1 Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201; and 2 Departement de Biologie Cellular et Moleculaire, Commissariate Energie Atomique, Saclay 91191, Gif Yvette, France
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The basolateral membrane sorting determinant of an inwardly rectifying potassium channel, Kir 2.3, is comprised of a unique arrangement of trafficking motifs containing tandem, conceivably overlapping, biosynthetic targeting and PDZ-based signals. In the present study, we elucidate a mechanism by which a PDZ interaction coordinates one step in a basolateral membrane sorting program. In contrast to apical missorting of channels lacking the entire sorting domain, deletion of the PDZ binding motif caused channels to accumulate into an endosomal compartment. Here, we identify a new human ortholog of a Caenorhabditis elegans PDZ protein, hLin-7b, that interacts with the COOH-terminal tail of Kir 2.3 in renal epithelia. hLin-7b associates with the channel as a part of a multimeric complex on the basolateral membrane similar to a basolateral membrane complex in C. elegans vulva progenitor cells. Coexpression of hLin-7b with Kir 2.3 dramatically increases channel activity by stabilizing plasma membrane expression. The discovery identifies one component of the sorting machinery and provides evidence for a retention mechanism in a hierarchical basolateral trafficking program.
polarity; membrane trafficking; intracellular sorting mechanism; protein-protein interaction; potassium; inward rectification; cortical collecting duct
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE POLARIZED EXPRESSION of disparate transport proteins on two distinct membrane domains is an essential prerequisite for the vectorial transport of water, solutes, and ions across epithelia. The renal cortical collecting duct (CCD), the site of potassium secretion in the kidney, provides a salient example. In these cells, the asymmetric expression of weakly inward-rectifying K+ channels on the apical membrane and more strongly rectifying K+ channels on the basolateral membrane may increase the fidelity of the secretory process, ensuring that potassium preferentially exits the cell across the apical membrane into the tubule lumen in concert with the demands of potassium homeostasis (10). The identification of a plausible gene candidate, Kir 2.3, for the basolateral K+ channel (39) provided the impetus to elucidate the basis for polarized membrane targeting of a native channel (21). In this effort, we discovered a novel sorting motif that is responsible for coordinating basolateral membrane expression of the strong inwardly rectifying K+ channel, Kir 2.3 (22).
In renal epithelial cells, most newly synthesized membrane proteins are thought to be sorted at the level of the trans-Golgi network (TGN) to the appropriate cellular domains (5, 24). In these cases, directed trafficking is dictated by signals located within the membrane protein structure, usually located within the cytoplasmic COOH-terminal domains (5). The sorting machinery in the TGN reads, interprets, and acts on the signals to target molecules to their correct destinations. Once delivered to the appropriate domain, many of these integral membrane proteins are effectively anchored at these polarized locales through interactions with the cytoskeleton or other membrane-associated proteins, completing the polarization program (25, 41). Several different basolateral sorting signals have been identified. The recent discovery of a novel form of the AP-1 clathrin adaptor complex (AP-1b), containing an epithelial-specific µIb-subunit (27), provided a plausible basolateral sorting mechanism for at least one class of these signals (8). Indeed, the capacity of the µ-adaptins to interact with a basolateral sorting motif that is colinear with tyrosine-based, clathrin-dependent endocytotic signal (2) suggests that the AP-1b complex may be recruited to vesicles in the TGN by specific membrane protein cargo containing these interaction motifs, marking them for basolateral membrane trafficking.
The basolateral sorting motif in the Kir 2.3 channel appears to be distinct from the classic tyrosine-based, clathrin-dependent endocytotic signal (22), suggesting interaction with targeting machineries other than AP-1b. Interestingly, the sorting signal is located at the extreme COOH terminus and juxtaposes or is colinear with a PDZ binding motif. PDZ domains, named after the three homologous proteins in which they were first discovered [PSD95, Dlg (Disc large), and ZO-1 (zona occludens)], are 80- to 90-amino acid residue protein interaction modules that generally recognize specific short motifs found at the extreme COOH-terminal domain of certain proteins. These domains have been strongly implicated in bringing multiprotein complexes together to localize expression on particular membrane domains or intracellular compartments (12).
In the present study, we evaluated the role of the Kir 2.3 PDZ binding motif within the broader sorting domain. Here, we identify and characterize an ortholog of a Caenorhabditis elegans PDZ protein, hLin-7b, that binds to the COOH-terminal tail of Kir 2.3. The discovery identifies one component of the sorting machinery and provides compelling evidence for a retention mechanism in a hierarchical basolateral trafficking program.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Yeast two-hybrid interaction trap library screen. The yeast two-hybrid interaction trap system was used according to established methods (11, 13) to identify kidney proteins that interact with the basolateral sorting signal in the extreme COOH terminus of Kir 2.3. Specifically, a sequential, two-step library screen with two different baits was performed to specifically capture proteins that interact with the PDZ binding motif in Kir 2.3. In the first stage of the screen, a lexA fusion protein of the Kir 2.3 COOH-terminal targeting domain (amino acids 417-445) was used as the bait (pJK202 containing a HIS3-selectable marker) to screen a conditionally expressed kidney cDNA library (Clontech) in the pJG4-5 plasmid (TRP4-selectable marker). Saccharomyces cerevisiae (EGY48, Mat a-ura3, his3, trp1, ura3, 3lexAop-leu2) were transfected with the wild-type bait and the kidney cDNA library and then plated onto one of two different selection plates. The number of transfectants was determined by plating yeast in a drop-out medium that selects for the presence of each plasmid (uracil-, histidine-, tryptophan-). In this system, interaction of library and bait proteins causes the transcriptional activation of two different reporter genes, leu2 and lacZ. Subsequently, interacting clones were identified by plating yeast onto medium that contains X-gal and lacks leucine, uracil, histidine, and tryptophan (2% galactose and 1% raffinose as carbon sources). Yeast that survived and turned blue were considered positive. Plasmid DNA from positive yeast clones was collected, and library pJG4-5 plasmids were isolated. In the second stage of the screen, positive library clones were screened with a lexA fusion of a mutant Kir 2.3 COOH-terminal domain that lacks the PDZ binding motif (443X). Library clones that scored positively in the first round with the wild-type bait but failed to interact with the truncated bait were considered true positives. These clones were sequenced using pJG4-5 primers.
DNA constructs. The vesicular stomatitis virus glycoprotein (VSV) epitope-tagged wild-type Kir 2.3 in the mammalian expression vector PCB6 was used as described previously (21). The PDZ ligand in Kir 2.3 was disrupted by introducing a premature stop codon at amino acid position 442 with PCR-based mutagenesis and cloned into the PCB6 vector. To specifically recognize hLin-7b in cells, we constructed an epitope-tagged version. The hemagglutinin protein (HA) epitope (YPYDVPDYA) was engineered into either the NH2 or COOH termini of hLin-7b by PCR and subcloned into the pcDNA3.1 vector (Invitrogen). Creation of wild-type and mutant Kir 2.3 COOH-terminal lexA fusion proteins was achieved by PCR-based mutagenesis and subsequent subcloning in frame with the lexA DNA binding domain in the pJK202 vector. Sequences were verified either by dye termination sequencing (ABI Prism) or with chain-terminating inhibitors using Sequenase.
Cell culture and transfections. COS cells were grown in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10 mM HEPES. Madin-Darby canine kidney (MDCK) cells were grown in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. For transfections, cells at ~70-80% confluence were washed once with serum-free DMEM and then incubated with 10 µl of Lipofectamine (GIBCO-BRL) and ~1 µg of DNA in OptiMEM (GIBCO-BRL) for 5 h. After the initial incubation with the transfection mix, cells were supplemented at a 1:1 ratio with DMEM containing 20% fetal bovine serum and 4 mM L-glutamine. Transfection medium was replaced 24 h after transfection with maintenance medium supplemented with 2 mM sodium butyrate to enhance protein expression.
Stably transfected MDCK cell lines were established as previously described (21). All vectors used provide neomycin resistance. Seventy-two hours after transfection, MDCK cells were placed in DMEM containing 500 µg/ml G418. Surviving clonal cell colonies were isolated within 12-18 days of initial neomycin selection, expanded, and maintained in DMEM containing 500 µg/ml G418.Immunoprecipitation and immunoblotting. COS cells, at 70-85% confluence, were transfected with epitope-tagged Kir 2.3 and hLin-7b constructs with Lipofectamine according to the manufacturer's specifications. Confluent, stably transfected MDCK cells expressing HA-hLin-7b and transiently transfected COS cells were incubated in growth medium containing 2 mM sodium butyrate for 24 h before harvesting. Forty-eight hours after transfection, cells were washed once with ice-cold PBS, harvested in cold lysis buffer (150 mM NaCl, 20 mM Tris pH 7.5, and 5 mM EDTA), pelleted (2,000 g for 5 min), and resuspended (~5× cell pellet volume) in lysis buffer containing 1% Triton X-100 and a protease inhibitor cocktail (10 µg/ml antipain, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml pepstatin A). Cells were then passed though a 27-gauge needle, rotated at 4°C for 1 h, and then centrifuged at 15,000 g for 15 min at 4°C. Soluble fractions from COS cells were precleared using 100 µl of a 50% Sepharose B slurry at 4°C for 2 h with rotation. Precleared COS cell lysates were then rotated overnight with 100 µl of a 10% protein A slurry with or without 2 µg of rabbit anti-HA antibody (Santa Cruz Biotechnology). MDCK cells were incubated overnight with agarose-conjugated anti-HA antibody (Santa Cruz Biotechnology) at 4°C. After being washed three times with lysis buffer containing 0.1% Triton, the immunoprecipitates from COS and MDCK cell lysates were eluted for 30 min at room temperature with SDS sample buffer and 0.1 M glycine (pH 3.5), respectively. Eluates were separated on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose (Amersham). For Western blotting, mouse anti-VSV (P5D4) (Sigma) and mouse anti-HA (Boehringer Mannheim) antibodies were used at 10 µg/ml and 1:1,000, respectively. Horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Jackson Laboratories) was used at 1:10,000 for detection with the enhanced chemiluminescence system (Amersham). The mouse anti-CASK (Transduction Laboratories) and mouse anti-syntrophin (9) antibodies were both used at 1:500 and detected with HRP-conjugated goat anti-mouse antibody (Sigma) at a 1:2,000 dilution.
Immunofluorescence and confocal microscopy. For immunolocalization studies, MDCK cells were plated onto Transwell filters (Costar) and grown until they formed a confluent monolayer. Confluent cells, incubated in medium supplemented with 2 mM sodium butyrate overnight, were washed twice with PBSCM (PBS + 1 mM CaCl2 + 1 mM MgCl2) and fixed for 30 min in 3.7% paraformaldehyde. Cells were then washed three times with PBSCM, quenched in PBSCM containing 50 mM NH4Cl for 30 min, and washed two times with PBSCM. MDCK cells were permeabilized with 0.1% Triton in PBSCM for 30 min, washed four times with PBSCM, and blocked with 1% BSA in PBSCM for 15 min. Cells were then incubated overnight at 4°C with the primary antibodies in PBSCM containing 0.1% BSA. The rabbit anti-HA (Santa Cruz Biotechnology) and mouse anti-VSV (P5D4) antibodies were used at 1:200 and 1:1,000, respectively. Mouse anti-CASK was used at 10 µg/ml. Cells were then washed three times with PBSCM and incubated with the secondary antibodies in 0.1% BSA-PBSCM for 30 min at room temperature. Alexa 488-conjugated goat anti-rabbit (Molecular Probes) and rhodamine-conjugated goat anti-mouse (Jackson Laboratories) antibodies were used at 1:100 and 1:80 dilutions, respectively. Labeled cells were rinsed four times with PBSCM and mounted onto slides with Vectashield glycerol (Vector Labs).
Colocalization of 442X channels with transferrin was performed using transferrin conjugated to biotin. Confluent MDCK cells were washed once with serum-free DMEM and then incubated for 1 h in serum-free DMEM containing 50 µg/ml biotin-conjugated transferrin (Sigma) at 37°C. Cells were then washed three times with PBS and fixed as described above. Fixed cells were washed three times with PBS and permeabilized with PBS containing 0.1% saponin for 30 min. Cells were then incubated with Texas red-conjugated streptavidin (Jackson Laboratories) at 3 µg/ml in PBS containing 0.1% saponin for 1 h at room temperature. Labeled cells were then washed three times with PBS and blocked with 1% BSA for 15 min. Immunolocalization of 442X channels was performed as above, except that the antibody incubations were performed in PBS containing 0.1% saponin. Fixation of male 129/SvEv mouse kidneys was achieved by perfusion via the heart. To clear blood from the kidneys, PBS was perfused for 2 min before a 5- or 30-min perfusion with paraformaldehyde (2%) and a 2-min perfusion with a cryoprotectant (10% EDTA in 0.1 M Tris). Twelve-micrometer-thick kidney sections were cut using a cryostat, placed on coverslips, coated with HistoGrip (Zymed), and stored at2-Nitrophenyl--D-galactopyranoside solution
-galactosidase assay and protein extraction from yeast.
To determine the relative strength of the interactions,
-galactosidase activity was measured at maximum velocity using the 2-nitrophenyl-
-D-galactopyranoside (ONPG) solution assay
according to established methods (26). Because the
interaction trap uses GAL1-inducible promoter to conditionally express
the AD prey protein, yeast were grown (A600 of
0.9-1.2) under conditions that induce (2% galactose and 1%
raffinose) or repress (2% glucose) the promoter. Yeast cultures were
pelleted (2,000 g) and resuspended in 750 µl of Z buffer
(in mM: 60 Na2HPO4, 40 NaH2PO4, 10 KCl, 1 MgSO4, and 50
-mercaptoethanol). For the assay, 100 µl of the Z buffer cell
suspension was added to 900 µl of Z buffer in glass culture tubes
containing 10 µl of 0.1% SDS and 20 µl of chloroform. Tubes were
vortexed for 15 s and equilibrated at 30°C for 15 min. After equilibration, 200 µl of stock 4 mg/ml ONPG was added to the samples and vortexed for 5 s. Reactions were stopped at 30 s to 4 min by adding 0.5 ml of 1 M Na2CO3 and centrifuged
to pellet debris, and the reaction product was measured by
spectrofluorometery. Miller units were calculated as previously
described (30).
Oocyte isolation and injection. Oocytes were isolated from female Xenopus laevis (NASCO; Fort Atkinson, WI). After anesthetization in 0.15% 3-aminobenzoate, an abdominal incision was made to perform a partial oophorectomy. Oocytes were manually dissected from the ovarian lobes and incubated in Ca2+-free OR2 medium (in mM: 82.5 NaCl, 2 KCl, 1 MgCl2, and 5 HEPES) containing collagenase (type IA, 2 mg/ml; Sigma) for ~2 h at room temperature to remove the follicular layer. After thorough washes in collagenase-free OR2 medium, oocytes were stored in a modified L15 medium (50% Leibovitz's medium, 10 mM HEPES, pH 7.4) at 19°C. Injection of healthy-looking Dumont stage V-VI oocytes was performed 12-14 h after isolation with a pneumatic PV829 picopump (World Precision Instruments). After injection of 0-50 ng of cRNA, oocytes were stored in L15 medium at 19°C and channel activity was assessed 1-3 days after injection.
Recording solutions and electrophysiology.
Whole cell oocyte currents were measured using a two-microelectrode
voltage clamp equipped with a bath-clamp circuit (OC-752B; Warner, New
Haven, CT) as described previously (39). Voltage-sensing and current-injecting electrodes had resistances of 0.5-1.5 M when back-filled with 3 M KCl. After a stable impalement was achieved, such that both electrodes measured the same membrane potential, pulse
protocols were conducted. A Macintosh Centris 650 computer equipped
with an Instrutech ITC16 analog-to-digital, digital-to-analog converter
and Pulse software (HEKA) was used for stimulation and data
acquisition. For analysis purposes, data were filtered at 1 kHz and
digitized online at 2 kHz to the hard disk using Pulse and IGOR
(WaveMetrics). A 45 mM K+ bathing solution [in mM: 45 KCl,
45 N-methyl-D-glucamine chloride, 1 CaCl2, 1 MgCl2, and 5 HEPES, pH 7.4] was used
for two-microelectrode voltage clamp experiments. To measure plasma
membrane stability, oocytes were incubated in 5 mM brefeldin A (BFA) or
vehicle (EtOH) at room temperature for the specified time.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PDZ ligand of Kir 2.3 is required for basolateral membrane
expression.
We found previously (22) that the extreme COOH terminus of
Kir 2.3 contains a signal that is both necessary and sufficient to
direct basolateral membrane expression. The unique domain juxtaposes or
is colinear with a consensus sequence (XS/T X I/V-COOH) for a type I
PDZ protein interaction (36), suggesting that the channel may associate with PDZ proteins in the kidney to coordinate polarized expression. To evaluate this hypothesis, we first determined the functional consequence of specifically removing the entire PDZ ligand,
the COOH-terminal four amino acids, on steady-state expression of Kir
2.3 in a polarized renal epithelial cell line. Confocal images of MDCK
cells stably expressing either the wild-type or truncated (442X) Kir
2.3 channels are shown in Fig. 1. In
contrast to the basolateral membrane expression of wild-type Kir 2.3 and the apical expression of the 438X mutant (22), the
442X truncated channel was distributed to a punctate, vesicle-like
compartment. Consistent with partial localization in recycling
endosomes, a fraction of 442X channels colocalized with transferrin in
a perinuclear vesicular compartment (Fig.
2) but not with PDI, an endoplasmic reticulum marker, or with a lysosomal marker (Lysotracker).
Collectively, these results indicate that the PDZ binding motif of Kir
2.3 is a critical determinant for basolateral membrane expression, a function conceivably achieved through an interaction with kidney PDZ
proteins.
|
|
Identification and structure of hLin-7b.
To begin to elucidate the mechanism for the PDZ-dependent expression of
Kir 2.3, kidney PDZ proteins that associate with the Kir 2.3 basolateral-sorting domain were identified using a directed two-stage
yeast two-hybrid interaction trap screen. From a screen of ~14
million kidney cDNAs, 116 library clones scored positively for
interaction with a lexA fusion to the COOH-terminal 29 amino acids of
Kir 2.3. Positive clones were clustered into common groups, based on
insert size and restriction digest patterns, and subjected to a second
round of screening using a lexA fusion to a mutant Kir 2.3 COOH
terminus lacking the PDZ binding motif (442X). Five cDNA clones that
interacted with the wild-type bait failed to interact with the mutant,
consistent with the presence of a PDZ domain (Fig.
3). Sequence analysis revealed that two
of the five clones were overlapping cDNAs encoding the same gene
product. A BLAST search (1) of public databases revealed
that the two overlapping cDNAs share a high degree of similarity to a
C. elegans protein called Lin-7 (18). Because
this is the second human Lin-7 homolog to be identified, we named it
hLin-7b. An amino acid alignment of hLin-7b, VELI2/MALS2 (the
corresponding mouse form; Refs. 4, 16), and
C. elegans Lin-7 is shown in Fig. 4A. Excluding the longer and
divergent NH2-terminal domain in the C. elegans
protein, hLin-7b shares 84% identity with the C. elegans
Lin-7. Supporting the existence of a family of related isoforms in
humans, the primary structure of hLin-7b is more similar to the
corresponding mouse homolog, Veli2/MALS2, than the other human Lin-7a
form (97% vs. 67% amino acid identity).
|
|
Recognition sequence for hLin-7b PDZ domain.
The basolateral membrane sorting signal of Kir 2.3 lies in close
proximity to and may even overlap with the PDZ binding motif. We
recently found (22) that Kir 2.3 431-441 channels,
harboring an internal deletion mutation that leaves the last four amino acids intact, are mistargeted to the apical membrane, consistent with
the removal of a basolateral sorting determinant. To determine whether
this upstream sorting domain influences hLin-7b interaction, the target
sequence for hLin-7b binding was delineated using the yeast two-hybrid
system. A series of alanine substitution mutations were created in
lexA-Kir 2.3 COOH terminus and tested for their ability to interact
with the AD-hLin-7b protein. All baits were expressed in yeast at
relatively equal levels as determined by Western blot analysis (Fig.
5B). To quantify the relative
strength of interaction, reporter activity (
-galactosidase) was
measured using the ONPG solution assay. Counting from the extreme
COOH-terminal amino acid of Kir 2.3, residues at positions 0,
2, and
3 were found to be absolutely required for interaction with hLin-7b. Alanine substitution at these sites specifically rendered the channel
unable to interact with hLin-7b (Fig. 5A), consistent with a
classic type I PDZ interaction. In contrast, point mutations made
further upstream, beyond the
3 residue, had no significant effect on
the interaction between lexA-Kir 2.3 and AD-hLin-7b proteins. The
result indicates that individual residues in the upstream sorting
determinant do not significantly influence Lin-7 interaction with Kir
2.3. In fact, replacement of the entire sorting domain
(431) with an 11-residue stretch of alanines
(431-41A) attenuated but did not eliminate binding of hLin-7b to
Kir 2.3 (Fig. 5A). Collectively, the observations provide
reason to speculate that the Lin-7 binding site may be distinct from
the biosynthetic sorting signal.
|
Coimmunoprecipitation of hLin-7b and Kir 2.3.
To authenticate the interaction of hLin-7b with Kir 2.3 in mammalian
cells, the full-length proteins were tested for association with one
another in COS cells by coimmunoprecipitation. For this purpose, Kir
2.3 and hLin-7b were tagged with the VSV G protein and HA epitopes,
respectively. Immunoblotted lysates from COS cells transiently
transfected with each of the tagged proteins were found to specifically
express a 29-kDa protein corresponding to HA-hLin-7b or a 53-kDa
protein corresponding to VSV-Kir 2.3 (Fig.
6, lanes
2).
|
hLin-7b promotes plasma membrane retention of Kir 2.3.
To investigate the functional consequences of hLin-7b interaction with
Kir 2.3, we examined macroscopic Kir 2.3 channel activity in
Xenopus oocytes using the two-microelectrode voltage clamp. Coinjection of hLin-7b cRNA significantly increased the macroscopic Kir
2.3 conductance, indicating an increase in active channel number,
single-channel conductance, or open probability. (Fig. 7, A and B). To
test whether the response involves an increase in plasmalemma
stability, oocytes were exposed to BFA or vehicle (EtOH) and
macroscopic currents were measured over 9 h at the time points
indicated in Fig. 7C. BFA is a fungal metabolite that inhibits transport of newly synthesized membrane proteins to the plasmalemma by blocking anterograde trafficking of vesicles from the
endoplasmic reticulum to the Golgi apparatus. Consequently, the
lifetime of active channels on the plasma membrane can be ascertained
by monitoring channel activity in the presence of BFA. Exposure to BFA
caused a progressive decrease in macroscopic channel activity, well
described by a single exponential with a half-life of 4.3 ± 0.85 h. The response occurs with a time course similar to that of
clathrin-dependent removal of other renal collecting duct channels,
epithelial Na+ channel (ENAc; Ref. 34) and
renal outer medullary K+ channel (ROMK; Ref.
42), as expressed in Xenopus oocytes. Although they are suggestive, it should be pointed out these residence time
measurements do not provide a strict quantification of endocytosis. It
is probable that recycling of endosomes back to the plasmalemma occurs,
causing an underestimate in the rate of endocytosis. Nevertheless, and
in dramatic contrast to these results without hLin-7b, Kir 2.3 channel
activity was not affected by BFA in oocytes coinjected with hLin-7b
(Fig. 7C). Collectively, these results are consistent with
our hypothesis that hLin-7b stabilizes Kir 2.3 channels in the plasma
membrane either by retention or by promoting recycling from early
endosomes.
|
hLin-7b associates with CASK in MDCK cells.
In C. elegans, Lin-7 directly interacts with Lin-2, forming
a part of a multimeric basolateral scaffold that coordinates
basolateral membrane expression of the Let-23 receptor
(18). To determine whether hLin-7b interacts with a
related protein in mammalian epithelia, MDCK cells were stably
transfected with HA-hLin-7b and examined for the presence of associated
proteins by HA immunoprecipitation. CASK, a mammalian ortholog of
Lin-2, copurified with HA-hLin-7b on HA antibody-agarose beads (Fig.
8A, lane
5). As controls, CASK could not be immunoprecipitated with
the HA antibody from wild-type cells (Fig. 8A,
lane 4) or with agarose beads alone from
HA-hLin-7b-transfected MDCK cells (Fig. 8A, lane
3). Supporting the idea that hLin-7b associates with a
specific PDZ complex, syntrophin, an unrelated basolateral membrane PDZ
protein (17), did not copurify with HA-hLin-7b (Fig.
8B, lane 5). Collectively, these data
convincingly establish that the Lin-7/Lin-2 (CASK) complex is conserved
in mammalian epithelia.
|
hLin-7b/CASK complex localizes to basolateral membranes of MDCK cells. To provide insight into the functional roles of the hLin-7b interaction with Kir 2.3, laser scanning confocal microscopy was used to examine the subcellular expression pattern of hLin-7b. As shown in Fig. 8C, the HA-hLin-7b protein localized to the basolateral membrane, similar to the distribution of Kir 2.3. The basolateral membrane expression pattern of hLin-7b was consistently observed in three different clonal lines of HA-hLin-7b-transfected MDCK cells. As predicted by the coimmunoprecipation studies, CASK also predominantly localized to the basolateral membrane in MDCK cells (Fig. 8D). The steady-state basolateral membrane expression of these proteins suggests that the Lin-7/Lin-2 complex acts to stabilize proteins like Kir 2.3 at the basolateral membrane.
Kir 2.3 and mLin-7/CASK complex localize to basolateral membrane of
CCD principal cells.
To test whether Kir 2.3 might interact with the Lin 7/CASK complex in
the kidney, immunolocalization studies were performed. First, the
specificity of the anti-Kir 2.3 antibody was tested by Western blot
analysis with COS cells that were transfected with either VSV-Kir 2.3 or a related collecting duct Kir channel, Kir 1.1, tagged with the FLAG
epitope. As shown in Fig.
9A, the anti-Kir 2.3 antibody exclusively detects an appropriately sized 59-kDa
protein in COS cells transiently transfected with VSV-Kir 2.3 (lane 2). In contrast, the anti-Kir 2.3 antibody did not
detect the FLAG-tagged ROMK. The anti-Kir 2.3 antibody also was found to recognize a single band corresponding to the predicted size of Kir
2.3 in the membrane fraction of kidney homogenates (Fig. 9B,
lane 2). Collectively, these results establish that the
anti-Kir 2.3 antibody specifically recognizes Kir 2.3.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We found recently (22) that the extreme COOH terminus
of the inwardly rectifying K+ channel Kir 2.3 is both
necessary and sufficient to direct basolateral membrane expression. In
the present study, we have elucidated one mechanism in a multistep
sorting process. Mutagenesis studies indicate that the COOH-terminal
sorting domain instructs at least two distinct functions. Kir 2.3 431-441 channels, harboring an internal deletion mutation that
leaves the archetypal PDZ binding site intact, are mistargeted to the
apical membrane (22), consistent with the removal of a
basolateral sorting determinant. In contrast, removal of the
neighboring PDZ binding site produced channels that localize within an
intracellular vesicle compartment. In this respect, the PDZ ligand is
required for efficient plasma membrane expression, dictating delivery,
retention, or both. Our discovery of an ortholog of a C. elegans PDZ protein, hLin-7b, that interacts with the
COOH-terminal tail of Kir 2.3 identifies one component of the sorting
machinery and provides compelling evidence for a retention mechanism in
a hierarchical basolateral trafficking program.
Lin-7 was originally identified in C. elegans (35) as member of a gene set (lin-7, lin-2, and lin-10) required for vulva progenitor cell (VPC) differentiation (18). Together, the products of these three genes, all containing PDZ domains, form a heterotrimeric protein complex that coordinates the basolateral membrane expression of Let-23, a tyrosine kinase receptor (18, 37). Oligomerization of the complex is specified by unique sites within each gene product, freeing the PDZ domains and other protein-protein interaction domains to recruit membrane proteins (4) and cytoskeletal elements (6) into a multiprotein complex. With the possibility for differential assembly, the modular design of the system may provide a mechanism for diversity (see below). In VPC, Lin-7 acts as the upstream scaffolding molecule, binding directly to the Let-23 receptor through a type 1 PDZ interaction and with Lin-2 via a unique NH2-terminal interaction (18, 35). Our discovery that the Kir 2.3 channel interacts with hLin-7b in mammalian renal epithelia suggests a similar mechanism for polarized basolateral membrane expression.
Orthologous gene products (Lin-7 = Veli/MALS; Lin-2 = CASK; Lin-10 = Mint/X11) were recently identified as a native tripartite complex in the mammalian brain (4), supporting the notion of an evolutionarily conserved mechanism for compartmentalizing proteins at specific membrane domains. On the basis of known binding capacities of each component of the complex, it has been suggested that the complex might act in two different steps of a membrane protein-targeting program in neurons (3, 4). Interestingly, KIF17, a neuron-specific molecular motor in neuronal dendrites, was recently found to interact with the PDZ domain of mLin-10 (Mint1/X11), providing a mechanism for selective transport of vesicles containing the complex (33). Lin-10 also interacts with Munc-18, suggesting a link to the SNARE machinery (4). Once docked, Lin-7, CASK, and Lin-10 would then act as a retention complex, recruiting different membrane proteins into clusters at the membrane via different types of PDZ interactions. For instance, the PDZ domain in CASK binds the synaptic adhesion molecule neurexin (4), potentially anchoring the complex at the presynaptic junction.
Although this is an intriguing hypothesis for protein targeting and retention in neurons, the biology of Lin-10 and Munc-18 in the kidney provides a reason to suspect a significant divergence in mammalian epithelia. The mammalian counterpart of Lin-10 is actually encoded by a family of proteins called the Mints or X11s (28). Although all three members of the Mint family share COOH-terminal PDZ and PTB domains, only the neuron-specific form, Mint-1, contains a CASK interaction domain (3), suggesting that the Mint module is dispensable for polarized targeting of the mammalian kidney. The alternative view that an undefined Mint isoform assembles with CASK in the kidney cannot be ruled out. However, because Munc-18 has been implicated in apical rather than basolateral membrane sorting (31), such a model would also necessitate a completely different intracellular routing mechanism than has been proposed for synaptic vesicle trafficking. In the absence of the Mint-1 module, the system loses the obvious link to vesicular trafficking. Subsequently, the Lin-2/Lin-7 complex may play a restricted role in retention of proteins at the plasmalemma.
Our data support such a Lin-7/CASK anchoring complex. As shown by coprecipitation, CASK interacts with hLin-7b in MDCK cells. As predicted for a retention complex, immunolocalization studies revealed that CASK and hLin-7b are largely coexpressed on the basolateral membrane of MDCK cells and renal epithelia (38). Our observation differs from the predominant intracellular localization of CASK in some neuronal cells, where it has been implicated in vesicular trafficking (33) or nuclear translocation (15). In contrast, CASK has been shown to bind the heparin sulfate proteoglycan syndecan on the basolateral membrane of mammalian epithelia via a type II PDZ interaction and with the actin/spectrin binding protein 4.1 (6). By linking extracellular matrix receptors and the cytoskeleton, the CASK complex provides a stable anchor to retain mLin-7 interacting proteins, like Kir 2.3, on the basolateral membrane.
Recent studies on the -amino butyric acid transporter (BGT-1)
support the view that mLin-7 regulates the surface density of
particular transport proteins on the basolateral membrane of renal
epithelia. Like Kir 2.3, the COOH-terminal tail of BGT-1 conforms to
the optimal binding motif for mLin-7 interaction (D/E S/T X I/V-COOH)
(29). Perego et al. (29) demonstrated that removing the PDZ ligand (BGT
5) disrupted mLin-7 association in MDCK
cells and dramatically increased the internalization of the transporter
from the plasmalemma. Significantly, BGT
5 transporters are
predominantly localized on the basolateral membrane despite the
increase in endocytotic retrieval. In this regard, the PDZ binding
domain in BGT-1 appears to operate solely as a retention signal without
effecting a basolateral trafficking step.
Although the general observations with BGT-1 are comparable with those with Kir 2.3, a distinct feature of the Kir 2.3 442X phenotype suggests disparate sorting mechanisms. In contrast to BGT-1, truncating the PDZ binding motif in the Kir 2.3 channel produced a predominant, if not exclusive, intracellular expression pattern. The observation indicates that the endocytic retrieval of the truncated channel might be more avid than the transporter; an intracellular routing step of the channel may be coordinated by a PDZ interaction or both. The two different, but not mutually exclusive, explanations should be considered.
Because PDZ interactions can control multiple intracellular routing and plasmalemma retention steps (7), the 442X phenotype simply provides insight into the nature of the compartment where the PDZ binding signal is first processed. Like BGT-1, the COOH-terminal domain of Kir 2.3 contains a di-leucine motif and a classic tyrosine-based internalization signal, supporting the potential for clathrin-mediated endocytosis from the plasmalemma as well as direct endosomal targeting from the trans-Golgi apparatus (2). Indeed, our observation that the truncated 442X channel partially colocalized with transferrin in perinuclear vesicles is consistent with trafficking into an endosomal recycling compartment (40). Unfortunately, the lack of free amino or carbohydrate groups in the small extracellular domain of Kir 2.3 precludes biotinylation targeting studies to easily determine how the 442X channel reaches the endosome. Accordingly, we cannot be certain whether the first PDZ-dependent processing step curbs endosomal trafficking from the TGN or from the plasmalemma. Moreover, we cannot exclude that possibility that docking and fusion of Kir2.3-containing endosomes occurs in a PDZ-dependent fashion. Clearly, all of these steps may normally control plasma membrane expression of the wild-type channel.
Nevertheless, our identification and characterization of Lin-7 as an interaction partner of Kir 2.3 illuminates one PDZ-dependent trafficking step. As mentioned above, steady-state expression of the Lin-7 complex on the basolateral membrane is consistent with a plasmalemma retention system. Furthermore, the effect of Lin-7 on Kir 2.3 in oocytes provides direct evidence for PDZ-dependent plasmalemma stabilization. Certainly, coexpression of Lin-7 dramatically increased the lifetime of active Kir 2.3 channels on the plasma membrane. Future studies will be required to elucidate the actual retention mechanism, particularly to determine whether Lin-7 promotes recycling from early endosomes or directly secures the channel on the basolateral membrane.
In conclusion, a functional PDZ binding motif juxtaposes or is colinear with the basolateral sorting signal in the Kir 2.3 channel. Our discovery that an ortholog of a C. elegans PDZ protein, hLin-7b, interacts with the COOH-terminal tail of Kir 2.3 identifies one component of the sorting machinery and provides compelling evidence for a retention mechanism in a multistep basolateral trafficking program.
![]() |
ACKNOWLEDGEMENTS |
---|
We acknowledge the expert technical assistance of Jie Liu. We thank D. S. Bredt for the anti-Kir 2.3 antibody and B. Margolis for the anti-Lin-7 antibody.
![]() |
FOOTNOTES |
---|
This study was supported in part by funding from the American Heart Association (P. A. Welling, Established Investigator), National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-54231 to P. A. Welling and DK-32839 to J. B. Wade, and a North American Treaty Organization travel award to J. Merot and P. A. Welling.
Address for reprint requests and other correspondence: P. A. Welling, Dept. of Physiology, Univ. of Maryland School of Medicine, 655 W. Baltimore St., Baltimore, Maryland 21201 (E-mail: pwelling{at}umaryland.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published September 21, 2001; 10.1152/ajpcell.00249.2001
Received 5 June 2001; accepted in final form 19 September 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Altschul, SF,
Gish W,
Miller W,
Myers EW,
and
Lipman DJ.
Basic local alignment search tool.
J Mol Biol
215:
403-410,
1990[ISI][Medline].
2.
Bonifacino, JS,
and
Dell'Angelica EC.
Molecular bases for the recognition of tyrosine-based sorting signals.
J Cell Biol
145:
923-926,
1999
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[ISI][Medline].
5.
Caplan, MJ.
Membrane polarity in epithelial cells: protein sorting and establishment of polarized domains.
Am J Physiol Renal Physiol
272:
F425-F429,
1997
6.
Cohen, AR,
Woods DF,
Marfatia SM,
Walther Z,
Chishti AH,
Anderson JM,
and
Wood DFW
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
7.
El-Husseini, AE,
Craven SE,
Chetkovich DM,
Firestein BL,
Schnell E,
Aoki C,
and
Bredt DS.
Dual palmitoylation of PSD-95 mediates its vesiculotubular sorting, postsynaptic targeting, and ion channel clustering.
J Cell Biol
148:
159-172,
2000
8.
Folsch, H,
Ohno H,
Bonifacino JS,
and
Mellman I.
A novel clathrin adaptor complex mediates basolateral targeting in polarized epithelial cells.
Cell
99:
189-198,
1999[ISI][Medline].
9.
Froehner, SC,
Murnane AA,
Tobler M,
Peng HB,
and
Sealock R.
A postsynaptic Mr 58,000 (58 K) protein concentrated at acetylcholine receptor-rich sites in Torpedo electroplaques and skeletal muscle.
J Cell Biol
104:
1633-1646,
1987[Abstract].
10.
Giebisch, G.
Renal potassium transport: mechanisms and regulation.
Am J Physiol Renal Physiol
274:
F817-F833,
1998
11.
Golemis, EA,
and
Khazak V.
Alternative yeast two-hybrid systems. The interaction trap and interaction mating.
Methods Mol Biol
63:
197-218,
1997[Medline].
12.
Gomperts, SN.
Clustering membrane proteins: it's all coming together with the PSD-95/SAP90 protein family.
Cell
84:
659-662,
1996[ISI][Medline].
13.
Gyuris, J,
Golemis EA,
Chertkov H,
and
Brent R.
Cdi, a human G1 and S phase protein phosphatase that associates with Cdk2.
Cell
75:
791-803,
1993[ISI][Medline].
14.
Hammerton, RW,
Krzeminski KA,
Mays RW,
Ryan TA,
Wollner DA,
and
Nelson WJ.
Mechanism for regulating cell surface distribution of Na+,K+-ATPase in polarized epithelial cells.
Science
254:
847-850,
1991[ISI][Medline].
15.
Hsueh, YP,
Wang TF,
Yang FC,
and
Sheng M.
Nuclear translocation and transcription regulation by the membrane-associated guanylate kinase CASK/LIN-2.
Nature
404:
298-302,
2000[ISI][Medline].
16.
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
17.
Kachinsky, AM,
Froehner SC,
and
Milgram SL.
A PDZ-containing scaffold related to the dystrophin complex at the basolateral membrane of epithelial cells.
J Cell Biol
145:
391-402,
1999
18.
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[ISI][Medline].
19.
Kamberov, E,
Makarova O,
Roh M,
Liu A,
Karnak D,
Straight S,
and
Margolis B.
Molecular cloning and characterization of Pals, proteins associated with mLin-7.
J Biol Chem
275:
11425-11431,
2000
21.
Le Maout, S,
Brejon M,
Merot J,
and
Welling PA.
Polarized expression of an epitope tagged CCD Kir channel, CCD-IRK3, in MDCK cells.
Proc Natl Acad Sci USA
94:
13329-13334,
1997
22.
Le Maout, S,
Welling PA,
Brejon M,
Olsen O,
and
Merot J.
Basolateral membrane expression of a K channel is directed by a novel cytoplasmic carboxyl-terminal domain.
Proc Natl Acad Sci USA
98:
10475-10480,
2001
23.
Lupas, A,
Van Dyke M,
and
Stock J.
Predicting coiled coils from protein sequences.
Science
252:
1162-1164,
1991[ISI][Medline].
24.
Matter, K,
and
Mellman I.
Mechanisms of cell polarity: sorting and transport in epithelial cells.
Curr Biol
6:
545-554,
1994.
25.
Mays, RW,
Nelson WJ,
and
Marrs JA.
Generation of epithelial cell polarity: roles for protein trafficking, membrane-cytoskeleton, and E-cadherin-mediated cell adhesion.
Cold Spring Harb Symp Quant Biol
60:
763-773,
1995[ISI][Medline].
26.
Mendelsohn, AR,
and
Brent R.
Applications of interaction traps/two-hybrid systems to biotechnology research.
Curr Opin Biotechnol
5:
482-486,
1994[Medline].
27.
Ohno, H,
Tomemori T,
Nakatsu F,
Okazaki Y,
Aguilar RC,
Foelsch H,
Mellman I,
Saito T,
Shirasawa T,
and
Bonifacino JS.
Mu1B, a novel adaptor medium chain expressed in polarized epithelial cells.
FEBS Lett
449:
215-220,
1999[ISI][Medline].
28.
Okamoto, M,
and
Sudhof TC.
Mints, Munc18-interacting proteins in synaptic vesicle exocytosis.
J Biol Chem
272:
31459-31464,
1997
29.
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
30.
Reynolds, A,
Lundblad V,
Dorris D,
and
Keaveney M.
Yeast vectors and assays for expression of cloned genes.
In: Current Protocols in Molecular Biology, edited by Ausubel FM,
Brent R,
Kingston RE,
Moore DD,
Seidman JG,
Smith JA,
and Struhl K.. New York: Wiley, 1997, p. 13.6.1-13.6.6.
31.
Riento, K,
Galli T,
Jansson S,
Ehnholm C,
Lehtonen E,
and
Olkkonen VM.
Interaction of Munc-18-2 with syntaxin 3 controls the association of apical SNAREs in epithelial cells.
J Cell Sci
111:
2681-2688,
1998
32.
Schultz, J,
Milpetz F,
Bork P,
and
Ponting CP.
SMART, a simple modular architecture research tool: identification of signaling domains.
Proc Natl Acad Sci USA
95:
5857-5864,
1998
33.
Setou, M,
Nakagawa T,
Seog DH,
and
Hirokawa N.
Kinesin superfamily motor protein KIF17 and mLin-10 in NMDA receptor-containing vesicle transport.
Science
288:
1796-1802,
2000
34.
Shimkets, RA,
Lifton RP,
and
Canessa CM.
The activity of the epithelial sodium channel is regulated by clathrin-mediated endocytosis.
J Biol Chem
272:
25537-25541,
1997
35.
Simske, JS,
Kaech SM,
Harp SA,
and
Kim SK.
LET-23 receptor localization by the cell junction protein LIN-7 during C. elegans vulval induction.
Cell
85:
195-204,
1996[ISI][Medline].
36.
Songyang, Z,
Fanning AS,
Fu C,
Xu J,
Marfatia SM,
Chishti AH,
Crompton A,
Chan AC,
Anderson JM,
and
Cantley LC.
Recognition of unique carboxyl-terminal motifs by distinct PDZ domains.
Science
275:
73-77,
1997
37.
Sternberg, PW,
Lesa G,
Lee J,
Katz WS,
Yoon C,
Clandinin TR,
Huang LS,
Chamberlin HM,
and
Jongeward G.
LET-23-mediated signal transduction during Caenorhabditis elegans development.
Mol Reprod Dev
42:
523-528,
1995[ISI][Medline].
38.
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
39.
Welling, PA.
Primary structure and functional expression of a cortical collecting duct Kir channel.
Am J Physiol Renal Physiol
273:
F825-F836,
1997[ISI][Medline].
40.
Yamashiro, DJ,
Tycko B,
Fluss SR,
and
Maxfield FR.
Segregation of transferrin to a mildly acidic (pH 6.5) para-Golgi compartment in the recycling pathway.
Cell
37:
789-800,
1984[ISI][Medline].
41.
Yeaman, C,
Grindstaff KK,
and
Nelson WJ.
New perspectives on mechanisms involved in generating epithelial cell polarity.
Physiol Rev
79:
73-98,
1999
42.
Zeng, WZ,
and
Huang C-L.
Evidence for endocytosis of ROMK1 via clathrin-coated vesicles in Xenopus oocytes (Abstract).
J Am Soc Nephrol
11:
41A,
2000.