Download PDZ proteins retain and regulate membrane transporters in

Am J Physiol Cell Physiol 288: C20 –C29, 2005;
doi:10.1152/ajpcell.00368.2004.
Invited Review
PDZ proteins retain and regulate membrane transporters
in polarized epithelial cell membranes
Bert Brône and Jan Eggermont
Laboratory of Physiology, Katholieke Universiteit Leuven, Campus Gasthuisberg O & N, Leuven, Belgium
retention of apical and basolateral membrane proteins; transducisomes; protein
complex formation
DIFFERENTIATION OF EPITHELIAL CELLS consists of two major steps:
apicobasal polarization and asymmetrical protein expression.
The first process leads to the delineation of apical and basolateral membrane domains that are separated by tight junctions
(63). The second step entails the asymmetrical sorting of
proteins to the polarized membrane domains (45). During the
past few years, it has emerged that PDZ proteins—their name
derives from the first three proteins in which PDZ domains
were identified: the postsynaptic density protein PSD-95/SAP90, the Drosophila septate junction protein Disc-large DLG,
and the epithelial tight junction protein zonula occludens
(ZO)-1 (30)—are crucial components in both processes (for
review, see Refs. 63, 64). In this review, we focus on the role
of PDZ proteins in the polarized expression and function of
membrane transporters (e.g., ion channels, exchangers, cotransporters) in epithelial cells, mainly of renal origin. Interestingly, the apicobasal differentiation process in epithelial
cells shares some striking similarities with the axodendritic
polarization in neurons, among which is the involvement of a
common set of PDZ proteins (26, 64).
Correct surface expression of membrane proteins in a polarized epithelial cell is a multistep process (45) involving 1)
biosynthetic sorting from the trans-Golgi network to the correct plasma membrane domain (apical or basolateral), 2) anchoring and/or retention at the plasma membrane, and 3)
Address for reprint requests and other correspondence: J. Eggermont,
Laboratory of Physiology, Campus Gasthuisberg O & N, Catholic University of Leuven, Herestraat 49, B-3000 Leuven, Belgium (E-mail:
[email protected]).
C20
endocytosis followed by either recycling to the plasma membrane or targeting for lysosomal degradation or delivery to the
opposing membrane domain (i.e., transcytosis). As discussed
below, PDZ proteins have been implied in one or more of these
steps for both apical and basolateral membrane proteins.
PDZ DOMAINS, PROTEINS, AND LIGANDS
The cellular structure and function strongly depend on the
coordinated cooperation of proteins. At the molecular level,
protein-protein interactions are driven by structurally conserved domains that specifically recognize peptide ligands in
other proteins (55). The most abundant protein interaction
domains in metazoan organisms are the PDZ domains (58).
The primary function of PDZ domains is to recognize and bind
to specific approximately five-amino acid motifs that occur at
the COOH terminus of target proteins. Depending on the target
sequence, PDZ domains are traditionally classified as class I
domains, which recognize the consensus sequence X⫺3(S/T)⫺2-X⫺1-␾0 (X ⫽ any amino acid; ␾ ⫽ hydrophobic
amino acid; ⫺3, ⫺2, ⫺1, and 0, the position relative to the
COOH-terminal residue) and in class II domains (target sequence: X⫺3-␾⫺2-X⫺1-␾0) (22). Thus amino acids at positions
0 (the COOH-terminal amino acid) and ⫺2 are critical determinants for PDZ domain binding. The relatively simple architecture of the ligand is such that a single PDZ domain can bind
to proteins with (slightly) different PDZ ligands. In addition, a
growing number of PDZ domains have been shown to recognize internal PDZ-binding motifs, so-called ␤-fingers that
structurally mimic a COOH-terminal end (21). The structural
basis of peptide binding and the classification of PDZ domain-
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Brône, Bert, and Jan Eggermont. PDZ proteins retain and regulate membrane
transporters in polarized epithelial cell membranes. Am J Physiol Cell Physiol 288:
C20 –C29, 2005; doi:10.1152/ajpcell.00368.2004.—The plasma membrane of epithelial cells is subdivided into two physically separated compartments known as
the apical and basolateral membranes. To obtain directional transepithelial solute
transport, membrane transporters (i.e., ion channels, cotransporters, exchangers,
and ion pumps) need to be targeted selectively to either of these membrane
domains. In addition, the transport properties of an epithelial cell will be maintained
only if these membrane transporters are retained and properly regulated in their
specific membrane compartments. Recent reports have indicated that PDZ domaincontaining proteins play a dual role in these processes and, in addition, that
different apical and basolateral PDZ proteins perform similar tasks in their respective membrane domains. First, although PDZ-based interactions are dispensable for
the biosynthetic targeting to the proper membrane domain, the PDZ network
ensures that the membrane proteins are efficiently retained at the cell surface.
Second, the close spatial positioning of functionally related proteins (e.g., receptors,
kinases, channels) into a signal transduction complex (transducisome) allows fast
and efficient control of membrane transport processes.
Invited Review
PDZ INTERACTIONS IN EPITHELIAL CELLS
containing proteins (simply called “PDZ proteins” in the following text) have been reviewed in two publications (22, 72).
On the basis of their structure, PDZ proteins can be divided
into three major groups: 1) PDZ-only proteins containing one
or more PDZ domains; 2) membrane-associated guanylate
kinase (MAGUK) domain-containing PDZ proteins; and 3) the
largest group, harboring PDZ proteins that contain other protein-interacting domains, e.g., SH3 domains, PH domains, and
L27 domains, in addition to their PDZ domains. Examples of
every group can be found in Fig. 1. The functional importance
of this structural classification is that the latter two classes of
PDZ proteins can easily participate in higher-order protein
networks because of their multiple binding sites.
In the past few years, a complex network of PDZ interactions involved in the apical expression of membrane proteins
has emerged (47). Much of this work has been performed in
kidney (e.g., proximal tubule), airways, and intestine and has
heavily focused on the effects of PDZ proteins on the expression and function of Na⫹/H⫹ exchanger type 3 (NHE3), cystic
fibrosis transmembrane conductance regulator (CFTR), and
Na⫹-Pi cotransporter IIa (Npt2 or NaPi IIa). NHE3 (82) and
Npt2 (48) are ion transporters in the apical membrane (brush
border) of the renal proximal tubule, whereas CFTR is an
apical Cl⫺ channel in multiple exocrinic tissues (e.g., airway,
intestine, pancreas, sweat gland) (67). All three transporters
contain a prototype COOH-terminal class I PDZ ligand
(NHE3: -S⫺3-T⫺2-H⫺1-M0; Npt2: -A⫺3-T⫺2-R⫺1-L0; CFTR:
-D⫺3-T⫺2-R⫺1-L0), which allows them to interact with a variety of PDZ proteins. As reviewed below, these PDZ interactions drastically influence the localization and function of the
transporters.
GOPC favors the intracellular retention and degradation
of CFTR. GOPC {Golgi-associated PDZ coiled-coil protein
(78), also called PIST [PDZ domain protein interacting specifically with TC10 (50)], CAL [CFTR-associated ligand (8)] and
FIG [fused in glioblastoma (7)]} is a scaffolding protein with
two predicted coiled-coiled domains and one PDZ domain
(Fig. 1) that binds to the COOH terminus of several membrane
proteins, including CFTR (8), frizzled (a plasma membrane
receptor) (78), and ClC-3B (an intracellular Cl⫺ channel) (15).
Endogenous and heterologously expressed GOPC is primarily
located at the Golgi apparatus and the trans-Golgi network
(TGN) (7, 8). Apparently, GOPC associates indirectly with the
Golgi by binding to syntaxin 6 via its second coiled-coil
domain (7). Syntaxin 6 is a SNARE (soluble N-ethylmale-
Fig. 1. The schematic structure of PDZ domain-containing
proteins that play a role in polarized expression of membrane
proteins. Alternative names of the PDZ proteins are shown in
parentheses. The interactions between PDZ proteins that occur
in epithelial cells are indicated by solid arrows, the specific
neuronal interactions by dashed arrows, and the link to the actin
cytoskeleton by dotted arrows. Only Munc-interacting protein
(Mint)2 and not Mint 3 has a Munc-interacting domain as
indicated by the dashed Munc-interacting domain (MID) sign.
4.1, protein 4.1-interacting domain or Hook domain; CAL,
CFTR-associated ligand (8); CaMK, calmodulin kinase II-like
domain; CAP70, CFTR accessory protein-70 (74); CASK,
calcium/calmodulin-dependent serine protein kinase (24); CID,
CASK-interacting domain; CC, coiled-coil domain; E3KARP,
NHE3 kinase A regulatory protein (81); EBP50, ezrin-radixinmoesin-binding phosphoprotein-50 (62); ERM, ezrin-radixinmoesin-binding domain; FIG, Fused in glioblastoma (7);
GOPC, Golgi-associated PDZ and coiled-coil motif-containing
protein (78); GuK, guanylate kinase-like domain; L27, L27
domain; MALS, mammalian Lin-7 (28); Mint, Munc18-1interacting protein (52, 53); NaPi-Cap1, Na⫹/phosphate cotransporter-CFTR accessory protein 1 (18); NHERF, Na⫹/H⫹
exchanger regulatory factor (81); P4.1, protein 4.1 (10); PDZ,
PDZ domain; PDZK1, PDZ containing 1 (32); PIST, PDZ
domain protein interacting specifically with TC10 (50); PTB,
phosphotyrosine-binding domain; SAP97, synapse-associated
protein 97 (36); SH3, SH3 domain; Veli, vertebrate Lin-7 (4).
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PDZ Proteins Involved in the Apical Expression
and Function of Membrane Proteins
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Invited Review
C22
PDZ INTERACTIONS IN EPITHELIAL CELLS
AJP-Cell Physiol • VOL
PDZ proteins that favor plasma membrane expression of CFTR
(e.g., NHERF-1); another possibility is a dynamic interaction
between GOPC and CFTR due to regulation of the PDZ
binding (e.g., phosphorylation).
NHERF-1 and -2 are involved in signaling complex assembly and anchoring in the apical membrane. Na⫹/H⫹ exchanger
regulatory factors (NHERF) are PDZ proteins that associate
with the apical brush-border membrane in polarized epithelial
cells. NHERF-1 was originally cloned as an essential cofactor
for the protein kinase A (PKA)-mediated inhibition of NHE3 in
renal proximal tubule cells (66, 77). The human ortholog of
NHERF-1 is also known as EBP50 (ezrin-radixin-moesinbinding phosphoprotein-50) (62). NHERF-2, also known as
E3KARP, is a structurally and functionally related isoform
(81). In mouse proximal tubule cells, NHERF-1 is strongly
expressed in microvilli, whereas NHERF-2 can be detected
weakly in the microvilli but is predominantly expressed at the
microvillar basis in the vesicle-rich domain (73). NHERF-1
and -2 contain two tandem PDZ domains (Fig. 1) that can bind
to a long list of ion channels (CFTR, ROMK K⫹ channels), ion
transporters (NHE3, Npt2), and receptors (␤2-adrenergic receptor; see Fig. 2) with a relative degree of specificity for
binding to the first or the second PDZ domain (75). NHERF
proteins also contain at their COOH terminus an ezrin-radixinmoesin (ERM)-binding domain that interacts with ezrin, a
scaffolding protein associated with the actin cytoskeleton. In
addition, ezrin functions as an A kinase anchoring protein
(AKAP): it interacts with the regulatory subunit (RII) of PKA,
thereby recruiting PKA to the PDZ complex. The multivalent
properties of NHERF (PDZ1, PDZ2, ERM-binding domain)
allow the formation of macromolecular complexes that anchor
membrane proteins to the apical actin cytoskeleton. Typical
examples include the NHE3-NHERF-1-ezrin and Npt2NHERF-1-ezrin complexes attached to the actin cytoskeleton
in the microvilli of proximal tubule cells in the kidney (73).
Another example of the scaffolding properties of NHERF is
found in airway and intestine epithelial cells with the formation
of apical CFTR-NHERF-1-ezrin or CFTR-NHERF-2-ezrin
complexes (49, 69). Finally, it should be mentioned that
although NHERF-1 and -2 share overlapping expression and
binding profiles, they do seem to fulfill unique properties with
respect to various membrane proteins, possibly due to the
partly different subcellular localization (73).
How do these NHERF-based complexes affect function and
expression of apical membrane transporters? In general, one
can discern three modes of action: 1) formation of transducisome complexes for efficient signal transduction, 2) enhancement of apical membrane expression, and 3) direct modulation
of the transporters.
First, because NHERF are multivalent scaffolds, they can
assemble functionally connected proteins (receptors, kinases,
transporters) into a spatially restricted unit (so-called transducisome), which allows tight control and efficient activation/
inhibition of membrane transport processes. For example, the
basal Na⫹/H⫹ exchange activity in brush-border membranes of
renal proximal tubule cells is indiscernible between wild-type
and NHERF-1-deficient mice. However, the cAMP-dependent
inhibition of NHE3 and the concomitant cAMP-stimulated
phosphorylation of NHE3 are observed only in NHERF-1expressing preparations (76). In addition, the stimulating effect
of glucocorticoids on NHE3 activity is seen only in the pres-
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imide-sensitive factor-activating protein receptor) protein localized in TGN and endosomes and is involved in vesicular
transport between TGN and endosomes (2, 34). However,
GOPC is also found in the plasma membrane when coexpressed in COS-7 cells with the plasma membrane protein
frizzled (78) or with a dominant-negative dynamin 2 that
blocks clathrin-mediated endocytosis (9). This suggests that
GOPC undergoes dynamic trafficking between the Golgi apparatus and the plasma membrane.
There is strong evidence that overexpression of GOPC
interferes with the surface expression of CFTR. Indeed, overexpression of GOPC (or CAL) in CFTR-expressing COS-7
cells reduces cAMP-activated Cl⫺ currents by decreasing the
number of channels in the plasma membrane. The reduced
surface expression of CFTR is caused both by a decreased rate
of insertion in the plasma membrane and by a decrease in
plasma membrane half-life of CFTR (8). Of note, CFTR
surface expression is restored to normal when GOPC and
NHERF-1, an apical PDZ protein (see NHERF-1 and -2 are
involved in signaling complex assembly anchoring in the apical membrane), are coexpressed. Furthermore, Cheng et al. (9)
showed that GOPC does not affect biosynthetic maturation of
CFTR in the Golgi apparatus but that it targets mature CFTR
for lysosomal degradation. Interestingly, the negative effects of
GOPC on CFTR are counteracted by coexpression with dominant-negative dynamin 2, in which case GOPC is trapped at
the cell periphery together with the dominant-negative dynamin 2. More evidence of a role for GOPC in modulating
protein expression stems from work on ClC-3, an intracellular
Cl⫺ channel that probably serves as an electrical shunt facilitating acidification of intracellular organelles by V-type H⫹
pumps. Different splice isoforms of the ClC-3 have been
identified (51). ClC-3A does not exhibit a PDZ ligand and is
located in late endosomes. In contrast, ClC-3B primarily resides in the Golgi apparatus and is identical to ClC-3A, except
for its COOH terminus, which contains a class I PDZ ligand
(S⫺3-T⫺2-T⫺1-L0) that interacts with GOPC as well as
NHERF-1 (15, 51). Overexpression of GOPC has a dual effect
on ClC-3B (15). First, ClC-3B becomes heavily concentrated
in the Golgi region colocalizing with GOPC. Second, it drastically reduces the expression level of ClC-3B, suggesting that
it promotes degradation of ClC-3B.
To conclude, GOPC is a Golgi/TGN-based PDZ protein that
reduces the apical membrane expression of CFTR. Although
its mode of action is not fully understood, one can think of two
mechanisms that are not mutually exclusive (9). One possibility is that GOPC interferes with the biosynthetic sorting of
CFTR at the level of TGN. By binding to CFTR, it may prevent
CFTR from being included in secretory transport vesicles
destined for the plasma membrane and redirect CFTR toward
the endosomal/lysosomal compartment. Alternatively, GOPC
may affect the endocytic recycling of CFTR by promoting the
lysosomal targeting of endocytosed CFTR. Regardless of the
mechanism, the net outcome would be enhanced lysosomal
degradation of CFTR and, consequently, reduced plasma membrane expression. However, it remains to be established that
the GOPC effects on CFTR are physiologically relevant rather
than an artifact due to heterologous overexpression. An intriguing question in the case of physiological relevance is how
CFTR normally escapes the negative regulation by GOPC. One
explanation could be competition between GOPC and other
Invited Review
PDZ INTERACTIONS IN EPITHELIAL CELLS
C23
ence of NHERF-2 (80), which recruits the serum- and glucocorticoid-induced protein kinase 1 (SGK1) in a PDZ-dependent interaction to the apical surface in close proximity to
NHE3. Remarkably, this activation could not be duplicated by
NHERF-1 (80). These results indicate that phosphorylation of
NHE3 occurs in a local complex in which the NHERF scaffold
efficiently approximates the kinases and their substrate NHE3.
Similarly, the CFTR-NHERF complex facilitates channel activation by cAMP/PKA. Indeed, the CFTR-NHERF complexes
contain not only PKA (recruited via ezrin) but also, in airway
epithelial cells, the ␤2-adrenergic receptor (␤2AR), which
binds to NHERF-1 via its COOH-terminal PDZ ligand (49,
69). Taken together, the NHERF-ezrin scaffold assembles a
macromolecular complex comprising ␤2AR, PKA, and CFTR
in which the close proximity between receptor, kinase, and ion
channel promotes efficient regulation of CFTR Cl⫺ currents by
␤2-agonists. Thus one function of NHERF-based protein complexes is the formation of multiprotein signaling complexes
(transducisomes) in apical membranes.
Second, NHERF proteins also affect membrane expression
and localization of apical proteins. This is most clearly illustrated for Npt2, which normally resides in the apical brushborder membrane of renal proximal tubule cells. However, in
kidneys from NHERF-1⫺/⫺ mice, Npt2 is redistributed to
intracellular vesicles, thereby reducing Npt2 expression in the
apical membrane (65). Consistent with the altered Npt2 localization, NHERF-1⫺/⫺ mice display a renal phosphate wasting
phenotype. Interestingly, NHERF-1 is not required for apical
localization of NHE3 in the proximal tubule, because NHE3
AJP-Cell Physiol • VOL
was expressed at normal levels in the apical brush-border
membrane of NHERF-1⫺/⫺ kidneys (65). In contrast,
NHERF-2 seems to affect the surface expression of NHE3 in a
dual way. NHERF-2 mediates the glucocorticoid and lysophosphatidic acid-induced increase in NHE3 activity by stimulating
exocytosis and plasma membrane delivery of NHE3 (37, 80).
On the other hand, elevated intracellular Ca2⫹ concentrations
diminish NHE3 activity and membrane expression through
oligomerization and endocytosis of NHE3, which requires the
formation of an NHERF-2-NHE3-␣-actinin-4 complex (31).
These data indicate that NHERF isoforms in epithelial cells
play a crucial role in the apical positioning of some (but clearly
not all) PDZ ligand-containing membrane proteins. However,
many of the underlying mechanisms remain to be resolved. Is
NHERF-1 required for apical sorting of Npt2, or does it
interfere with the anchoring or endocytic turnover of Npt2 at
the apical membrane? The latter possibility is an attractive
hypothesis, because a major regulatory step for Npt2 expression in the apical membrane is endocytosis followed by either
recycling to the plasma membrane or sorting to lysosomes for
degradation (48). In addition, such a mechanism would be
consistent with the evidence that NHERF-1 promotes endocytic recycling of CFTR. Another question prompted by these
studies is what determines the apical localization of NHE3 in
the absence of NHERF-1. Is there functional redundancy
between PDZ proteins, and can NHERF-2 and/or PDZK1
substitute for NHERF-1? Consistent with this possibility is the
recent observation that in mouse proximal tubules, both
NHERF-1 and NHERF-2 interact with NHE3, whereas only
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Fig. 2. Putative interaction pattern of PDZ proteins involved in apical expression of membrane proteins in epithelia. The PDZ domain-containing proteins (shown
in ovals) interact with various membrane transporters (rounded rectangles) and membrane receptors (rectangles). Additional links to signaling or cytoskeletal
molecules are shown. The dashed lines show mutual interactions between PDZ proteins. The shaded circles and the corresponding arrow indicate a putative
common role for PDZ interactions in the subcellular location of the transporters. ␤2AR, ␤2-adrenergic receptor (20, 49, 69); CFEX, Cl⫺ formate exchanger (17);
ClC3B, Cl⫺ channel type 3 splice form B (15); CFTR, cystic fibrosis transmembrane conductance regulator (8, 49, 69); D-AKAP2, dual specific A-kinase
anchoring protein 2 (16); ezrin (see Refs. 49, 69); GOPC, Golgi-associated PDZ and coiled-coil motif-containing protein (9, 15); MAP17, membrane-associated
protein (59); NHE3, Na⫹ proton exchanger (17, 73, 77); NHERF, Na⫹/H⫹ exchanger regulatory factor (17); Npt2, type II Na⫹-dependent phosphate
cotransporter (17, 73); OCTN1, organic cation transporter (17); P2Y1, purinergic receptor (20); PDZK1, PDZ containing 1 (17); PKA, protein kinase A; PTH1R,
parathyroid hormone receptor (20, 41); ROMK, renal outer medullary K⫹ channel (79); SGK1, serum- and glucocorticoid-induced protein kinase 1 (80); SR-BI,
scavenger receptor type BI (33); URAT1, renal specific urate anion exchanger (17).
Invited Review
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PDZ INTERACTIONS IN EPITHELIAL CELLS
AJP-Cell Physiol • VOL
Npt2 in intracellular vesicles in NHERF-1-deficient proximal
tubule cells (65) is also consistent with a role for NHERF-1 in
the recycling of internalized Npt2 to the brush-border membrane. To conclude, there are several lines of evidence indicating that PDZ-based interactions with NHERF-1 promote
surface expression of a variety of membrane proteins (CFTR,
Npt2, ␤2AR), possibly by affecting the postendocytic sorting
step. However, it should be kept in mind that in several
experimental systems, NHERF-1 plays only a minor role or no
role in the apical localization of CFTR (1).
A third mode of interaction between NHERF proteins and
apical transport proteins is direct modulation of the transport
process. For example, addition of recombinant NHERF-1 containing the two PDZ domains (PDZ1-PDZ2) to excised membrane patches from a lung submucosal gland cell stimulates the
open probability of CFTR single channels (61). This effect
requires the presence of two intact PDZ domains in a single
peptide and can be competed by the addition of single PDZ
domains. In addition, PKC phosphorylation of the second PDZ
domain in NHERF-1 disrupts the PDZ2-CFTR interaction and
precludes the stimulatory effect of NHERF-1 on CFTR open
probability (60). One interpretation of these experiments is that
the functional CFTR channel is a homodimeric complex and
that multivalent PDZ proteins such as NHERF-1 promote or
stabilize the homodimeric conformation by capturing the
COOH termini of two CFTR molecules.
PDZK1 is a docking station for apical membrane proteins.
PDZK1 (also called CAP70 and NaPi-Cap1) is a PDZ-only
protein containing four PDZ domains and is expressed in
kidney, intestine, and liver (18, 32). Renal expression of
PDZK1 is restricted to the proximal tubule, where it localizes
to the apical brush-border membrane (microvilli and intermicrovillar clefts) (18). Interestingly, PDZK1 seems to be devoid
of an intrinsic apical sorting signal because apical positioning
requires the interaction with other proteins such as MAP17, an
apical membrane protein that forms a PDZ-dependent complex
with PDZK1 (59).
Murer and colleagues (16, 17, 25) have proposed that in
proximal tubule cells, PDZK1 and NHERF-1 form an extended
network underneath the brush-border membrane that serves as
a docking station for apical proteins. Because of its multivalent
binding properties, PDZK1 is a crucial stabilizer of this protein
network. First, because of its four PDZ domains, PDZK1 can
simultaneously bind several membrane transporters via their
COOH-terminal PDZ ligands: Npt2 (NaPi IIa), the solute
carrier SLC17A1 (NaPi I), NHE3, the organic cation transporter (OCTN1), the Cl⫺ formate exchanger (CFEX), and the
urate anion exchanger (URAT1) (17). Second, PDZK1 forms
heteromeric complexes with NHERF-1 (17), which anchors the
network to the apical cytoskeleton. In addition, several types of
signaling proteins are recruited by the PDZK1/NHERF-1 scaffold. G protein-coupled receptors, such as the purinergic P2Y1
receptor or the parathyroid hormone I receptor (PTHIR) receptor, bind to NHERF via a PDZ-based interaction (20, 41).
Furthermore, PKA is positioned in the apical network via
various AKAP such as ezrin and D-AKAP2, which binds to
PDZK1 (16). So, PDZK1, in combination with NHERF, establishes an extensive network of transport and signaling proteins beneath the brush-border membrane of renal proximal
tubule cells, but further exploration of the precise physiological
relevance of the NHERF-1/PDZK1 is required.
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NHERF-1 associates with Npt2 (73). Alternatively, PDZ-independent mechanisms, e.g., direct association with the apical
cytoskeleton, could be involved in the apical sorting and/or
retention of NHE3.
Whether PDZ-based interactions are also required for the
apical expression of CFTR is a controversial issue. The PDZ
ligand of CFTR was initially proposed by Stanton and colleagues (46, 47) to be an apical targeting signal because a
truncation mutant of CFTR lacking the three COOH-terminal
amino acids (CFTR-⌬TRL) had lost its apical positioning in
Madin-Darby canine kidney (MDCK) cells and human airway
epithelial cells and distributed nearly equally in the apical and
basolateral membrane. Because deletion of the PDZ ligand also
abolishes CFTR binding to NHERF-1, it was concluded that
apical polarization of CFTR requires an interaction with
NHERF-1 (47). In a subsequent study, Stanton and colleagues
(70) showed that there is no difference in the initial polarized
targeting between newly synthesized wild-type CFTR and
CFTR-⌬TRL when stably transfected in MDCK cells. In
contrast, Lukacs and colleagues (1) showed that disruption of
the CFTR-NHERF complex (by COOH-terminal epitope tagging of CFTR, COOH-terminal deletion of CFTR, or overexpression of a dominant-negative NHERF mutant) does not
interfere at all with the apical localization of CFTR in polarized
epithelia derived from the trachea, pancreatic duct, intestine,
and distal tubule of the kidney. However, a common point in
the observations of both groups is that the COOH-terminal
PDZ ligand does not function as a sorting signal for the (initial)
biosynthetic targeting of CFTR from the trans-Golgi network
to the apical membrane.
Still, controversy exists about the role of PDZ proteins in the
apical expression of CFTR. Deletion of the PDZ ligand reduced the half-life of CFTR in the apical membrane, and it also
diminished the recycling of endocytosed CFTR back to the
plasma membrane (70). It was therefore concluded that the
PDZ ligand in CFTR functions as an apical endocytic recycling
motif and that PDZ-based interactions, e.g., with NHERF-1,
affect the endocytic turnover of CFTR by promoting the
reinsertion of internalized CFTR in the plasma membrane (70).
In contrast, truncation of the PDZ ligand in the COOH terminus of CFTR did not affect the apical expression when membrane half-life was measured in pancreatic duct cells and baby
hamster kidney (BHK) cells (1). However, a role for PDZ
interactions in the “recycling” mechanism would also explain
the functional antagonism between NHERF-1 and GOPC, with
the former increasing apical positioning of CFTR by promoting
endocytic recycling and the latter decreasing apical positioning
by promoting lysosomal targeting of endocytosed CFTR (8).
Furthermore, similar experiments on the endocytic turnover of
G protein-coupled receptors suggest that the postendocytic
sorting step could be a key action point for NHERF-1. Recycling to the plasma membrane of endocytosed ␤2AR depends
on an intact PDZ ligand in the ␤2AR COOH terminus (-D⫺3S⫺2-S⫺1-L0) and requires an interaction between the receptor
and NHERF-1 (5). Moreover, transplantation of the COOHterminal PDZ ligand of ␤2AR (-D⫺3-S⫺2-S⫺1-L0) to the ␦-opioid receptor changes the postendocytic sorting of the opioid
receptor. The endocytosed mutant ␦-opioid receptor is rapidly
recycled to the plasma membrane instead of being targeted to
the lysosomal compartment for degradation as found in the
wild-type ␦-opioid receptor (14). Finally, the accumulation of
Invited Review
PDZ INTERACTIONS IN EPITHELIAL CELLS
Finally, it should be mentioned that other functions have
been attributed to PDZK1. First, similar to what has been
described for NHERF-1, addition of recombinant PDZK1 increases the open probability of CFTR single channels (74).
This stimulation depends on an intact PDZ ligand in CFTR and
on the presence of two functional PDZ domains (PDZ3-PDZ4)
in PDZK1, which is consistent with the hypothesis that multivalent PDZ proteins such as PDZK1 and NHERF-1 favor a
more active dimer configuration of CFTR (61, 74). Second,
expression of the scavenger receptor class B type I (SR-BI) in
hepatocytes critically depends on PDZK1 (33). PDZK1⫺/⫺
mice show strongly reduced levels of hepatic SR-BI and
consequently have increased plasma levels of high-density
lipoprotein particles.
An evolutionary conserved PDZ protein complex associates
with the basolateral membrane. Research on the LET-23
epithelial growth factor receptor (EGFR) of the nematode
Caenorhabditis elegans revealed an important role of the
heterotrimeric PDZ complex Lin-10-Lin-2-Lin-7 in the basolateral localization of the LET-23 receptor in vulval precursor
cells. In these processes, Lin-7 accounts for the binding to
LET-23 via a PDZ-based interaction (for review, see Refs. 29,
64). Three laboratories (3, 4, 29) independently discovered that
the mammalian homologs of Lin-10, Lin-2 and Lin-7 interact
with each other and can be found in an evolutionary conserved,
tripartite complex. The three mammalian homologs of Lin-10
are called Munc-interacting proteins (Mint1–3) for their interaction with the synaptic protein Munc18-1 or X11 (X11␣-␥)
(52, 53). The mammalian Lin-2 homolog CASK is a MAGUK
protein and was found to bind to neurexins, a family of
neuronal cell surface molecules (24). Three mammalian Lin-7
counterparts were identified and were given various names:
mLin7a, mLin7b, and mLin7c for mammalian Lin-7 (3, 27);
Veli-1, Veli-2, and Veli-3 for vertebrate Lin-7 (4); and
MALS1, 2, and 3 for mammalian Lin-7 (28, 43). For simplicity, we refer to them as Veli-1, Veli-2, and Veli-3.
At the core of the heterotrimeric PDZ complex is CASK, a
MAGUK protein with multiple protein interaction domains
(see Fig. 1), among which are two L27 domains, one PDZ
domain, a calmodulin kinase (CaMK)-like domain, a protein
4.1-binding or Hook domain, and a guanylate kinase domain.
L27 domains are conserved interaction modules that form
heterodimers (and dimers of dimers) with L27 domains in other
proteins, thereby providing the molecular basis for the formation of an extended protein network (13, 40). It has been shown
for both the C. elegans and the rat proteins that the second L27
domain (L27C) in CASK heterodimerizes with the L27 domain
in Veli, whereas the first L27 domain (L27N) forms a complex
with the L27 domain of SAP97, another MAGUK PDZ protein
(23, 36). In addition, CASK can bind to Mint1 via its NH2terminal CaMK-like domain. However, CASK is unable to
form complexes with either Mint2 or Mint3 because both Mint
isoforms lack the critical interaction domain (42). In view of
the different expression profiles of Mint1 (neuronal) and Mint3
(ubiquitous), this implies that heterotrimeric Veli-CASK-Mint
complexes are found in the brain, where they are involved in
AJP-Cell Physiol • VOL
synapse formation (64). In contrast, renal epithelial cells contain dimeric Veli-CASK complexes that are localized to the
basolateral membrane (42, 68). Finally, the Hook domain
connects CASK to protein 4.1, an actin-binding protein in the
cortical actin cytoskeleton (10). In contrast, Velis are small
proteins with a relatively simple structure; in addition to one
L27 domain, with which they bind to CASK, they also contain
one PDZ domain. Basolateral location of Veli in renal epithelia
depends on an intact L27 domain, indicating that Veli is
recruited to the basolateral membrane via binding to CASK
(68). Thus, in epithelial cells, the Veli-CASK core complex
forms a large basolateral protein network that is anchored to
the actin cytoskeleton and that exposes protruding PDZ domains that are free to interact with other proteins. In addition,
other PDZ proteins such as SAP97, a MAGUK protein, can be
recruited to this complex (36).
Velis retain transporters in the basolateral membrane in
renal epithelial cells. Velis are reported to bind via a PDZbased interaction to several basolateral membrane transporters,
such as the strong inward rectifier K⫹ channel (Kir2.3) and the
epithelial ␥-aminobutyric acid (GABA) transporter (BGT-1)
(54, 57). Both proteins contain a bona fide class I PDZ ligand:
-E⫺3-S⫺2-A⫺1-I0 (Kir2.3), -E⫺3-T⫺2-H⫺1-L0 (BGT-1). As in
the apical expression process of membrane transporters, the
PDZ ligand of these basolateral transporters is required for
efficient retention in the basolateral membrane rather than for
the initial sorting process to the basolateral membrane. First,
BGT-1 with a truncated PDZ motif (deletion of the 5 COOHterminal amino acids) is still targeted to the basolateral membrane of MDCK cells, but its surface expression is reduced
because of increased internalization in an endosomal recycling
compartment (57). Moreover, injection of a small synthetic
peptide (17-mer) that corresponds to the BGT-1 COOH terminus and binds in vitro to Veli-1 also relocates BGT-1 from the
basolateral surface to an intracellular vesicular compartment.
These observations are therefore consistent with a Veli-1dependent retention in the basolateral membrane, although a
role for other PDZ proteins cannot be formally excluded.
Second, Veli-2-mediated plasma membrane retention has also
been reported for Kir2.3 channels expressed in MDCK cells
(54). The cytosolic COOH terminus of the Kir2.3 protein
contains two partially overlapping motifs: a biosynthetic sorting signal (amino acids 431– 442) and a class I PDZ ligand
(amino acids 442– 445) (38). Deletion of the COOH-terminal
15 amino acids (431– 445) or of amino acids 431– 441 results
in apical missorting of the channel (38). In contrast, selective
deletion of the PDZ ligand (amino acids 442– 445) causes
channels to accumulate in intracellular vesicles that partially
overlap with recycling endosomes (54). Furthermore, it was
shown that Veli-2 and Kir2.3 interact in a PDZ-dependent way
and that Kir2.3, Veli-2, and CASK colocalize at the basolateral
membrane of the renal cortical collecting duct, which is consistent with the basolateral presence of a Kir2.3-Veli-2-CASK
complex. A functional correlate of such a complex was obtained by coexpression of Kir2.3 and Veli-2 in Xenopus oocytes, which results in an increased K⫹ current due to a
Veli-2-promoted membrane retention of Kir2.3 (54). A third
line of evidence in favor of Veli-mediated basolateral membrane retention stems from work on the Drosophila ShakerB
K⫹ channel (44). ShakerB contains a COOH-terminal class I
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PDZ Proteins Affecting Basolateral Expression
of Membrane Proteins
C25
Invited Review
C26
PDZ INTERACTIONS IN EPITHELIAL CELLS
OUTLOOK
The available data indicate that PDZ proteins play a crucial
role in the localization and function of apical and basolateral
membrane ion channels and transporters in epithelial cells.
However, to fully appreciate the role of PDZ proteins, one
should also consider the aspects discussed next.
Dynamics of PDZ Interactions and PDZ Complexes
As described above, Npt2 is part of an apical PDZK1/
NHERF-1-based protein network that extends all along the
microvillus of proximal tubule cells. Yet, after PTH stimulation, Npt2 is endocytosed via invaginations at the base of the
microvilli (71). Thus, in PTH-stimulated proximal tubule cells,
Npt2 proteins that are located at the microvillar tip must travel
down the microvillus to the intermicrovillar cleft at the base.
However, there is evidence that a PDZ network restricts the
lateral mobility of bound membrane proteins (6). This prompts
the question of whether Npt2 endocytosis requires prior disruption of the Npt2-PDZK1 and Npt2-NHERF-1 complexes so
that NpT2 is free to move laterally in the brush-border membrane and, if so, how this disruption is triggered, e.g., by
phosphorylation (see Regulation of PDZ interactions). Alternatively, it has recently been shown for CFTR that PDZ
interactions are dynamic and do not result in immobilization of
CFTR in the plasma membrane (19).
Second, in MDCK cells, there is evidence for a dynamic
interaction between Veli-1 and ␤-catenin that contains a class
I PDZ ligand (-D⫺3-T⫺2-D⫺1-L0) (56). The progressive translocation of Veli-1 from the cytosol to the junctional domain
during polarization of MDCK cells requires the presence of
␤-catenin in the junctional domain and depends on a PDZbased interaction between Veli-1 and ␤-catenin. Furthermore,
only Veli-1 proteins that have been translocated to the junctional domain are able to retain BGT-1 transporters in the
basolateral membrane (also a PDZ-dependent effect). Because
Veli-1 has only one PDZ domain, this implies that Veli-1 must
first disengage from ␤-catenin before it can interact with
AJP-Cell Physiol • VOL
basolateral membrane proteins such as BGT-1 or Kir2.3, but at
present it is not known what regulates the Veli-1-␤-catenin
interaction.
Recent work on Veli-CASK-SAP97 complexes in brain also
hints at a dynamic rearrangement of these PDZ complexes
(39). The Kir2.2 K⫹ channel has a COOH-terminal class I PDZ
ligand (-E⫺3-S⫺2-E⫺1-I0), which binds to PDZ domains in Veli
and SAP97. In neurons, Kir2.2 seems to be incorporated in two
different PDZ complexes dependent on the partnering PDZ
protein. Kir2.2 is bound in the first complex to SAP97, which
also recruits CASK and the CASK-associated proteins Veli and
Mint1. Kir2.2 is in the second complex attached to Veli-1 or -3,
which then associates with CASK and the CASK-associated
SAP97. It has been proposed that these two complexes serve
different functions: the Kir2.2-SAP97-based complex would
be involved in membrane trafficking of the channel, whereas
the Kir2.2-Veli complex would be involved in membrane
anchoring. This would require a rearrangement of the PDZ
interactions whereby Kir2.2, upon arrival in the plasma membrane, is transferred from the PDZ domain of SAP97 to the
PDZ domain of Veli-1 or -3 (39). However, at this time, it is
not clear what regulates or triggers such a dynamic rearrangement. It remains to be established whether dynamic rearrangement of CASK/Veli complexes is also involved in the basolateral expression of membrane proteins in epithelial cells.
Regulation of PDZ Interactions
Because typical class I PDZ ligands contain a serine or
threonine at position ⫺2, PDZ ligands can in principle be
phosphorylated, which opens the perspective for regulation of
PDZ interactions. This was clearly shown for the interaction
between ␤2AR and NHERF-1, which is under the control of G
protein-regulated kinase (GRK). GRK-mediated phosphorylation of serine at position ⫺2 (Ser411) in the COOH terminus of
␤2AR precluded the NHERF-1-dependent recycling of the
receptor to the plasma membrane (5). Moreover, a serine to
aspartate mutation at position ⫺2, which mimics the effect of
phosphorylation, inhibited both complex formation with
NHERF-1 and recycling of internalized mutant receptors. Similarly, serine phosphorylation of the Kir2.3 PDZ ligand
(-ESAI) by PKA has been shown to disrupt Kir2.3 interaction
with the neuronal PDZ protein PSD-95, which could potentially alter the postsynaptic anchoring of Kir2.3 or change the
gating characteristics of the channel (11). Whether phosphorylation of the Kir2.3 PDZ ligand also occurs in renal epithelial
cells and whether this would affect basolateral expression is
still an open question.
Furthermore, phosphorylation outside the PDZ ligand has
also been reported to modulate PDZ complex composition. For
example, PKA phosphorylation of the CFTR R domain inhibited binding of CFTR to NHERF-1, but it had no effect on the
␤2AR-NHERF-1 interaction (49). Clearly, phosphorylationdependent PDZ interactions allow for variations in PDZ complexes as a function of specific extracellular stimuli or developmental stage. Consequently, regulation of PDZ interactions
should be kept in mind when addressing the physiological
modulation of transport proteins incorporated into a PDZ
network.
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PDZ ligand (-E⫺3-T⫺2-D⫺1-V0) and, after expression in
MDCK cells, is sorted to the basolateral membrane, where it
forms a complex with Veli and CASK (44). Deletion of the
COOH-terminal 45 amino acids disrupts the basolateral sorting. However, a mutant in which only the PDZ ligand is
removed (deletion of the COOH-terminal 3 amino acids) is still
sorted to the basolateral membrane, but this mutant again
accumulates in intracellular vesicles. From these results, one
can conclude that 1) the COOH-terminal PDZ ligands in
BGT-1, Kir2.3, and ShakerB do not function as biosynthetic
sorting signals for the basolateral membrane; 2) these membrane transporters are incorporated in a basolateral Veli/CASK
complex; and 3) interaction with Veli promotes basolateral
membrane retention of BGT-1, Kir2.3, and ShakerB.
Whether the basolateral Veli-CASK network is also required
for the spatial assembly of signal transduction complexes is
less clear. In neurons, PKA is recruited to the Veli-CASK
complex via SAP97, a MAGUK protein that binds AKAP79/
150 (12). However, it remains to be established whether
SAP97, which associates with the basolateral Veli/CASK complex in epithelial cells (36), also recruits PKA to the basolateral
membrane and, if so, what the functional consequences are.
Invited Review
PDZ INTERACTIONS IN EPITHELIAL CELLS
Spatial Limitations of a PDZ Network
Functional Competition Between PDZ Proteins
As mentioned above, the surface expression of CFTR is
reduced by overexpression of GOPC in COS-7 cells and can be
rescued again by the coexpression of GOPC and NHERF-1,
suggesting a functional competition between the Golgi-associated GOPC and the plasma membrane-associated NHERF-1,
with the outcome of the competition being determined by the
relative amounts of either protein (8). A relevant question
therefore is whether such a situation also occurs in vivo, i.e.,
whether antagonizing pairs of PDZ proteins can form an on-off
switch for PDZ-bound proteins that could be regulated by
changes in expression level or phosphorylation of one of the
PDZ proteins. Another potential mechanism for functional
competition stems from the multimeric composition of PDZ
complexes, which renders them sensitive to dominant-negative
effects. Indeed, overexpression of a dominant-negative CASK
isoform in MDCK cells results in the mislocalization of endogenous SAP97 (35). Again, it remains to be clarified whether
dominant-negative isoforms of PDZ proteins occur in vivo
(e.g., as splice variants or because of gene mutations) and what
their physiological or pathophysiological role would be.
CONCLUSIONS
PDZ proteins play a crucial role in the correct positioning of
membrane proteins in polarized epithelial cells. On the basis of
the above evidence discussed in this review, we propose that
PDZ proteins form an extended protein network underneath the
apical and basolateral membrane that is linked to both the
membrane and the cytoskeleton. The apical network consists of
a NHERF1-PDZK1 scaffold that associates with the brushborder membrane of proximal tubule cells. At the basolateral
side, there is evidence for a Veli-CASK scaffold in MDCK
cells and collecting duct cells. Such a protein network would
then constitute a large “Velcro strip” because of the protruding
PDZ domains. A variety of membrane proteins (e.g., ion
channels, transporters, receptors) is then recruited to this scaffold via PDZ-based interactions with two functional conseAJP-Cell Physiol • VOL
quences. First, although PDZ-based interactions are dispensable for the biosynthetic targeting to the proper membrane
domain, the PDZ network ensures that the membrane proteins
are efficiently retained at the cell surface, either by downregulating the endocytosis or by promoting surface recycling of
endocytosed transporters. Second, the close spatial positioning
of functionally related proteins (e.g., receptors, kinases, channels) into a signal transduction complex allows fast and efficient control of membrane transport processes. In this respect,
PDZ scaffolds function as social networks in humans: You
need an invitation ticket (i.e., a biosynthetic sorting signal) to
get in, but once entered, you are firmly anchored in the network
(via PDZ-based interactions), and you get a lot of functional
bonuses (i.e., transducisomes).
GRANTS
Research in our laboratory is supported by the Forton Foundation (Koning
Boudewijn Stichting, Belgium) and the Fund for Scientific Research-Flanders
(FWO-Vlaanderen).
REFERENCES
1. Benharouga M, Sharma M, So J, Haardt M, Drzymala L, Popov M,
Schwappach 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 –22089, 2003.
2. Bock JB, Klumperman J, Davanger S, and Scheller RH. Syntaxin 6
functions in trans-Golgi network vesicle trafficking. Mol Biol Cell 8:
1261–1271, 1997.
3. Borg JP, Straight SW, Kaech SM, de Taddeo-Borg M, Kroon DE,
Karnak D, Turner RS, Kim SK, and Margolis B. Identification of an
evolutionarily conserved heterotrimeric protein complex involved in protein targeting. J Biol Chem 273: 31633–31636, 1998.
4. Butz S, Okamoto M, and Sudhof TC. A tripartite protein complex with
the potential to couple synaptic vesicle exocytosis to cell adhesion in
brain. Cell 94: 773–782, 1998.
5. Cao TT, Deacon HW, Reczek D, Bretscher A, and von Zastrow M. A
kinase-regulated PDZ-domain interaction controls endocytic sorting of the
␤2-adrenergic receptor. Nature 401: 286 –290, 1999.
6. Cha B, Kenworthy A, Murtazina R, and Donowitz M. The lateral
mobility of NHE3 on the apical membrane of renal epithelial OK cells is
limited by the PDZ domain proteins NHERF1/2, but is dependent on an
intact actin cytoskeleton as determined by FRAP. J Cell Sci 117: 3353–
3365, 2004.
7. 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 N-ethylmaleimide-sensitive fusion protein (NSF)
attachment protein receptor) protein syntaxin 6. J Biol Chem 276: 29456 –
29465, 2001.
8. 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.
9. 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.
10. Cohen AR, Woods DF, Marfatia SM, Walther Z, Chishti AH, Anderson JM, and Wood DF. Human CASK/LIN-2 binds syndecan-2 and
protein 4.1 and localizes to the basolateral membrane of epithelial cells.
J Cell Biol 142: 129 –138, 1998.
11. Cohen NA, Brenman JE, Snyder SH, and Bredt DS. Binding of the
inward rectifier K⫹ channel Kir 2.3 to PSD-95 is regulated by protein
kinase A phosphorylation. Neuron 17: 759 –767, 1996.
12. Colledge M, Dean RA, Scott GK, Langeberg LK, Huganir RL, and
Scott JD. Targeting of PKA to glutamate receptors through a MAGUKAKAP complex. Neuron 27: 107–119, 2000.
13. Feng W, Long JF, Fan JS, Suetake T, and Zhang M. The tetrameric
L27 domain complex as an organization platform for supramolecular
assemblies. Nat Struct Mol Biol 11: 475– 480, 2004.
288 • JANUARY 2005 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 17, 2017
A PDZ scaffold such as the NHERF-1-PDZK1 network can
in principle bind a whole array of ion channels, transporters,
receptors, and kinases. However, this does not necessarily
imply that all of these proteins are close neighbors in the cell
and therefore can functionally interact. Indeed, biochemical
and yeast two-hybrid experiments indicate that Npt2, NHE3,
and D-AKAP2 all bind to PDZK1 (17). However, NHE3 and
Npt2 are localized in the microvilli (brush border), whereas
D-AKAP2 is found at the base of microvilli and in the
subapical compartment (16). The different subcellular localization therefore renders it unlikely that D-AKAP2 is involved
in the regulation of transport activity of either Npt2 or NHE3.
However, because Npt2 endocytosis is stimulated by PKA and
endocytosis occurs at the base of microvilli, it may well be that
D-AKAP2 plays a role either in the endocytosis at the microvillar base or in the sorting from the subapical compartment
to the lysosomes. Thus the biochemical analysis of complex
composition should always be complemented by a morphological analysis to find out whether the different PDZ-bound
proteins have the same spatial distribution within the cell.
C27
Invited Review
C28
PDZ INTERACTIONS IN EPITHELIAL CELLS
AJP-Cell Physiol • VOL
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
canalicular multispecific organic anion transporter-related transporter.
Cancer Res 58: 2741–2747, 1998.
Lee S, Fan S, Makarova O, Straight S, and Margolis B. A novel and
conserved protein-protein interaction domain of mammalian Lin-2/CASK
binds and recruits SAP97 to the lateral surface of epithelia. Mol Cell Biol
22: 1778 –1791, 2002.
Lee-Kwon W, Kawano K, Choi JW, Kim JH, and Donowitz M.
Lysophosphatidic acid stimulates brush border Na⫹/H⫹ exchanger 3
(NHE3) activity by increasing its exocytosis by an NHE3 kinase A
regulatory protein-dependent mechanism. J Biol Chem 278: 16494 –
16501, 2003.
Le Maout S, Welling PA, Brejon M, Olsen O, and Merot J. Basolateral
membrane expression of a K⫹ channel, Kir 2.3, is directed by a cytoplasmic COOH-terminal domain. Proc Natl Acad Sci USA 98: 10475–10480,
2001.
Leonoudakis D, Conti LR, Radeke CM, McGuire LMM, and Vandenberg CA. A multiprotein trafficking complex composed of SAP97,
CASK, Veli, and Mint1 is associated with inward rectifier Kir2 potassium
channels. J Biol Chem 279: 19051–19063, 2004.
Li Y, Karnak D, Demeler B, Margolis B, and Lavie A. Structural basis
for L27 domain-mediated assembly of signaling and cell polarity complexes. EMBO J 23: 2723–2733, 2004.
Mahon MJ, Donowitz M, Yun CC, and Segre GV. Na⫹/H⫹ exchanger
regulatory factor 2 directs parathyroid hormone 1 receptor signalling.
Nature 417: 858 – 861, 2002.
Margolis B, Borg JP, Straight S, and Meyer D. The function of PTB
domain proteins. Kidney Int 56: 1230 –1237, 1999.
Misawa H, Kawasaki Y, Mellor J, Sweeney N, Jo K, Nicoll RA, and
Bredt DS. Contrasting localizations of MALS/LIN-7 PDZ proteins in
brain and molecular compensation in knockout mice. J Biol Chem 276:
9264 –9272, 2001.
Moreno J, Cruz-Vera LR, Garcia-Villegas MR, and Cereijido M.
Polarized expression of Shaker channels in epithelial cells. J Membr Biol
190: 175–187, 2002.
Mostov K, Su T, and ter Beest M. Polarized epithelial membrane traffic:
conservation and plasticity. Nat Cell Biol 5: 287–293, 2003.
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.
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.
Murer H, Hernando N, Forster I, and Biber J. Regulation of Na/Pi
transporter in the proximal tubule. Annu Rev Physiol 65: 531–542, 2003.
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 ␤2 adrenergic receptor, CFTR, and ezrin/radixin/moesinbinding phosphoprotein 50 is regulated by PKA. Proc Natl Acad Sci USA
100: 342–346, 2003.
Neudauer CL, Joberty G, and Macara IG. PIST: a novel PDZ/coiledcoil domain binding partner for the rho-family GTPase TC10. Biochem
Biophys Res Commun 280: 541–547, 2001.
Ogura T, Furukawa T, Toyozaki T, Yamada K, Zheng YJ, Katayama
Y, Nakaya H, and Inagaki N. ClC-3B, a novel ClC-3 splicing variant that
interacts with EBP50 and facilitates expression of CFTR-regulated ORCC.
FASEB J 16: 863– 865, 2002.
Okamoto M and Sudhof TC. Mints, Munc18-interacting proteins in
synaptic vesicle exocytosis. J Biol Chem 272: 31459 –31464, 1997.
Okamoto M and Sudhof TC. Mint 3: a ubiquitous mint isoform that does
not bind to munc18-1 or -2. Eur J Cell Biol 77: 161–165, 1998.
Olsen O, Liu H, Wade JB, Merot J, and Welling PA. Basolateral
membrane expression of the Kir 2.3 channel is coordinated by PDZ
interaction with Lin-7/CASK complex. Am J Physiol Cell Physiol 282:
C183–C195, 2002.
Pawson T, Raina M, and Nash P. Interaction domains: from simple
binding events to complex cellular behavior. FEBS Lett 513: 2–10, 2002.
Perego C, Vanoni C, Massari S, Longhi R, and Pietrini G. Mammalian
LIN-7 PDZ proteins associate with ␤-catenin at the cell-cell junctions of
epithelia and neurons. EMBO J 19: 3978 –3989, 2000.
288 • JANUARY 2005 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 17, 2017
14. Gage RM, Kim KA, Cao TT, and von Zastrow M. A transplantable
sorting signal that is sufficient to mediate rapid recycling of G proteincoupled receptors. J Biol Chem 276: 44712– 44720, 2001.
15. Gentzsch M, Cui L, Mengos A, Chang XB, Chen JH, and Riordan JR.
The PDZ-binding chloride channel ClC-3B localizes to the Golgi and
associates with cystic fibrosis transmembrane conductance regulator-interacting PDZ proteins. J Biol Chem 278: 6440 – 6449, 2003.
16. Gisler SM, Madjdpour C, Bacic D, Pribanic S, Taylor SS, Biber J, and
Murer H. PDZK1: II. an anchoring site for the PKA-binding protein
D-AKAP2 in renal proximal tubular cells. Kidney Int 64: 1746 –1754,
2003.
17. 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.
18. 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.
19. Haggie PM, Stanton BA, and Verkman AS. Increased diffusional
mobility of CFTR at the plasma membrane after deletion of its C-terminal
PDZ binding motif. J Biol Chem 279: 5494 –5500, 2004.
20. Hall RA, Ostedgaard LS, Premont RT, Blitzer JT, Rahman N, Welsh
MJ, and Lefkowitz RJ. A C-terminal motif found in the ␤2-adrenergic
receptor, P2Y1 receptor and cystic fibrosis transmembrane conductance
regulator determines binding to the Na⫹/H⫹ exchanger regulatory factor
family of PDZ proteins. Proc Natl Acad Sci USA 95: 8496 – 8501, 1998.
21. Harris BZ, Hillier BJ, and Lim WA. Energetic determinants of internal
motif recognition by PDZ domains. Biochemistry 40: 5921–5930, 2001.
22. Harris BZ and Lim WA. Mechanism and role of PDZ domains in
signaling complex assembly. J Cell Sci 114: 3219 –3231, 2001.
23. Harris BZ, Venkatasubrahmanyam S, and Lim WA. Coordinated
folding and association of the LIN-2, -7 (L27) domain: an obligate
heterodimerization involved in assembly of signaling and cell polarity
complexes. J Biol Chem 277: 34902–34908, 2002.
24. Hata Y, Butz S, and Sudhof TC. CASK: a novel dlg/PSD95 homolog
with an N-terminal calmodulin-dependent protein kinase domain identified by interaction with neurexins. J Neurosci 16: 2488 –2494, 1996.
25. Hernando N, Wagner CA, Gisler SM, Biber J, and Murer H. PDZ
proteins and proximal ion transport. Curr Opin Nephrol Hypertens 13:
569 –574, 2004.
26. Horton AC and Ehlers MD. Neuronal polarity and trafficking. Neuron
40: 277–295, 2003.
27. Irie M, Hata Y, Deguchi M, Ide N, Hirao K, Yao I, Nishioka H, and
Takai Y. Isolation and characterization of mammalian homologues of
Caenorhabditis elegans Lin-7: localization at cell-cell junctions. Oncogene 18: 2811–2817, 1999.
28. Jo K, Derin R, Li M, and Bredt DS. Characterization of MALS/Velis-1,
-2, and -3: a family of mammalian LIN-7 homologs enriched at brain
synapses in association with the postsynaptic density-95/NMDA receptor
postsynaptic complex. J Neurosci 19: 4189 – 4199, 1999.
29. Kaech SM, Whitfield CW, and Kim SK. The LIN-2/LIN-7/LIN-10
complex mediates basolateral membrane localization of the C. elegans
EGF receptor LET-23 in vulval epithelial cells. Cell 94: 761–771, 1998.
30. Kennedy MB. Origin of PDZ (DHR, GLGF) domains. Trends Biochem
Sci 20: 350, 1995.
31. Kim JH, Lee-Kwon W, Park JB, Ryu SH, Yun CH, and Donowitz M.
Ca2⫹-dependent inhibition of Na⫹/H⫹ exchanger 3 (NHE3) requires an
NHE3-E3KARP-␣-actinin-4 complex for oligomerization and endocytosis. J Biol Chem 277: 23714 –23724, 2002.
32. 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.
33. 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.
34. Kuliawat R, Kalinina E, Bock J, Fricker L, McGraw TE, Kim SR,
Zhong J, Scheller R, and Arvan P. Syntaxin-6 SNARE involvement in
secretory and endocytic pathways of cultured pancreatic ␤-cells. Mol Biol
Cell 15: 1690 –1701, 2004.
35. Lee K, Belinsky MG, Bell DW, Testa JR, and Kruh GD. Isolation of
MOAT-B, a widely expressed multidrug resistance-associated protein/
Invited Review
PDZ INTERACTIONS IN EPITHELIAL CELLS
AJP-Cell Physiol • VOL
70. 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.
71. Traebert M, Roth J, Biber J, Murer H, and Kaissling B. Internalization
of proximal tubular type II Na-Pi cotransporter by PTH: immunogold
electron microscopy. Am J Physiol Renal Physiol 278: F148 –F154, 2000.
72. Van Ham M and Hendriks W. PDZ domains-glue and guide. Mol Biol
Rep 30: 69 – 82, 2003.
73. Wade JB, Liu J, Coleman RA, Cunningham R, Steplock DA, LeeKwon W, Pallone TL, Shenolikar S, and Weinman EJ. Localization
and interaction of NHERF isoforms in the renal proximal tubule of the
mouse. Am J Physiol Cell Physiol 285: C1494 –C1503, 2003.
74. Wang S, Yue H, Derin RB, Guggino WB, and Li M. Accessory protein
facilitated CFTR-CFTR interaction, a molecular mechanism to potentiate
the chloride channel activity. Cell 103: 169 –179, 2000.
75. Wang S, Raab RW, Schatz PJ, Guggino WB, and Li M. Peptide
binding consensus of the NHE-RF-PDZ1 domain matches the C-terminal
sequence of cystic fibrosis transmembrane conductance regulator (CFTR).
FEBS Lett 427: 103–108, 1998.
76. Weinman EJ, Steplock D, and Shenolikar S. NHERF-1 uniquely transduces the cAMP signals that inhibit sodium-hydrogen exchange in mouse
renal apical membranes. FEBS Lett 536: 141–144, 2003.
77. 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.
78. Yao R, Maeda T, Takada S, and Noda T. Identification of a PDZ
domain containing Golgi protein, GOPC, as an interaction partner of
frizzled. Biochem Biophys Res Commun 286: 771–778, 2001.
79. Yoo D, Flagg TP, Olsen O, Raghuram V, Foskett JK, and Welling PA.
Assembly and trafficking of a multiprotein ROMK (Kir 1.1) channel
complex by PDZ interactions. J Biol Chem 279: 6863– 6873, 2004.
80. 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.
81. Yun CH, 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. Proc Natl Acad Sci USA 94: 3010 –3015, 1997.
82. Yun CH, Tse CM, Nath SK, Levine SA, Brant SR, and Donowitz M.
Mammalian Na⫹/H⫹ exchanger gene family: structure and function studies. Am J Physiol Gastrointest Liver Physiol 269: G1–G11, 1995.
288 • JANUARY 2005 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on June 17, 2017
57. Perego C, Vanoni C, Villa A, Longhi R, Kaech SM, Frohli E, Hajnal
A, Kim SK, and Pietrini G. PDZ-mediated interactions retain the epithelial GABA transporter on the basolateral surface of polarized epithelial
cells. EMBO J 18: 2384 –2393, 1999.
58. Ponting CP. Evidence for PDZ domains in bacteria, yeast, and plants.
Protein Sci 6: 464 – 468, 1997.
59. Pribanic S, Gisler SM, Bacic D, Madjdpour C, Hernando N, Sorribas
V, Gantenbein A, Biber J, and Murer H. Interactions of MAP17 with
the NaPi-IIa/PDZK1 protein complex in renal proximal tubular cells. Am J
Physiol Renal Physiol 285: F784 –F791, 2003.
60. Raghuram V, Hormuth H, and Foskett JK. A kinase-regulated mechanism controls CFTR channel gating by disrupting bivalent PDZ domain
interactions. Proc Natl Acad Sci USA 100: 9620 –9625, 2003.
61. Raghuram V, Mak DOD, and Foskett JK. Regulation of cystic fibrosis
transmembrane conductance regulator single-channel gating by bivalent
PDZ-domain-mediated interaction. Proc Natl Acad Sci USA 98: 1300 –
1305, 2001.
62. 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.
63. Roh MH and Margolis B. Composition and function of PDZ protein
complexes during cell polarization. Am J Physiol Renal Physiol 285:
F377–F387, 2003.
64. Rongo C. Disparate cell types use a shared complex of PDZ proteins for
polarized protein localization. Cytokine Growth Factor Rev 12: 349 –359,
2001.
65. Shenolikar S, Voltz JW, Minkoff CM, Wade JB, and Weinman EJ.
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 USA 99: 11470 –11475, 2002.
66. Shenolikar S and Weinman EJ. NHERF: targeting and trafficking
membrane proteins. Am J Physiol Renal Physiol 280: F389 –F395, 2001.
67. Sheppard DN and Welsh MJ. Structure and function of the CFTR
chloride channel. Physiol Rev 79, Suppl: S23–S45, 1999.
68. Straight SW, Karnak D, Borg JP, Kamberov E, Dare H, Margolis B,
and Wade JB. mLin-7 is localized to the basolateral surface of renal
epithelia via its NH2 terminus. Am J Physiol Renal Physiol 278: F464 –
F475, 2000.
69. Sun F, Hug MJ, Lewarchik CM, Yun CH, 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.
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