhibition of NHE3 (10). A macromolecular
complex is formed that includes NHE3, E3KARP and membrane bound myristoylated cGK-II
(cGMP activated kinase type II), which likely phosphorylates and thus inhibits NHE3.
Ca/PKC-mediated regulation of NHE3
NHE3 is inhibited by Ca2+ mobilizing agents such as carbachol or ionomycin (21) through the
action of PKC. In contrast to the inhibition of NHE3 by PKA, PKC-mediated inhibition is caused
by a decrease in NHE3 membrane expression (49;50). The involvement of E3KARP in these
processes has been elucidated in four consecutive papers (53;69;75;76).
In PS120/NHE3 fibroblasts, Ca2+-mediated inhibition of NHE3 has been shown to occur via
NHE3 removal from the plasma membrane. This endocytosis specifically requires E3KARP
(53;69) (figure 3, left panel). E3KARP, which is constitutively bound to NHE3, recruits -actinin-4
and PKC in a Ca2+-dependent manner (53;69). This leads to the clustering of NHE3, E3KARP,
-actinin-4, PKC
and possibly other proteins (53;76), and subsequent internalization of the
complexes.
Consistent with the findings in heterologous expression systems are data derived from native
intestine. Treatment of rabbit ilea with carbachol leads to inhibition of Na-absorption and to a
24% decrease of total brush border NHE3 which is accompanied by an increase of endosomal
NHE3 from 22% to 38% (53;75;76). The decrease of brush border NHE3 protein abun-
dance is reflected by an increase of the NHE protein abundance in the detergent resistant fraction. The formation of these complexes requires the activation of c-src, which is also
present in the detergent resistant fraction (76).
IKEPP has recently been found to bind to NHE3 and to mediate Ca2+-induced stimulation of
NHE3 in PS120/NHE3/IKEPP cells (125). This was accompanied by a shift of NHE3 and IKEPP
containing complexes to smaller sizes. At present the physiological relevance of this finding is
not clear, especially since Ca2+ is thought to inhibit NHE3 in the intestine.
Page 16 of 51
16
Glucocorticoid-mediated stimulation of NHE3
Glucocorticoids stimulate ileal Na+ absorption, and one of the mechanisms is upregulation of
NHE3 expression by transcriptional (122) and posttranslational mechanisms (121). In cell culture, the posttranslational activation of Na+/H+ exchange activity requires E3KARP (i.e. E3KARP
cannot be substituted by NHERF) and SGK1 (serum glucocorticoid inducible kinase, isoform 1)
(16;121). Recently S663 of NHE3 has been identified as the substrate for SGK1 (113). The
SGK1-mediated upregulation of NHE3 function also involves PI3 kinase, because it can be
blocked with LY294002 (121). Because PI3 kinase is known to influence the surface expression
of NHE3 (50), it is tempting to hypothesize that SGK1 upregulates NHE3 via an increase in
NHE3 surface expression, but this has not been explored yet.
Coupling of NHE3 with SLC26A3 (DRA)
Two decades ago, elegant studies in isolated brush border membrane vesicles provided evidence that the coupling of intestinal brush border membrane Na+/H+ and Cl-/HCO3- exchange
may occur through the intracellular pH (56;57). Due to lack of appropriate techniques to influence and measure intracellular pH in the appropriate fashion in the native intestinal epithelium,
proof for this concept has not yet been provided. Furthermore, a physical interaction of both
transporters may augment pH-mediated regulation or may be a prerequisite for a regulatory interaction, as has been described for CFTR and the SLC26A3 and SLC26A6 anion exchangers
(58;59). This has prompted us to study whether SLC26A3 (DRA) also has a PDZ interaction motif, and whether it interacts with the same PDZ domain protein(s) as NHE3. Based on the interaction of both NHE3 and SLC26A3 (DRA) with the second PDZ domain of E3KARP (66) and
based on the ability of E3KARP to form dimers (31;103) we have proposed a model (figure 4), in
which NHE3 and SLC26A3 (DRA) are physically coupled to each other by a NHERF or E3KARP
dimer (66). Since then it has become clear that SLC26A3 (DRA) in vitro not only interacts with
E3KARP but also with NHERF (Natour and Lamprecht, unpublished) and PDZK1 (97), both of
Page 17 of 51
17
which are expressed at much higher levels than E3KARP in the native intestine. Thus the exact
composition of SLC26A3 (DRA) (and NHE3) containing complexes requires further study.
Interaction of NHE3 and CFTR
Agonist-stimulated intestinal anion secretion occurs concomitantly with inhibition of electroneutral salt absorption, and in the intestinal tract of CFTR-deficient mice, cAMP-mediated inhibition
of electroneutral NaCl absorption is absent (20). This finding is surprising, because cAMP inhibits NHE3 in cells that do not express CFTR (124).
Two recent studies may shed some light on this puzzle. In PS120/NHE3 fibroblasts, the expression and the cAMP-mediated activation of CFTR results in a markedly stronger inhibition of
NHE3 by forskolin than found in its absence (1). Luminal NHE3 activity in mouse pancreatic
ducts was more strongly inhibited by forskolin perfusion than in ducts from CFTR mutant mice
(1). The inhibition of NHE3 by CFTR in PS120 fibroblasts required the C-terminal PDZ binding
motif of CFTR. Likewise, NHE3 could only be immunoprecipitated from mouse pancreas that
expressed CFTR containing the C-terminal PDZ binding motif (1).
Bargorda et al recently demonstrated that in a renal cell line which endogenously expresses
CFTR, the heterologous expression of NHE3 results in a decrease in the maximal activation of
CFTR by PKA (4). Likewise, antisense-mediated suppression of CFTR resulted in a decrease in
the NHE3 inhibition by cAMP. Again, this reciprocal regulation appeared to occur in a multiprotein complex, likely consisting of CFTR, NHE3, Ezrin, E3KARP, and possibly other proteins (4).
The former study may explain the findings in the CFTR knockout mice and suggests that in cells
which coexpress CFTR and NHE3 endogenously, a PDZ dependent interaction of the two transporters augments the PKA-mediated switch from an absorbing to a secreting cell.
However, the question remains where such coexpression takes place. Overall expression levels
for CFTR in the intestinal mucosa are much lower than for NHE3, and CFTR is predominantly
located in the crypts and basal parts of the villi, whereas NHE3 is most strongly expressed in the
Page 18 of 51
18
surface cells. Especially in colonic surface cells, where cAMP-induced inhibition of NHE3 is also
absent in CFTR-deficient mice (3), the theory of a structural interaction of NHE3 and CFTR as
the basis for this inhibition is at present difficult to reconcile with the assumed number of both
proteins in the brush border membrane.
In the jejunal mid villus region, where CFTR and NHE3 are coexpressed within the same cell,
cAMP activation of CFTR has been shown to mediate anion secretion resulting in cell shrinkage
(33). NHE3 inhibition by CFTR-activation induced cell shrinkage was suggested as an explanation for the observed lack of NHE3 inhibition in CFTR-deficient intestine. Thus, alternative explanations have been brought forth, which require the functional activity of CFTR in the membrane
rather than a physical interaction of CFTR and NHE3 (33).
Data from knockout animals
Two different knockout mice of NHERF (78;104), and one type of knockout mice of PDZK1 (62),
E3KARP 4, and CAL (120) have been generated.
Female NHERF knockout mice generated by Shenolikar have a lower body weight, reduced life
span and decreased bone mineral density compared to their heterozygous or wild type littermates (104). Both male and female knockout animals show increased urinary phosphate excretion and altered NaPi-IIa expression in the proximal tubule (104). The expression of NHE3 in the
proximal tubule (82) as well as the enterocyte (18) is unaltered, but Na/H exchange in the proximal tubule is not inhibited by cAMP any more (82). In the enterocyte, cAMP-mediated inhibition
of Na/H exchange is still present both in the ileum (82) and colon (18), but in the colon to a reduced extent. Interestingly, cAMP-mediated activation of CFTR-dependent anion current is also
reduced (18). As a consequence ileal fluid production is decreased in NHERF knockout mice
4
Hogema et al., Dept. of Biochemistry, Erasmus Medical Center, Rotterdam, The Netherlands
Page 19 of 51
19
(Hogema et al, manuscript in preparation). All in all, these mice should be less susceptible to
enterotoxin-mediated diarrheal disease, but this has not been tested yet.
The NHERF knockout mice generated by Morales (78), on the other hand, do not show increased mortality of the females, although they also have decreased serum phosphate levels.
The kidneys do not show macroscopic or microscopic alterations. There is strong downregulation of ezrin and activated ERM (Ezrin-Radixin-Moesin) proteins, which is more pronounced in
the intestine than in the kidney. These animals have disorganized intestinal microvilli and dispersed actin-rich terminal webs. The number of goblet cells is increased. Intestinal ion transport
studies in these animals have not been reported yet.
E3KARP knockout mice have also recently been generated, and various organ systems are in
the process of being studied. In the intestinal tract, cAMP-dependent activation of anion secretion, cAMP-mediated inhibition of NHE3, and forskolin-inhibition of fluid absorption in isolated
small intestinal loops is not altered (Hogema et al, manuscript in preparation), but differences in
Ca2+-mediated inhibition of NHE3 are similar to those described in heterologous expression systems (Cinar et al, manuscript in preparation), i.e. the inhibitory effect of increased intracellular
Ca2+ is lost in the knock out animals.
PDZK1 knockout mice do not have a gross anatomical phenotype (62), but have elevated levels
of cholesterol due to a defect in membrane expression of the scavenger receptor (63). When
these mice are challenged with a high phosphate diet, they show decreased NaPi-IIa expression
and increased renal phosphate excretion compared to the wild type animals (9).
Intestinal ion transport has recently been studied in PDZK1 knockout mice (19). They show
slightly diminished forskolin stimulated duodenal anion secretion. In the small intestinal mucosa,
basal Na absorption is reduced by about 50%, the immediate inhibitory effect of forskolin is lost,
but some inhibition does occur at later stages. In colonic surface cells5 basal NHE3 activity was
5
Different from the mouse, PDZK1 expression is minimal in human and rabbit colon (97;114).
Page 20 of 51
20
reduced by about 50% and the immediate inhibitory effect of forskolin was lost. NHE3 abundance in the brush border membrane was decreased by approximately 30% compared to wild
type littermates, while NHE3 mRNA expression was significantly increased and CFTR expression was not significantly altered. The data suggest that a complex regulatory interaction of
PDZK1 and NHE3 takes place in the intestine, affecting agonist-mediated NHE3 regulation and
BBM protein abundance in an as yet incompletely understood fashion.
Thus, the first data that emerge from studies in the native intestine underline the importance of
PDZ domain-interaction in the regulation of intestinal electrolyte transport. They also demonstrate, however, that the situation is more complex than in heterologous coexpression systems,
in which one or two components of a potential multiprotein complex are strongly overexpressed,
but others may be underrepresented. Presently, only relatively little experimental knowledge has
been obtained from knockout studies, and methods have varied between different groups. Yet, it
appears that NHERF, E3KARP, and PDZK1 have more cell and signal specific functions than
could have been anticipated from heterologous expression systems. It follows that the signaling
complexes for CFTR and NHE3 are likely different in different organs and under different circumstances. New and exciting findings on the role of this family of proteins in native tissues are
likely to come up in the near future.
Summary and future perspectives
Since the cloning of NHERF by Weinman in 1995 and the discovery that it is a necessary cofactor for cAMP-mediated inhibition of NHE3 in renal brush border membranes (117) much progress has been made in the field. E3KARP, PDZK1, IKEPP, CAL and Shank2 have been identified as additional PDZ adapter proteins that interact with some or all major ion transporting proteins in the gastrointestinal tract, i.e. CFTR, NHE3 and the SLC26 transporters SLC26A3 (DRA)
and SLC26A6 (PAT1). Functional characterization has expanded from a quite simple model of a
stationary complex that facilitates phosphorylation of a single transporter protein in response to a
Page 21 of 51
21
single signal, to much more complex models such as signal complex formation affecting several
different signals, clustering of one or several transport proteins, and regulation of membrane traffic of various transporter proteins.
The coordinated action of different transport proteins in the same membrane, as well as in the
contralateral membrane, is of particular interest during vectorial ion transport in intestinal epithelia. Therefore, several questions are of particular interest for future studies in the intestine:
1.
Despite the existence of the models presented in this review, the actual components of
the PDZ-adapter mediated multiprotein complexes in the intestinal brush border membrane during regulated transport are unknown, and likely contain more proteins than our current models
predict. The isolation of such complexes from native intestine is extremely difficult and fraught
with many uncertainties. Unfortunately, intestinal cell lines, unless genetically manipulated, uniformly express NHE3 and SLC26A3 (DRA) at low levels. However, ingenious methods have
been developed to search for protein interaction partners, as well as to study the interaction and
its functional consequences in heterologous expression systems. The recent advance of transgenic mouse generation with cell-specific or inducible expression/knockout of a protein, of multiple-knockout mice, and of si-RNA based approaches in vivo, allows to test the applicability of the
derived models to the situation in native organs.
2.
In the past, absorptive and secretory functions within the intestinal epithelium were
thought to be separated along the crypt-villus axis (100). Current models on complex formation
of intestinal ion transporters require CFTR, NHE3, and the SLC26 anion exchangers in the same
cellular membrane. At least in some small intestinal villus cells, there is evidence that this may
be so (33). According to current models, the switch from the absorptive to the secretory state
has to be accompanied by a dramatic reorganization of the multiprotein complexes within an enterocyte. How is, in this situation, the binding affinity between PDZ-adapters and transporters
determined and how is it regulated? First reports have been published on the secondmessenger dependent regulation of the affinity between PDZ-adapter and ligand (15;17;72;93).
Page 22 of 51
22
Crystallographic studies have revealed the structural basis for PDZ-domain-ligand interactions
and the extension of such studies to ligand-PDZ adapter interactions during regulation of intestinal transport processes is urgently needed (65a).
3.
Drug targeting of PDZ-domain-ligand interactions holds great therapeutic promise (24).
Secretory diarrhea, the number one killer among gastrointestinal disease worldwide, is mediated
by stimulation of anion secretion and inhibition of salt absorption, and, as detailed above, both
processes are thought to be mediated by multiprotein complex formation and to involve a common set of adapter- and anchor proteins. Targeting of specific PDZ-domain-ligand interactions
may theoretically interfere with both processes and thus prevent electrolyte and fluid loss. But
even much simpler approaches, such as an interruption of PDZ-domain-ligand interactions that
retain transport proteins in the cell interior, or in the membrane, may be of great benefit in cystic
fibrosis or diarrheal diseases.
Page 23 of 51
23
Reference List
1. Ahn W, Kim KH, Lee JA, Kim JY, Choi JY , Moe OW, Milgram SL, Muallem S, and
Lee MG. 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, 2001.
2. Ameen NA, Ardito T, Kashgarian M, and Marino CR. A unique subset of rat and human intestinal villus cells express the cystic fibrosis transmembrane conductance regulator. Gastroenterology 108: 1016-1023, 1995.
3. Bachmann, O., B. Riederer, G. Ziesmann, W.H. Colledge, M.P. Manns, and U.
Seidler. cAMP-dependent inhibition of surface cell NHE3and mucosal PDZ-domain binding protein expression levels are reduced in CFTR deficient murine colon. Gastroenterology 128 (Suppl. 2): A-45, 2005. (Abstract)
4. Bagorda A, Guerra L, Di Sole F, Hemle-Kolb C, Cardone RA, Fanelli T, Reshkin SJ,
Gisler SM, Murer H, and Casavola V. Reciprocal Protein Kinase A Regulatory Interactions between Cystic Fibrosis Transmembrane Conductance Regulator and Na+/H+ Exchanger Isoform 3 in a Renal Polarized Epithelial Cell Model. J Biol.Chem. 277: 2148021488, 2002.
5. Benharouga M, Sharma M, So J, Haardt M, Drzymala L, Popov M, Schwapach B,
Grinstein S, Du K, and Lukacs GL. The Role of the C Terminus and Na+/H+ Exchanger
Regulatory Factor in the Functional Expression of Cystic Fibrosis Transmembrane Conductance Regulator in Nonpolarized Cells and Epithelia. J Biol.Chem. 278: 22079, 2003.
Page 24 of 51
24
6. Bertrand CA and Frizzell RA. The role of regulated CFTR trafficking in epithelial secretion. Am.J.Physiol. 285: C1-C18, 2003.
7. Birrane G, Chung J, and Ladias JAA. Novel Mode of Ligand Recognition by the Erbin
PDZ Domain. J Biol.Chem. 278: 1399-1402, 2003.
8. Boeckers TM, Kreutz MR, Winter C, Zuschratter W, Smalla KH, Sanmarti-Vila L,
Wex H, Langnaese K, Bockmann J, Garner CC, and Gundelfinger ED. Proline-Rich
Synapse-Associated Protein-1/Cortactin Binding Protein 1 (ProSAP1/CortBP1) Is a PDZDomain Protein Highly Enriched in the Postsynaptic Density. J.Neurosci. 19: 6506-6518,
1999.
9. Capuano P, Bacic D, Stange G, Hernando N, Kaissling B, Pal R, Kocher O, Biber J,
Wagner CA, and Murer H. Expression and regulation of the renal Na/phosphate cotransporter NaPi-IIa in a mouse model deficient for the PDZ protein PDZK1. Pflugers
Arch. 449: 392-405, 2005.
10. 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, and Donowitz M. cGMP
Inhibition of Na+/H+ Antiporter 3 (NHE3) Requires PDZ Domain Adapter NHERF2, a
Broad Specificity Protein Kinase G-anchoring Protein. J Biol.Chem. 280: 16642-16650,
2005.
11. Charest A, Lane K, McMahon K, and Housman DE. Association of a Novel PDZ Domain-containing Peripheral Golgi Protein with the Q-SNARE (Q-soluble NEthylmaleimide-sensitive Fusion Protein (NSF) Attachment Protein Receptor) Protein
Syntaxin 6. J Biol.Chem. 276: 29456-29465, 2001.
Page 25 of 51
25
12. Cheng J, Moyer BD, Milewski M, Loffing J, Ikeda M, Mickle JE, Cutting GR, Li M,
Stanton BA, and Guggino WB. A Golgi-associated PDZ Domain Protein Modulates
Cystic Fibrosis Transmembrane Regulator Plasma Membrane Expression. J Biol.Chem.
277: 3520-3529, 2002.
13. Cheng J, Wang H, and Guggino WB. Modulation of Mature Cystic Fibrosis Transmembrane Regulator Protein by the PDZ Domain Protein CAL. J Biol.Chem. 279: 1892-1898,
2004.
14. Cheng J, Wang H, and Guggino WB. Regulation of Cystic Fibrosis Transmembrane
Regulator Trafficking and Protein Expression by a Rho Family Small GTPase TC10. J
Biol.Chem. 280: 3731-3739, 2005.
15. Chetkovich DM, Chen L, Stocker TJ, Nicoll RA, and Bredt DS. Phosphorylation of the
Postsynaptic Density-95 (PSD-95)/Discs Large/Zona Occludens-1 Binding Site of Stargazin Regulates Binding to PSD-95 and Synaptic Targeting of AMPA Receptors.
J.Neurosci. 22: 5791-5796, 2002.
16. Chun J, Kwon T, Lee E, Suh PG, Choi EJ, and Sun KS. The Na+/H+ exchanger regulatory factor 2 mediates phosphorylation of serum- and glucocorticoid-induced protein kinase 1 by 3-phosphoinositide-dependent protein kinase 1. Biochem Biophys.Res.Commun. 298: 207-215, 2002.
17. Chung HJ, Huang YH, Lau LF, and Huganir RL. Regulation of the NMDA Receptor
Complex and Trafficking by Activity-Dependent Phosphorylation of the NR2B Subunit
PDZ Ligand. J.Neurosci. 24: 10248-10259, 2004.
Page 26 of 51
26
18.
Cinar, A, M Chen, B Riederer, B Hogema, H de Jonge, M Donowitz, EJ Weinman, O
Kocher, U Seidler. Differential effects of PDZ-adapterprotein NHERF1, E3KARP and
PDZK1 knockout on the regulation of NHE3 transport activity in native murine colonic epithelium. Pflugers Arch. 2006 (Abstract online); http://www.physiologischegesellschaft.de/kongress/abstracts/2006/12899.pdf [19th March 2006]
19. Cinar, A., B. Hillesheim, B. Tuo, B. Riederer, M. Manns, O. Kocher, and U. Seidler.
The PDZ-Domain Binding Protein PDZk1/cap70 Is Involved in the Regulation Of Murine
Intestinal NHE3 and CFTR Function. Gastroenterology 128 (Suppl. 2): A-368,
2005.(Abstract)
20. Clarke LL and Harline MC. CFTR is required for cAMP inhibition of intestinal Na+ absorption in a cystic fibrosis mouse model. Am. J. Physiol. 270: G259-G267, 1996.
21. Cohen ME, Wesolek J, McCullen J, Rys Sikora K, Pandol S, Rood RP, Sharp GW,
and Donowitz M. Carbachol- and elevated Ca2+-induced translocation of functionally active protein kinase C to the brush border of rabbit ileal Na+ absorbing cells. J.Clin.Invest.
88: 855-863, 1991.
22. Custer M, Spindler B , Verrey F, Murer H, and Biber J. Identification of a new gene
product (diphor-1) regulated by dietary phosphate. Am. J. Physiol. 273: F801-F806,
1997.
23. Deliot N, Hernando N , Horst-Liu Z, Gisler SM, Capuano P, Wagner CA, Bacic D,
O'Brien S, Biber J, and Murer H. Parathyroid hormone treatment induces dissociation
of type IIa Na+-Pi cotransporter-Na+/H+ exchanger regulatory factor-1 complexes.
Am.J.Physiol. 289: C159-C167, 2005.
Page 27 of 51
27
24. Dev KK. Making protein interactions druggable: TARGETING PDZ DOMAINS. Nat Rev
Drug Discov 3: 1047-1056, 2004.
25. Devor DC, Singh AK, Lambert LC, DeLuca A, Frizzell RA, and Bridges RJ. Bicarbonate and Chloride Secretion in Calu-3 Human Airway Epithelial Cells. J.Gen.Physiol.
113: 743-760, 1999.
26. Donowitz M, Janecki A, Akhter S, Cavet ME, Sanchez F, Lamprecht G, Zizak M,
Kwon WL, Khurana S, Yun CH, and Tse CM. Short-term regulation of NHE3 by EGF
and protein kinase C but not protein kinase A involves vesicle trafficking in epithelial cells
and fibroblasts. Ann.N.Y.Acad.Sci. 915: 30-42, 2000.
27. Donowitz M, Tai YH, and Asarkof N. Effect of serotonin on active electrolyte transport
in rabbit ileum, gallbladder, and colon. Am J Physiol 239: G463-G472, 1980.
28. Donowitz M, Cha B, Zachos NC, Brett CL, Sharma A, Tse CM, and Li X. NHERF family and NHE3 regulation. J. Physiol. 567: 3-11, 2005.
28a. Donowitz M, Milgram S, Murer H, Kurachi Y, Yun C, Weinman E. Coming out of the
NHERF family. J. Physiol. 567: 1, 2005.
29. Du Y, Weed SA, Xiong WC, Marshall TD, and Parsons JT. Identification of a Novel
Cortactin SH3 Domain-Binding Protein and Its Localization to Growth Cones of Cultured
Neurons. Mol.Cell.Biol. 18: 5838-5851, 1998.
30. Fernandez-Salazar MP, Pascua P, Calvo JJ, Lopez MA, Case RM, Steward MC, and
San Roman JI. Basolateral anion transport mechanisms underlying fluid secretion by
mouse, rat and guinea-pig pancreatic ducts. J. Physiol. 556: 415-428, 2004.
Page 28 of 51
28
31. Fouassier L, Yun CC, Fitz JG, and Doctor RB. Evidence for ezrin-radixin-moesinbinding phosphoprotein 50 (EBP50) self-association through PDZ-PDZ interactions. J
Biol Chem 275: 25039-25045, 2000.
32. Gadsby DC and Nairin AC. Control of CFTR Channel Gating by Phosphorylation and
Nucleotide Hydrolysis. Physiol.Rev. 79: 77-107, 1999.
33. Gawenis L, Franklin C, Simpson J, Palmer B, Walker N, Wiggins T, and Clarke L.
cAMP inhibition of murine intestinal Na+/H+ exchange requires CFTR-mediated cell
shrinkage of villus epithelium. Gastroenterology 125: 1148-1163, 2003.
34. Gisler SM, Pribanic S, Bacic D, Forrer P, Gantenbein A, Sabourin LA, Tsuji A, Zhao
ZS, Manser E, Biber J, and Murer H. PDZK1: I. A major scaffolder in brush borders of
proximal tubular cells. Kidney Int. 64: 1733-1745, 2003.
35. Gisler SM, Stagljar I, Traebert M, Bacic D, Biber J, and Murer H. Interaction of the
Type IIa Na/Pi Cotransporter with PDZ Proteins. J Biol.Chem. 276: 9206-9213, 2001.
36. Greeley T, Shumaker H, Wang Z, Schweinfest CW, and Soleimani M. Downregulated
in adenoma and putative anion transporter are regulated by CFTR in cultured pancreatic
duct cells. Am. J. Physiol. 281: G1301-G1308, 2001.
37. Grubb BR and Boucher RC. Pathophysiology of Gene-Targeted Mouse Models for Cystic Fibrosis. Physiol.Rev. 79: 193-214, 1999.
Page 29 of 51
29
38. Guerra L, Fanelli T, Favia M, Riccardi SM, Busco G, Cardone RA, Carrabino S,
Weinman EJ, Reshkin SJ, Conese M, and Casavola V. Na+/H+ Exchanger Regulatory
Factor Isoform 1 Overexpression Modulates Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Expression and Activity in Human Airway 16HBE14o- Cells and
Rescues F508 CFTR Functional Expression in Cystic Fibrosis Cells. J Biol.Chem. 280:
40925-40933, 2005.
39. Hall RA, Spurney RF, Premont RT, Rahman N, Blitzer JT, Pitcher JA, and Lefkowitz
RJ. G Protein-coupled Receptor Kinase 6A Phosphorylates the Na+/H+ Exchanger Regulatory Factor via a PDZ Domain-mediated Interaction. J Biol.Chem. 274: 24328-24334,
1999.
40. Han WS, Kim KH, Jo MJ, Lee JH, Yang J, Doctor RB, Moe OW, Lee J, Kim E, and
Lee MG. Shank2 Associates with and Regulates Na+/H+ Exchanger 3. J Biol.Chem. 281:
1461-1469, 2006.
41. Harris BZ and Lim WA . Mechanism and role of PDZ domains in signaling complex assembly. J.Cell Sci. 114: 3219-3231, 2001.
42. He J, Lau AG, Yaffe MB, and Hall RA. Phosphorylation and Cell Cycle-dependent
Regulation of Na+/H+ Exchanger Regulatory Factor-1 by Cdc2 Kinase. J Biol.Chem. 276:
41559-41565, 2001.
43. Hillier BJ, Christopherson KS, Prehoda KE, Bredt DS, and Lim WA. Unexpected
Modes of PDZ Domain Scaffolding Revealed by Structure of nNOS-Syntrophin Complex.
Science 284: 812-815, 1999.
Page 30 of 51
30
44. Hu MC, Fan L, Crowder LA, Karim-Jimenez Z, Murer H, and Moe OW. Dopamine
Acutely Stimulates Na+/H+ Exchanger (NHE3) Endocytosis via Clathrin-coated Vesicles.
DEPENDENCE ON PROTEIN KINASE A-MEDIATED NHE3 PHOSPHORYLATION. J
Biol.Chem. 276: 26906-26915, 2001.
45. Huang P, Trotter K, Boucher RC, Milgram SL, and Stutts MJ. PKA holoenzyme is
functionally coupled to CFTR by AKAPs. Am J Physiol. 278: C417-C422, 2000.
46. Ikemoto M, Arai H, Feng D, Tanaka K, Aoki J, Dohmae N, Takio K, Adachi H, Tsujimoto M, and Inoue K. Identification of a PDZ-domain-containing protein that interacts
with the scavenger receptor class B type I. PNAS 97: 6538-6543, 2000.
47. Ishiguro H, Naruse S , San Roman JI, Case M, and Steward MC. Pancreatic ductal
bicarbonate secretion: past, present and future. JOP 2: 192-197, 2001.
48. Ishiguro H, Steward MC, Wilson RW, and Case RM. Bicarbonate secretion in interlobular ducts from guinea-pig pancreas. J Physiol. 495: 179-191, 1996.
49. Janecki AJ, Montrose MH, Zimniak P, Zweibaum A, Tse CM, Khurana S, and Donowitz M. Subcellular redistribution is involved in acute regulation of the brush border
Na+/H+ exchanger isoform 3 in human colon adenocarcinoma cell line Caco-2. Protein
kinase C-mediated inhibition of the exchanger. J Biol Chem 273: 8790-8798, 1998.
50. Janecki AJ, Janecki M, Akhter S, and Donowitz M. Basic Fibroblast Growth Factor
Stimulates Surface Expression and Activity of Na+/H+ Exchanger NHE3 via Mechanism
Involving Phosphatidylinositol 3-Kinase. J Biol.Chem. 275: 8133-8142, 2000.
Page 31 of 51
31
51. Kere J, Lohi H, and Hoglund P. Genetic Disorders of Membrane Transport III. Congenital chloride diarrhea. Am J Physiol. 276: G7-G13, 1999.
52. Khurana S, Nath SK, Levine SA, Bowser JM, Tse CM, Cohen ME, and Donowitz M.
Brush Border Phosphatidylinositol 3-Kinase Mediates Epidermal Growth Factor Stimulation of Intestinal NaCl Absorption and Na/H Exchange. J Biol.Chem. 271: 9919-9927,
1996.
53. Kim JH, Lee-Kwon W, Park JB, Ryu SH, Yun CHC, and Donowitz M. Ca2+-dependent
Inhibition of Na+/H+ Exchanger 3 (NHE3) Requires an NHE3-E3KARP-alpha-Actinin-4
Complex for Oligomerization and Endocytosis. J Biol.Chem. 277: 23714-23724, 2002.
54. Kim JY, Han WS, Namkung W, Lee JH, Kim KH, Shin H, Kim E, and Lee MG. Inhibitory Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Aniontransporting Activities by Shank2. J Biol.Chem. 279: 10389-10396, 2004.
55. Kirk KL. New paradigms of CFTR chloride channel regulation. Cell Mol Life Sci 57: 623634, 2000.
56. Knickelbein R, Aronson PS, Atherton W, and Dobbins JW. Sodium and chloride
transport across rabbit ileal brush border. I. Evidence for Na-H exchange. Am.J.Physiol.
245: G504-G510, 1983.
57. Knickelbein R, Aronson PS, Schron CM, Seifter J, and Dobbins JW. Sodium and
chloride transport across rabbit ileal brush border. II. Evidence for Cl-HCO3 exchange
and mechanism of coupling. Am.J.Physiol. 249: G236-G245, 1985.
Page 32 of 51
32
58. Ko SB, Zeng W, Dorwart MR, Luo X, Kim KH, Millen L, Goto H, Naruse S, Soyombo
A, Thomas PJ, and Muallem S. Gating of CFTR by the STAS domain of SLC26 transporters. Nat.Cell Biol. 6: 343-350, 2004.
59. Ko SB, Shcheynikov N , Choi JY, Luo X, Ishibashi K, Thomas PJ, Kim JY, Kim KH,
Lee MG, Naruse S, and Muallem S. A molecular mechanism for aberrant CFTRdependent HCO3- transport in cystic fibrosis. The EMBO Journal 21: 5662-5672, 2002.
60. Kocher O, Comella N, Gilchrist A, Pal R, Tognazzi K, Brown LF, and Knoll JH.
PDZK1, a novel PDZ domain-containing protein up-regulated in carcinomas and mapped
to chromosome 1q21, interacts with cMOAT (MRP2), the multidrug resistance-associated
protein. Lab.Invest. 79: 1161-1170, 1999.
61. Kocher O, Comella N, Tognazzi K, and Brown LF. Identification and partial characterization of PDZK1: a novel protein containing PDZ interaction domains. Lab.Invest. 78:
117-125, 1998.
62. Kocher O, Pal R, Roberts M, Cirovic C, and Gilchrist A. Targeted Disruption of the
PDZK1 Gene by Homologous Recombination. Mol Cell Biol 23: 1175-1180, 2003.
63. Kocher O, Yesilaltay A, Cirovic C, Pal R, Rigotti A, and Krieger M. Targeted Disruption of the PDZK1 Gene in Mice Causes Tissue-specific Depletion of the High Density
Lipoprotein Receptor Scavenger Receptor Class B Type I and Altered Lipoprotein Metabolism. J Biol.Chem. 278: 52820-52825, 2003.
64. Kunzelmann K and Mall M. Electrolyte Transport in the Mammalian Colon: Mechanisms
and Implications for Disease. Physiol.Rev. 82: 245-289, 2002.
Page 33 of 51
33
65. Kunzelmann K and McMorran B. First Encounter: How Pathogens Compromise Epithelial Transport. Physiology 19: 240-244, 2004.
65a.
Ladias JAA. Structural Insights into the CFTR-NHERF Interaction. J Membr Biol 192:
79-88, 2003.
66. Lamprecht G, Heil A, Baisch S, Lin-Wu E, Yun CC, Kalbacher H, Gregor M, and
Seidler U. 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, 2002.
67. Lamprecht G, Weinman EJ, and Yun CH. The role of NHERF and E3KARP in the
cAMP-mediated inhibition of NHE3. J Biol Chem 273: 29972-29978, 1998.
68. Lau AG and Hall RA. Oligomerization of NHERF-1 and NHERF-2 PDZ domains: differential regulation by association with receptor carboxyl-termini and by phosphorylation .
Biochemistry 40: 8572-8580, 2001.
69. Lee-Kwon W, Kim JH, Choi JW, Kawano K, Cha B, Dartt DA, Zoukhri D, and Donowitz M. Ca2+-dependent inhibition of NHE3 requires PKC which binds to E3KARP to
decrease surface NHE3 containing plasma membrane complexes. Am.J.Physiol. 285:
C1527-C1536, 2003.
70. Lee MG, Choi JY, Luo X, Strickland E, Thomas PJ, and Muallem S. Cystic fibrosis
transmembrane conductance regulator regulates luminal Cl-/HCO3- exchange in mouse
submandibular and pancreatic ducts. J Biol Chem 274: 14670-14677, 1999.
Page 34 of 51
34
71. Lee MG, Wigley WC, Zeng W, Noel LE, Marino CR, Thomas PJ, and Muallem S.
Regulation of Cl-/HCO3- exchange by cystic fibrosis transmembrane conductance regulator expressed in NIH 3T3 and HEK 293 cells. J Biol Chem 274: 3414-3421, 1999.
72. Leonard AS, Yermolaieva O, Hruska-Hageman A, Askwith CC, Price MP, Wemmie
JA, and Welsh MJ. cAMP-dependent protein kinase phosphorylation of the acid-sensing
ion channel-1 regulates its binding to the protein interacting with C-kinase-1. PNAS 100:
2029-2034, 2003.
73. Li C, Roy K, Dandridge K, and Naren AP. Molecular Assembly of Cystic Fibrosis
Transmembrane Conductance Regulator in Plasma Membrane. J Biol.Chem. 279:
24673-24684, 2004.
74. Li J, Dai Z, Jana D, Callaway DJE, and Bu Z. Ezrin Controls the Macromolecular Complexes Formed between an Adapter Protein Na+/H+ Exchanger Regulatory Factor and
the Cystic Fibrosis Transmembrane Conductance Regulator. J Biol.Chem. 280: 3763437643, 2005.
75. Li X, Galli T, Leu S, Wade JB, Weinman EJ, Leung G, Cheong A, Louvard D, and
Donowitz M. Na+-H+ exchanger 3 (NHE3) is present in lipid rafts in the rabbit ileal brush
border: a role for rafts in trafficking and rapid stimulation of NHE3. J Physiol (Lond) 537:
537-552, 2001.
76. Li X, Zhang H, Cheong A, Leu S, Chen Y , Elowsky CG, and Donowitz M. 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
(Lond) 556: 791-804, 2004.
Page 35 of 51
35
77. McWilliams RR, Gidey E, Fouassier L, Weed SA, and Doctor RB. Characterization of
an ankyrin repeat-containing Shank2 isoform (Shank2E) in liver epithelial cells. Biochem
J 380: 181-191, 2004.
78. Morales FC, Takahashi Y, Kreimann EL, and Georgescu MM. Ezrin-radixin-moesin
(ERM)-binding phosphoprotein 50 organizes ERM proteins at the apical membrane of
polarized epithelia. PNAS 101: 17705-17710, 2004.
79. Mount DB and Romero MF. The SLC26 gene family of multifunctional anion exchangers. Pflugers Arch. 447: 710-721, 2004.
80. Moyer BD, Denton J, Karlson KH, Reynolds D, Wang S, Mickle JE, Milewski M, Cutting GR, Guggino WB, Li M, and Stanton BA. A PDZ-interacting domain in CFTR is an
apical membrane polarization signal. J Clin Invest. 104: 1353-1361, 1999.
81. Moyer BD, Duhaime M, Shaw C, Denton J, Reynolds D, Karlson KH, Pfeiffer J,
Wang S, Mickle JE, Milewski M, Cutting GR, Guggino WB, Li M, and Stanton BA.
The PDZ-interacting Domain of Cystic Fibrosis Transmembrane Conductance Regulator
Is Required for Functional Expression in the Apical Plasma Membrane. J Biol.Chem.
275: 27069-27074, 2000.
82. Murtazina, R., O. Kovbasnjuk, D. Steplock, B.M. Hogema, E.J. Weinman, H.R. de
Jonge, and M. Donowitz. Two Photon Microscopic Measurement Of Brush Border (bb)
Nhe Activity Demonstrates That Nherf1 Is Necessary for cAMP Inhibition Of NHE3 in
Mouse Proximal Tubule But Not in Ileum. Gastroenterology 128 (Suppl. 2): A-367, 2005.
(Abstract)
Page 36 of 51
36
83. Nakamura T, Shibata N, Nishimoto-Shibata T, Feng D, Ikemoto M, Motojima K, Iso,
Tsukamoto K, Tsujimoto M, and Arai H. Regulation of SR-BI protein levels by phosphorylation of its associated protein, PDZK1. PNAS 102: 13404-13409, 2005.
84. Naren AP, Cobb B, Li C, Roy K, Nelson D, Heda GD, Liao J, Kirk KL, Sorscher EJ,
Hanrahan J, and Clancy JP. A macromolecular complex of beta 2 adrenergic receptor,
CFTR, and ezrin/radixin/moesin-binding phosphoprotein 50 is regulated by PKA. PNAS
100: 342-346, 2003.
85. Neudauer CL, Joberty G, and Macara IG. PIST: a novel PDZ/coiled-coil domain binding
partner for the rho-family GTPase TC10. Biochem Biophys.Res.Commun. 280: 541-547,
2001.
86. Novak I and Greger R . Properties of the luminal membrane of isolated perfused rat
pancreatic ducts. Effect of cyclic AMP and blockers of chloride transport. Pflugers Arch.
411: 546-553, 1988.
87. O'Reilly C, Winpenny J, Argent B, and Gray M. Cystic fibrosis transmembrane conductance regulator currents in guinea pig pancreatic duct cells: Inhibition by bicarbonate
ions. Gastroenterology 118: 1187-1196, 2000.
88. Ostedgaard LS, Randak C, Rokhlina T, Karp P, Vermeer D, Ashbourne E, and
Welsh MJ. Effects of C-terminal deletions on cystic fibrosis transmembrane conductance
regulator function in cystic fibrosis airway epithelia. PNAS 100: 1937-1942, 2003.
89. Penkert RR, DiVittorio HM, and Prehoda KE. Internal recognition through PDZ domain
plasticity in the Par-6-Pals1 complex. Nat Struct Mol Biol 11: 1122-1127, 2004.
Page 37 of 51
37
90. Peterson FC, Penkert RR, Volkman BF, and Prehoda KE. Cdc42 regulates the Par-6
PDZ domain through an allosteric CRIB-PDZ transition. Mol Cell 13: 665-676, 2004.
91. Poulsen JH, Fischer H, Illek B, and Machen TE. Bicarbonate Conductance and pH
Regulatory Capability of Cystic Fibrosis Transmembrane Conductance Regulator. PNAS
91: 5340-5344, 1994.
92. Puschett JB, Whitbred J, Ianosi-Irimie M, Vu HV, Rabon E, Robinson J, and Deininger P. Molecular effects of volume expansion on the renal sodium phosphate cotransporter. Am J Med.Sci. 326: 1-8, 2003.
93. Raghuram V, Hormuth H, and Foskett JK. A kinase-regulated mechanism controls
CFTR channel gating by disrupting bivalent PDZ domain interactions. PNAS 100: 96209625, 2003.
94. Raghuram V, Mak DO, and Foskett JK. Regulation of cystic fibrosis transmembrane
conductance regulator single-channel gating by bivalent PDZ-domain-mediated interaction. PNAS 98: 1300-1305, 2001.
95. Reczek D, Berryman M , and Bretscher A. Identification of EBP50: A PDZ-containing
phosphoprotein that associates with members of the ezrin-radixin-moesin family. J.Cell
Biol. 139: 169-179, 1997.
96. Redecker P, Gundelfinger ED, and Boeckers TM. The Cortactin-binding Postsynaptic
Density Protein ProSAP1 in Non-neuronal Cells. J.Histochem.Cytochem. 49: 639-648,
2001.
Page 38 of 51
38
97. Rossmann H, Jacob P, Baisch S, Hassoun R, Meier J, Natour D, Yahya K, Yun C,
Biber J, Lackner KJ, Fiehn W, Gregor M, Seidler U, and Lamprecht G. The CFTR
associated protein CAP70 interacts with the apical Cl/HCO3 exchanger DRA in rabbit
small intestinal mucosa. Biochemistry 44: 4477-4487, 2005.
98. Schultheis PJ, Clarke LL, Meneton P, Miller ML, Soleimani M, Gawenis LR, Riddle
TM, Duffy JJ, Doetschman T, Wang T, Giebisch G, Aronson PS, Lorenz JN, and
Shull GE. Renal and intestinal absorptive defects in mice lacking the NHE3 Na+/H+ exchanger. Nat Genet 19: 282-285, 1998.
99. Scott RO, Thelin WR, and Milgram SL. A Novel PDZ Protein Regulates the Activity of
Guanylyl Cyclase C, the Heat-stable Enterotoxin Receptor. J Biol.Chem. 277: 2293422941, 2002.
100. Sellin, J.H. Intestinal electrolyte absorption and secretion. In: Gastrointestinal and liver
disease, edited by M. Feldman, B.F. Scharschmidt, and M.H. Sleisenger. W.B. Saunders, 1997, p. 1451-1471.
101. Sheng M and Kim E. The Shank family of scaffold proteins. J.Cell Sci. 113: 1851-1856,
2000.
102. Sheng M and Sala C. PDZ domains and the organization of supramolecular complexes.
Annu.Rev.Neurosci. 24: 1-29, 2001.
103. Shenolikar S, Minkoff CM, Steplock DA, Evangelista C, Liu M, and Weinman EJ. Nterminal PDZ domain is required for NHERF dimerization. FEBS Lett. 489: 233-236,
2001.
Page 39 of 51
39
104. Shenolikar S, Voltz JW, Minkoff CM, Wade JB, and Weinman EJ. Targeted disruption
of the mouse NHERF-1 gene promotes internalization of proximal tubule sodiumphosphate cotransporter type IIa and renal phosphate wasting. PNAS 99: 11470-11475,
2002.
105. Sheppard DN and Welsh MJ. Structure and Function of the CFTR Chloride Channel.
Physiol.Rev. 79: 23-45, 1999.
106. Short DB, Trotter KW, Reczek D, Kreda SM, Bretscher A, Boucher RC, Stutts MJ,
and Milgram SL. An apical PDZ protein anchors the cystic fibrosis transmembrane conductance regulator to the cytoskeleton. J Biol Chem 273: 19797-19801, 1998.
107. Simpson JE, Gawenis LR, Walker NM, Boyle KT, and Clarke LL. Chloride conductance of CFTR facilitates basal Cl-/HCO3- exchange in the villous epithelium of intact murine duodenum. Am J Physiol 288: G1241-G1251, 2005.
108. Spiegel S, Phillipper M, Rossmann H, Riederer B, Gregor M, and Seidler U. Independence of apical Cl-/HCO3- exchange and anion conductance in duodenal HCO3- secretion. Am J Physiol 285: G887-G897, 2003.
109. Sun F, Hug JM, Bradbury NA, and Frizzell RA. Protein kinase A associates wit cystic
fibrosis transmembrane conductance regulator via an interaction with ezrin. J Biol Chem
275: 14360-14366, 2000.
Page 40 of 51
40
110. Sun F, Hug MJ, Lewarchik CM, Yun C-HC, Bradbury NA, and Frizzell RA. E3KARP
Mediates the Association of Ezrin and Protein Kinase A with the Cystic Fibrosis Transmembrane Conductance Regulator in Airway Cells. J Biol.Chem. 275: 29539-29546,
2000.
111. Swiatecka-Urban A, Duhaime M, Coutermarsh B, Karlson KH, Collawn J, Milewski
M, Cutting GR, Guggino WB, Langford G, and Stanton BA. PDZ Domain Interaction
Controls the Endocytic Recycling of the Cystic Fibrosis Transmembrane Conductance
Regulator. J Biol.Chem. 277: 40099-40105, 2002.
112. Vaandrager AB. Structure and function of the heat-stable enterotoxin receptor/guanylyl
cyclase C. Mol Cell Biochem 230: 73-83, 2002.
113. Wang D, Sun H, Lang F, and Yun CC. Activation of NHE3 by dexamethasone requires
phosphorylation of NHE3 at Ser663 by SGK1. Am.J.Physiol. 289: C802-C810, 2005.
114. Wang S, Yue H, Derin RB, Guggino WB, and Li M. Accessory protein facilitated CFTRCFTR interaction, a novel molecular mechanism to potentiate the chloride channel activity. Cell 103: 169-179, 2000.
115. Wang Z, Wang T, Petrovic S, Tuo B, Riederer B, Barone S, Lorenz JN, Seidler U,
Aronson PS, and Soleimani M. Renal and intestinal transport defects in Slc26a6-null
mice. Am.J.Physiol. 288: C957-C965, 2005.
116. Weinman EJ, Steplock D, Donowitz M, and Shenolikar S. NHERF associations with
sodium-hydrogen exchanger isoform 3 (NHE3) and ezrin are essential for cAMPmediated phosphorylation and inhibition of NHE3. Biochemistry 39: 6123-6129, 2000.
Page 41 of 51
41
117. Weinman EJ, Steplock D, Wang Y, and Shenolikar S. Characterization of a protein
cofactor that mediates protein kinase A regulation of the renal brush border membrane
Na+-H+ exchanger. J.Clin.Invest. 95: 2143-2149, 1995.
118. Wheat VJ, Shumaker H, Burnham C, Shull GE, Yankaskas JR, and Soleimani M.
CFTR induces the expression of DRA along with Cl-/HCO3- exchange activity in tracheal
epithelial cells. Am J Physiol. 279: C62-C71, 2000.
119. Yao R, Ito C, Natsume Y, Sugitani Y, Yamanaka H, Kuretake S, Yanagida K, Sato A,
Toshimori K, and Noda T. Lack of acrosome formation in mice lacking a Golgi protein,
GOPC. PNAS 99: 11211-11216, 2002.
120. Yeo CJ, Barry K, Gontarek JD, and Donowitz M. Na+/H+ exchange mediates mealstimulated ileal absorption. Surgery 116: 388-394, 1994.
121. Yun CC, Chen Y, and Lang F. Glucocorticoid Activation of Na+/H+ Exchanger Isoform 3
Revisited. THE ROLES OF SGK1 AND NHERF2. J Biol.Chem. 277: 7676-7683, 2002.
122. Yun CHC, Gurubhagavatula S, Levine SA, Montgomery JL, Brant SR, Cohen ME,
Cragoe EJ, Jr., Pouyssegur J, Tse CM, and Donowitz M. Glucocorticoid stimulation of
ileal Na+ absorptive cell brush border Na+/H+ exchange and association with an increase
in message for NHE-3, an epithelial Na+/H+ exchanger isoform. J.Biol.Chem. 268: 206211, 1993.
123. Yun CHC, Lamprecht G, Foster DV, and Sidor A. 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, 1998.
Page 42 of 51
42
124. Yun CHC, Oh S, Zizak M, Steplock D, Tsao S, Tse CM, Weinman EJ, and Donowitz
M. cAMP-mediated inhibition of the epithelial brush border Na+/H+ exchanger, NHE3, requires an associated regulatory protein. PNAS 94: 3010-3015, 1997.
125. Zachos, N., X. Li, C. Hodson, B. Cha, Y. Chen, S.L. Milgram, and M. Donowitz. Brush
Border (bb) PDZ Proteins PDZk1 and Ikepp (intestinal and Kidney Epithelial PDZ Domain
Protein) Differentially Regulate NHE3 in Response To Elevated Ca2+: Inhibition With
PDZk1 and Stimulation With Ikepp. Gastroenterology 128 (Suppl. 2): A-32, 2005 (Abstract).
126. Zachos NC, Tse M, and Donowitz M. Molecular physiology of intestinal Na+/H+ exchange. Annu.Rev.Physiol. 67: 411-443, 2005.
127. Zizak M, Lamprecht G, Steplock D, Tariq N, Shenolikar S, Donowitz M, Yun CHC,
and Weinman EJ. cAMP-induced Phosphorylation and Inhibition of Na+/H+ Exchanger 3
(NHE3) Are Dependent on the Presence but Not the Phosphorylation of NHE Regulatory
Factor. J.Biol.Chem. 274: 24753-24758, 1999.
Page 43 of 51
43
Figure legends
Figure 1: The role of the PDZ adapter proteins NHERF and CAL in membrane traffic of CFTR
The PDZ interaction motif of CFTR is required for reinsertion of CFTR from early endosomes/recycling endosomes back into the plasma membrane; the PDZ adapter protein is
NHERF or possibly another PDZ adapter protein such as PDZK1. CAL is predominantly localized in the Golgi. It directs CFTR for lysosomal degradation and thereby decreases total cellular
as well as plasma membrane CFTR. NHERF counteracts the effect of CAL. A constitutively active form of the small rho-GTPase TC10, which binds to the second coiled-coil motif of CAL, also
counteracts the effect of CAL. TC10 may act as a molecular switch between CFTR recycling and
CFTR degradation.
Page 44 of 51
44
Figure 2: PDZ interaction of CFTR
Several models for the role of PDZ adapter proteins in the activation of CFTR have been developed. A: CFTR channel open probability is increased by the formation of CFTR dimers through
interaction with common PDZ adapter proteins like NHERF (93;94) or PDZK1 (114). NHERF
primarily binds with its first PDZ domain to one CFTR molecule. After activation of Ezrin ( )
NHERF binds with its ERM binding domain to the N-terminal domain of Ezrin ( ). This increases
the affinity of the second PDZ domain for CFTR ( ) facilitating the formation of dimers (74). B:
Similar to figure 3 Ezrin may function as an A kinase anchoring protein (AKAP) allowing PKA to
phosphorylate CFTR in a complex of CFTR, E3KARP or NHERF, Ezrin, and PKA
(45;106;109;110). The models shown in panel A and B may coexist. C: CFTR and SLC26A3
(DRA) or SLC26A6 (PAT1) function is increased by interaction of their R- and STAS domains.
This complex is further stabilized by the interaction of both proteins with a common PDZ adapter
protein (58;59).
Figure 3: Involvement of NHERF and E3KARP in PKC and PKA regulation of NHE3
E3KARP but not NHERF facilitates the formation of a multiprotein complex including NHE3,
-
to the complex is Ca2+ dependent.
-
Actinin-4 and PKC . Binding of
-Actinin-4 and PKC
Actinin-4 also binds to Actin linking the complex to the cytoskeleton. The downstream targets of
PKC are unknown.
E3KARP or NHERF facilitate the formation of a multiprotein complex including NHE3 and Ezrin.
In that complex Ezrin is believed to act as an A kinase anchoring protein (AKAP) for PKA type II.
Stimulation of PKA results in the phosphorylation of NHE3 at S552 and S605, ultimately leading
to the inhibition of NHE3, which may involve a decrease in NHE3 transport rate as well as a decrease of the amount of NHE3 in the plasma membrane.
Page 45 of 51
45
Figure 4: Working model for the interaction of NHE3 and DRA
Intestinal Na+/H+ and Cl-/HCO3- exchange are functionally coupled by the intracellular or subapical pH (56;57). NHE3 interacts with the second PDZ domain of E3KARP or NHERF (123). DRA
also interacts with the second PDZ domain of E3KARP (66). A common complex may be formed
by the dimerization of two E3KARP or NHERF molecules through their PDZ domains
(31;68;103).
Page 46 of 51
46
Tables
Table 1: Overview of the PDZ adapter proteins discussed
PDZ adapter protein
Synonym/ ortholog
Multimerization
Phosphorylated
Knockout
NHERF (117)
(NHERF-1)
Mouse, rat and rabbit:
NHERF (117)
(31;68;103)
S289, S301
and S162
A: (104)
B: (78)
Human: EBP50 (95)
E3KARP (124)
(NHERF-2)
NHERF-2
(68;121)
Probably not
but see (16)
**
PDZK1 (61)
Mouse: CAP70 (114) or
NaPi-Cap1 (35)
Heterotypic
oligomerization
to NHERF (34)
(83)
(62;63)
(NHERF-3)
Rabbit: CAP70 (97)
Rat: diphor-1 (22) or
PDZ-dc1 (92) or
CLAMP (46)
IKEPP (99)
(NHERF-4)
Mouse: NaPi-Cap2 (35)
n.d.
n.d.
Shank2*
(CortBP1) (54)
CortBP1* (29)
n.d.
n.d.
(12)
n.d.
ProSAP1* (8)
Shank2E* (77)
CAL (12)
GOPC (119)
(119)
PIST (85)
FIG (11)
*CortBP1, ProSAP1 and Shank2E are splice variants of Shank2; Shank2 discribed by (54) is
actually CortBP1.
** Hogema et al., Dept. of Biochemistry, Erasmus Medical Center, Rotterdam, The Netherlands
GOPC: Golgi associated PDZ and coiled-coil domain containing
PIST: PDZ domain protein interacting specifically with TC10
FIG: fused in glioblastoma
n.d. not done
Page 47 of 51
47
Page 48 of 51
Endocytosis:
• Dynamin-dependent
• PDZ-independent
Membrane insertion:
• PDZ-dependent
EBP50
(PDZK1)
CAL
Early Endosomes/
recycling endosomes
TC10
Lysosome
Golgi
CAL
Figure 1
Page 49 of 51
Regulation by PKC
Regulation by PKA
NHE3
NHE3
Plasmamembrane
S552 + S605
E3KARP
but not
NHERF
PKC
E3KARP
or
NHERF
Ezrin
-Actinin-4
Dynamin ?
AP2 ?
Actin
Synaptojanin ?
Clathrin ?
>> NHE3-inhibition by
increased endocytosis
PKA
Actin
Figure 2
Page 50 of 51
CFTR
B
CFTR
CFTR
R-Dom
3
NHERF
E3KARP/NHERF
2
Ezrin
Ezrin (closed)
1
PKA
Ezrin (open)
Actin
CFTR
C
CFTR
SLC26A3
or
CFTR
R-Dom
SLC26A6
STAS
A
NHERF
Ezrin
Actin
PDZ adapter protein;
(e.g. NHERF or E3KARP)
Figure 3
Page 51 of 51
Cl
Na
DRA
HCO3
NHE3
pHi
H+
Dimerization mediated by
PDZ domain interaction ?
Ezrin
PKA
Figure 4