From the Mayo Clinic Scottsdale, S. C. Johnson Medical Research Center, Scottsdale, Arizona 85259
Received for publication, October 29, 2002
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ABSTRACT |
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ClC chloride channels are widely distributed in
organisms across the evolutionary spectrum, and members of the
mammalian family play crucial roles in cellular function and are
mutated in several human diseases (Jentsch, T. J., Stein, V.,
Weinreich, F., and Zdebik, A. A. (2002) Physiol. Rev.
82, 503-568). Within the ClC-3, -4, -5 branch of the family that are
intracellular channels, two alternatively spliced ClC-3 isoforms were
recognized recently (Ogura, T., Furukawa, T., Toyozaki, T., Yamada, K.,
Zheng, Y. J., Katayama, Y., Nakaya, H., and Inagaki, N. (2002)
FASEB J. 16, 863-865). ClC-3A resides in late endosomes
where it serves as an anion shunt during acidification. We show here
that the ClC-3B PDZ-binding isoform resides in the Golgi where it
co-localizes with a small amount of the other known PDZ-binding
chloride channel, CFTR (cystic fibrosis transmembrane conductance
regulator). Both channel proteins bind the Golgi PDZ protein, GOPC
(Golgi-associated PDZ and coiled-coil motif-containing protein).
Interestingly, however, when overexpressed, GOPC, which is thought to
influence traffic in the endocytic/secretory pathway, causes a large
reduction in the amounts of both channels, probably by leading them to
the degradative end of this pathway. ClC-3B as well as CFTR also binds EBP50 (ERM-binding phosphoprotein 50) and PDZK1, which are concentrated at the plasma membrane. However, only PDZK1 was found to promote interaction between the two channels, perhaps because they were able to
bind to two different PDZ domains in PDZK1. Thus while small
portions of the populations of ClC-3B and CFTR may associate and
co-localize, the bulk of the two populations reside in different organelles of cells where they are expressed heterologously or endogenously, and therefore their cellular functions are likely to be
distinct and not primarily related.
Chloride channels mediate a broad range of cellular functions.
Among the several different families of proteins that constitute these
channels, the neurotransmitter ligand-gated Recently, a splice variant of human ClC-3 was discovered that has a
PDZ1 domain-binding C
terminus (6). When heterologously expressed, this form, termed ClC-3B,
was detectable mainly in a general intracellular localization. However,
when co-expressed with the PDZ protein, EBP50, a small amount of the
channel protein was detected on the surface at the leading edge of
spreading cells. Furthermore, EBP50-mediated association with the other
known PDZ-binding chloride channel, CFTR, was reported as was an
ability of CFTR to activate ClC-3B. The CFTR chloride channel is
distinct from the other families of chloride channels and belongs to
the large family of ABC transport proteins (7). CFTR resides in the
apical membrane of epithelial cells where it is crucial to ion and
fluid secretion and reabsorption (8). Hence the possibility of ClC-3B
trafficking to the plasma membrane and either being influenced by CFTR
when it is present or potentially substituting for it when it is absent
or dysfunctional in cells of CF patients is of great importance to
understanding or influencing the disease. For this reason we have
carefully examined the subcellular localization of heterologously and
endogenously expressed ClC-3B as well as its possible PDZ
protein-mediated interaction with CFTR. We found that ClC-3B is
localized to the Golgi and differs in its location from the late
endosomal isoform ClC-3A and from most CFTR, present at the plasma
membrane. Both ClC-3B and CFTR interact with PDZ domains of
GOPC,2 EBP50, and PDZK1 and
PDZK1 promotes the association of the two PDZ-binding chloride
channels. Despite this the only co-localization of CFTR and ClC-3B that
we detected is at the Golgi where a small amount of CFTR resides.
ClC-3A and ClC-3B were found to interact with each other, most likely
as heterodimers as described for other ClC channels.
Plasmid Construction--
Full-length cDNAs of ClC-3A and
ClC-3B were amplified by PCR using a human pancreas cDNA library
(Clontech) as template. PCR fragments were first
introduced into the EcoRV site of pBluescript and then
subcloned into pcDNA3. A c-Myc tag was added to the ClC-3B open
reading frame at the N terminus; ClC-3A was tagged at its N terminus
with amino acid residues 723-732 of the MRP1 protein as an epitope
recognized by mouse monoclonal antibody 42.4 (9).
Partial cDNA fragments encoding different PDZ domains of EBP50,
PDZK1, and GOPC were subcloned into pGEX-5X-3 (Amersham Biosciences) to
generate glutathione S-transferase (GST) fusion proteins.
The boundaries of the expressed polypeptides containing different PDZ
domains were amino acid residues 1-129 for PDZ domain 1 of EBP50,
amino acid residues 133 to the C-terminal end for PDZ domain 2 of
EBP50, amino acid residues 1-140 for PDZ domain 1 of PDZK1, amino acid
residues 127-237 for PDZ domain 2 of PDZK1, amino acid residues
231-371 for PDZ domain 3 of PDZK1, amino acid residues 368 to the
C-terminal end for PDZ domain 4 of PDZK1, and amino acid residues
281-363 for the PDZ domain of GOPC. A cDNA fragment encoding the
C-terminal amino acid residues 1440-1480 of CFTR was subcloned into
pGEX-5X-3 vector to produce a fusion of GST and the C terminus of
CFTR.
Cell Culture and Transfection--
BHK-21, HEK293T, Calu-3,
Capan-1, CFPAC-1, T84, Caco-2, MDCK, and PANC-1 cells were obtained
from the American Type Culture Collection (ATCC) and grown at 37 °C
in 5% CO2. BHK-21 cells were transiently transfected with
LipofectAMINE Plus Reagent (Invitrogen) or stably transfected using
calcium phosphate (10). For transient expression cells were transfected
with cDNAs of CFTR, ClC-3A, ClC-3B, EBP50, or PDZK1 subcloned into
pcDNA3. GOPC was transiently expressed using the plasmid
pKH3-PIST (11). For transient cotransfection experiments
equivalent amounts of individual plasmid DNAs were employed. For stable
transfections pNUT vector was contransfected with ClC-3A in pcDNA3
or ClC-3B in pcDNA3. Stably transfected cells were selected with
500 µM methotrexate in the growth medium, individual
clones were isolated and ClC-3A or ClC-3B expression was analyzed by
Western blotting. HEK293T cells were transiently transfected using
LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions.
Cell Lysis and Membrane Preparation--
Cells were washed in
ice-cold phosphate-buffered saline and lysed with Nonidet P-40 lysis
buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM
Tris, pH 7.4, 10 mM NaMoO4) at 4 °C for 30 min. Protease inhibitors were added to Nonidet P-40 lysis buffer prior
before use as described earlier (12). Cell lysates were centrifuged at
maximal speed in an Eppifuge at 4 °C, and supernatants were collected for further experiments. Scrapings from human bronchus were
extracted with 2× Nonidet P-40 buffer and sonicated briefly before
centrifugation. Scrapings from colon were first ground in a homogenizer
in extraction buffer and then processed exactly as the bronchial
scrapings. The preparation of membranes from cultured cells was
performed exactly as described earlier (13).
Primary Antibodies--
Mouse monoclonal antibodies recognizing
ClC-3A and ClC-3B were raised against conjugated peptides derived from
C-terminal sequences of ClC-3A (RHMAQTANQDPASIMFN) and ClC-3B
(KQHVEPLAPPWHYNKKR). Initial screens of hybridomas were performed by
peptide ELISA, and followed by secondary screening by Western blotting
of lysates or membranes from cells heterologously expressing ClC-3A or
ClC-3B. The derived monoclonal antibodies 34.1 and 69.16 recognized
ClC-3A and ClC-3B, respectively. Additionally, ClC-3A and ClC-3B were detected with antibodies 42.4 and 9E10 directed against their N-terminal tags. CFTR was detected with antibody 596, 528, or 570, FLAG-tagged PDZK1 with antibody anti-FLAG M2 (Sigma), HA-tagged GOPC,
and HA-tagged EBP50 with the antibody 16B12 (Babco). The antibodies
from the following manufacturers were used to detect proteins by
immunofluorescence: Giantin, Babco; Golgin, Molecular Probes, GM130, BD
Transduction Laboratories; Immunoblotting, Immunoprecipitation, and Pull-down
Experiments--
Immunoblotting and immunoprecipitations were
performed as described earlier (14). For immunoblots and
immunoprecipitations either cell lysates or membranes solubilized in
1% Nonidet P-40 buffer were used. For pull-down assays GST fusion
proteins were overproduced in bacterial strain BL-21 and immobilized on
glutathione-Sepharose 4B beads (Amersham Biosciences). After incubation
with cell lysates or solubilized membranes, bound proteins were washed
three or four times and eluted with electrophoresis sample buffer.
Deglycosylation--
Proteins were deglycosylated with
endoglycosidase H (Roche Molecular Biochemicals) or
N-glycosidase F (Glyko) according to the instructions of the manufacturers.
Confocal Immunofluorescence--
BHK-21 and HEK293T cells were
grown on collagen-coated chamber slides (BD PharMingen) and fixed in
4% paraformaldehyde for 10 min, washed with phosphate-buffered saline,
permeabilized in 0.1% saponin in phosphate-buffered saline, and
blocked with 1% bovine serum albumin and 5% normal goat serum in
phosphate-buffered saline. Primary antibodies were added for at least
1 h in the same buffer. Secondary antibodies were either goat
anti-mouse or goat anti-rabbit IgG conjugated to Alexa 488 or Alexa
568. In addition anti-ClC-3A and ClC-3B antibodies were directly
labeled with Alexa 488 or Alexa 568 according to the instructions of
the manufacturer (Molecular Probes). Directly labeled anti-HA (Alexa 594 or 488) and anti-FLAG antibodies (fluorescein isothiocyanate or
Cy3) were purchased from Babco and Sigma, respectively.
Calu-3 cells were grown in a liquid air interface on Transwell Clear
inserts (Costar) for several weeks. Cell layers were fixed in 4%
paraformaldehyde for 10 min. Frozen sections were thawed and treated
with 1% sodium borohydride for 10 min and exposed to 1% SDS for 5 min
to improve antibody labeling. Detection of endogenously expressed
ClC-3B was facilitated using the Alexa 488 Signal-Amplification kit for
mouse antibodies from Molecular Probes. RhoB was expressed as a GFP
fusion using pEGFP-Endo (Clontech). Cells were
examined on a LSM510 confocal microscope from Zeiss.
Cloning and Expression of ClC-3A and B cDNAs--
Our interest
was attracted by the report by Ogura et al. (6) of a
PDZ-binding isoform of the ClC-3 chloride channel because of its
potential relation to CFTR. To be able to investigate this we cloned
cDNAs corresponding to ClC-3A and ClC-3B by PCR from a total human
pancreas cDNA pool. The protein sequences coded for by these
cDNAs with their principal features are shown in Fig.
1A. As originally shown by
Ogura et al. (6), the two forms are identical except at
their C-terminal ends where insertion of an additional exon makes
ClC-3B longer than ClC-3A by 47 residues and makes their sequences
differ at several other positions before the end of ClC-3A. The final
four amino acids of ClC-3B fit the consensus for binding to Class I PDZ
proteins (15). Consensus extracytoplasmic N-glycosylation
sites are indicated between helices B and C, and L and M, assigned by
alignment with the bacteria ClCs for which three-dimensional structures
have been obtained (16).
pcDNA3 plasmids with different epitope tags at the N termini of
ClC-3A and ClC-3B were transfected into BHK-21 and HEK293 cells so that
the proteins could be detected with antibodies to the tags. Both
transient and stable BHK transfectants exhibited a heterogenous larger
band of ~130 kDa and a more distinct smaller band of ~90 kDa (Fig.
1B). The ClC-3A bands detected with the mAb 42.4 to an MRP1
epitope (9) and ClC-3B bands with mAb 9E10 to the c-Myc tag appear very
similar as expected since they differ in size by only 47 amino acids.
Comparison of transient and stable expression in BHK cells revealed
that the ratio of the smaller to the larger bands was higher in the
former as if complete maturation had not yet occurred. This appeared
also to be the case in HEK293 cells where both bands appeared nearly
identical to those in transiently expressing BHK-21 cells.
Endoglycosidase digestion provided further characterization of these
electrophoretic bands (Fig. 1C). N-glycosidase F
caused the 130-kDa and 90-kDa bands to collapse to a band slightly
smaller than the latter in the case of both isoforms. Endoglycosidase H
had a similar effect on the 90-kDa bands, indicating they are glycosylated species. Their decrease in size corresponds to ~5 kDa,
consistent with the removal of two core oligosaccharide chains, probably from the two consensus N-glycosylation sites in the
protein sequence (Fig. 1A). The 130-kDa bands were
unaffected by endoglycosidase H as expected if they had acquired
complex oligosaccharide chains. Thus both forms of ClC-3 are
N-glycosylated membrane proteins that would be synthesized
on membrane-bound ribosomes and transported to the Golgi where the
glycosyl transferases that assemble complex oligosaccharide chains are located.
Differential Localization of ClC-3A and ClC-3B--
In previous
work, ClC-3A has been most definitely localized to late endosomes or
lysosomes (4) and to synaptic vesicles in the brain (3). These same
studies provided evidence that its presence contributes to the rate of
acidification of these compartments (3, 4). When expressed in BHK cells
ClC-3A co-localizes extensively with the late endosomal marker RhoB, and to a lesser extent with the early endosomal marker EEA-1 (Fig. 2A). This localization is
distinct from that of calnexin in the ER and several Golgi markers
including giantin, GM130, and Heterodimerization of ClC-3A and ClC-3B--
Since ClC-3A and B
are identical except for their C termini and all ClC channels are
dimers (1), heterodimerization might be expected. To assess whether
this occurred, we determined if they could be co-immunoprecipitated
from BHK cells in which they were co-expressed. Fig.
3A shows that each isoform was
present in an immunoprecipitate of the other member of the pair.
Interestingly, in these cells the intracellular localizations of both
were partially shifted toward that of the other (Fig. 3B).
Thus the characteristic endosomal pattern of ClC-3A is also exhibited
by a small amount of ClC-3B when the two are expressed together.
Conversely, the Golgi pattern of ClC-3B alone is displayed by ClC-3A in
many cells on co-expression of the two. Localization to the Golgi seems
to dominate over the endosomal location when heterodimerization occurs since there are clearly more cells with a predominant Golgi
localization of ClC-3A than cells showing a endosomal location of
ClC-3B (not shown). Homodimers of both ClC-3 proteins are expected to
also form and retain their individual locations.
Endogenous ClC-3B Expression and Localization in
Epithelia--
Since we were primarily interested in its possible
relationship to CFTR and relevance to cystic fibrosis, we examined the presence of ClC-3B in epithelial cells. First epithelial scrapings from
human bronchus and colon were homogenized in detergent (Nonidet P-40)
and immunoprecipitated with a mAb to ClC-3B, 69.16. After SDS-PAGE
analysis under fully reducing conditions, a Western blot was probed
with the same antibody (Fig.
4A). Distinct ClC-3B bands appeared in colonic samples from all three individuals tested and one
bronchial sample; the other two bronchial samples gave weak signals but
these observations clearly confirm ClC-3B expression in epithelial
tissue affected in cystic fibrosis. We then examined expression in a
number of epithelial cell lines and found positive signals of variable
intensity in Western blots of lysates of all (Fig. 4B).
ClC-3B appears most highly expressed in two of the lines, which also
contain large amounts of CFTR viz. Calu-3 derived from submucosal
glands and Capan-1, a pancreatic ductal cell line. Confocal
immunofluorescence of Calu-3 cells grown on permeable supports at an
air-liquid interface revealed ClC-3B in intracellular vesicular
structures (Fig. 4C) as when it was heterologously
expressed. This distinct population of vesicles is similar to those
stained with the Golgi markers, giantin and golgin and entirely
distinct from several apical and basolateral markers which clearly
demarcate these portions of the cell surface. Thus, as when
heterologously expressed, the principal residences of ClC-3B (Golgi)
and CFTR (apical) are different.
ClC-3B Interactions with PDZ Proteins--
The PDZ binding
capacity of ClC-3B led to the idea that it might associate with and
even be influenced by CFTR, possibly via EBP50 (6). We tested the
ability of ClC-3B to bind to the individual PDZ domains of three
different PDZ proteins, EBP50, PDZK, and GOPC, known to interact with
CFTR (17-19). GST fusions with each of the PDZ domains were incubated
with detergent lysates of BHK cells expressing CFTR and ClC-3B. Both
channel proteins bound preferentially to the first PDZ domain of EBP50
and to a much lesser extent to the second (Fig.
5A). This result is different from that of Ogura et al. (6) who reported that ClC-3B
preferred PDZ2 of EBP50 and hence that ClC-3B and CFTR might be coupled by EBP50. The C-terminal sequences of both channels indicate they should bind class I PDZ domains (15). When similar experiments were
performed with the four PDZ domains of PDZK1, ClC-3B appeared to bind
only the first (Fig. 5B). CFTR, however, bound strongly to
both domains 1 and 3. Hence at least in principle, a ternary complex could form with ClC-3B at PDZ1 and CFTR at PDZ3 of PDZK1. Since
we found that ClC-3B resides primarily in the Golgi it seemed reasonable that it might bind to the Golgi-associated coiled-coil PDZ
protein, GOPC, which binds CFTR (19). Fig. 5C confirms that the single PDZ domain of GOPC binds ClC-3B as well as CFTR.
Since the three PDZ proteins interact with both channel proteins, we
examined the influence of each of the PDZ proteins on the localization
of ClC-3B and CFTR (Fig. 6). Consistent
with the results of Cheng et al. (19), EBP50 overexpression
had little effect on the expression of CFTR, which is already at the
cell surface. ClC-3B, however, was shifted from its presence just in the Golgi to somewhat more peripheral locations in the cell but was not
detectible in the plasma membrane. PDZK1 overexpression similarly had
little effect on CFTR, which is reasonable because PDZK1 and most CFTR
are located in nearly the same place even when not co-expressed. On
ClC-3B, however, PDZK1 had a stronger impact than EBP50, causing even
more of that channel to move toward the cell periphery, although
apparently not entirely to the plasma membrane. GOPC overexpression has
a much more dramatic effect on both channels, causing them to become
condensed with it in a focal Golgi location. The apparent large
reduction in the amount of both channel proteins on co-expression with
GOPC but not EBP50 or PDZK1 is confirmed by Western blots of whole cell
lysates (Fig. 7). While these findings
clearly show that the Golgi PDZ protein, GOPC, interacts strongly with
the Golgi chloride channel, ClC-3B, this interaction cannot be entirely
responsible for their co-localization since CFTR also is bound by GOPC
but resides mostly at the cell surface.
ClC-3B Interaction with CFTR--
Since PDZK1 bound the two
channel proteins at different PDZ domains it seemed possible that it
might couple them. To test this possibility two types of experiments
were performed. In the first, both ClC-3B and CFTR could be
co-immunoprecipitated with PDZK1 (Fig.
8A). Second, we compared the
ability of a GST fusion with the C-terminal 40 amino acids of CFTR to
pull-down ClC-3B from lysates of cells in which it was overexpressed
alone or together with PDZK1 or one of the other PDZ proteins. Fig.
8B shows that interaction between the CFTR tail and ClC-3B
is greatly enhanced by PDZK1. The low level of association without
overexpressed PDZK1 may reflect the action of endogenous PDZ proteins
or interaction between these two integral membrane proteins mediated by
other means. In this assay, GOPC did not appear to cause increased
association (Fig. 8B) despite its strong self-association
via its C-terminal coiled-coil domain (11, 20). Similarly, EBP50, which
also self-associates, did not appear to mediate interaction of ClC-3B with the tail of CFTR (Fig. 8B). This result is consistent
with the fact that both channel proteins are bound primarily by the same PDZ domain of EBP50 (Fig. 5A). Overall, these results
show that PDZK1 can mediate interactions between ClC-3B and CFTR.
However, in our experiments the two channels are seen to co-localize
only in the Golgi where a very small portion of cellular CFTR resides (Fig. 9). Although PDZK1 seems to cause
some movement of ClC-3B from the Golgi, the co-localization with CFTR
at the surface shown by Ogura et al. (6) was not detected in
our experiments. This does not exclude the possibility that it may
occur under different conditions. Nevertheless, the most consistent
observation is of the co-localization of much of the ClC-3B pool with a
small proportion of the total CFTR pool in a Golgi compartment.
Since CFTR and ClC-3B, members of every different protein
families, are both chloride channels with C termini that bind class I
PDZ domains, it is reasonable to ask what may be the significance, if
any, of this common feature. This is of particular interest from the
perspective of the proposal of Ogura et al. (6) that the two
channel proteins may interact via the PDZ protein, EBP50, enabling CFTR
to regulate the ClC-3B channel. The sites of localization of CFTR in
cells are reasonably well established (21-23) as are certain aspects
of its trafficking (22, 23). The bulk of the protein resides at the
apical plasma membrane of epithelial cells in which it is endogenously
expressed with small but detectible amounts associated with
intracellular membranes in the secretory pathway (22). When expressed
heterologously in non-polar mammalian cells, most of the mature protein
is at the plasma membrane but significant amounts are intracellular,
most in the ER (14). These major features of CFTR localization have
been observed by many investigators and are amply illustrated in Figs.
4C, 6, and 9. The only novel feature of CFTR localization
arose from our focus on the Golgi because of the finding that ClC-3B is
present primarily at the Golgi. A small but readily detectable amount of CFTR, when heterologously expressed, co-localized with ClC-3B at the
Golgi (Fig. 9). This is worthy of note only because most investigators
have not reported on CFTR in the Golgi and those who have (22)
emphasized a lesser amount there than in the ER. Indeed, in Calu-3
epithelial cells in which CFTR is endogenously expressed, only weak
signals are detected in any intracellular compartment (Fig. 4).
In contrast to CFTR, localization of the newly discovered ClC-3B has
not previously been extensively characterized. The original work of
Ogura et al. (6) showed only that most of the protein was
intracellular with a small amount appearing at the leading surface
of spreading cells in which EBP50 was also overexpressed. We have now
shown that virtually all of the ClC-3B either heterologously expressed
in BHK or HEK 293 cells or endogenously expressed in Calu-3 epithelial
cells resides in the Golgi, separate from but nearly contiguous with
ClC-3A, which is in late endosomes (Fig. 2). Both ClC-3 isoforms must
at least transit the Golgi since they acquire complex
N-linked oligosaccharide chains (Fig. 1C). ClC-3A
has been found to reside in endosomal compartments (3, 4). The fact
that ClC-3B remains Golgi-associated whereas ClC-3A does not would seem
likely due to the PDZ-binding capacity of the former. Several
Golgi-associated PDZ proteins are known including the GRASPs (24),
Mints (25), GIPC (26), and GOPC (27). We focused on GOPC since Cheng
et al. (19) recently have shown that it binds and influences
the localization and turnover of CFTR. We confirmed these findings with
CFTR and found that overexpression of GOPC had a similar influence on
ClC-3B (Figs. 6 and 7). Although the mechanisms involved are not yet
clear, these responses of the two PDZ-binding channel proteins is
consistent with a growing body of evidence that GOPC plays an important
role in vesicular trafficking in the secretory and endocytic pathways.
It was discovered by its binding to the C terminus of a member of the
frizzled family of cell surface WNT receptors and co-localized with a
portion of the population of these receptors at the Golgi (27).
Independently, the second coiled-coil domain of GOPC was found to bind
TC-10, a member of the Rho-GTPase family involved in the regulation of the endocytic pathway (11). The same domain also interacts with the
Golgi membrane protein, syntaxin 6, suggesting a relationship of GOPC
with SNARE-mediated membrane recognition and fusion (20). Very recently
the PDZ domain of GOPC was found to interact with a neurodegenerative
mouse mutant of the PDZ-binding GluR While GOPC, which is known to self-associate via its coiled-coil
domains (11, 27) binds and co-localizes with some CFTR and ClC-3B
molecules, we did not find evidence that it promoted association
between them (Fig. 8). Under special circumstances favoring
dimerization instead of interactions with components of the trafficking
machinery, GOPC could theoretically promote association of ClC-3B and
CFTR. However, its influence seems more likely to be on the fate of
each individually under normal circumstances, in that both are severely
knocked down by its overexpression. Other Golgi PDZ proteins, including
those mentioned above, also have been proposed to play roles in the
trafficking and biosynthetic sorting of other PDZ-binding membrane
proteins (24-26) and may also with these chloride channels. How such a
multiplicity of potential interactions within a specific organelle are
regulated is not yet understood.
Similarly, PDZ protein binding alone clearly does not entirely
determine the major localizations of CFTR and ClC-3B in cells since, in
addition to GOPC, they both bind EBP50 and PDZK1, which reside
primarily at the cell surface. Interactions of CFTR with these promotes
its endocytic recycling and, hence, residence in the apical membrane of
epithelial cells (30). CFTR and ClC-3B bind to different PDZ domains of
PDZK1 and it is able to promote association of the two channels (Fig.
8). PDZK1, which normally has a very similar localization as CFTR,
causes some redistribution of ClC-3B toward more peripheral regions of
cells (Figs. 6 and 9), but we have been unable to detect any in the
plasma membrane on co-expression with either PDZK1 or EBP50. Neither
PDZK1 nor EBP50 knocked down the amount of ClC3B or CFTR as GOPC did.
As mentioned above, while promotion of cell surface retention of CFTR
is attributed to interaction with PDZ proteins such as EBP50 at
that location (30), this clearly does not occur with ClC-3B. Additional
factors must be at play in determining the primary steady-state
localization of the two channel proteins.
Although ClC-3A was not the primary focus of our experiments it was
necessary to precisely correlate its localization with that of a number
of markers of different intracellular membrane compartments to clearly
distinguish it from ClC-3B. This result (Fig. 2) showed a late
endosomal localization in excellent agreement with the findings of
Strobrawa et al. (3) and Li et al. (4). This is
significant for at least two reasons. First, there have been several
claims of heterologously expressed ClC-3A at the cell surface (31, 32).
However, these findings when made with functional assays of channel
activity may have reflected endogenous channels (33) and when made by
immunofluorescence in some cases used a commercial antibody that
recognized proteins other than ClC-3A (1). Li et al. (4)
have reported that small amounts of an N-terminally truncated version
of ClC-3A does reach the cell surface. Both swelling (31) and
calcium/calmodulin kinase (32) activated chloride channels attributed
to ClC-3A in some of these studies are not altered in cells from mice
in which the ClC-3 gene was knocked out (3). Second, since
there is now evidence that ClC-3A functions as an anion shunt to
increase the rate of endosomal acidification (3, 4), ClC-3B, which is identical in sequence except at the C terminus, probably has a similar
function in Golgi membranes.
Overall, our present study has clearly established that the ClC-3B
isoform is a Golgi channel where it may function as an anion shunt
during acidification as ClC-3A does in late endosomes (3, 4). While
this possibility remains to be rigorously tested, if confirmed it would
also fit well with this general function of the other members of the
ClC-3, -4, -5 branch of the mammalian ClC family (1). Our findings do
not preclude the possibility that a small amount of ClC-3B might
traffic to the cell surface and perhaps even be influenced by CFTR
under some circumstances as suggested by Ogura et al. (6).
Mohammad-Panah et al. (34) have reported immunolocalization
of ClC-4 to the apical surface of epithelial cells in intestinal
crypts. Appearance of ClC-3B at the cell surface could require the
involvement of a
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-aminobutyric acid
(GABA) and glycine and voltage-gated ClC classes are the largest and
best characterized (1). The ClC family is especially diverse with
members in all organisms including at least nine in mammals. Mutations
in several of these are disease-causing (2). ClC-1, ClC-2, and ClC-K
channels within one branch of the mammalian family reside in the plasma
membrane of cells in different tissue types whereas ClC-3 through ClC-7
form another large branch and reside primarily in membranes of
intracellular organelles with the highly homologous ClC-3, -4, -5 channels occupying the endosomal/lysosomal compartments. These three
may all provide negative charge shunts that increase the rate of
acidification of these compartments by proton ATPases (1, 3, 4). A similar function has been suggested for bacterial ClC channels in
extreme acid resistance response (5).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Adaptin, Santa Cruz Biotechnology;
Calnexin, StressGen; EEA1, BD Transduction Laboratories; ZO-1,
Zymed Laboratories Inc.; NaK-ATPase, ABR. Antibodies
against MUC1 and NKCC were generously provided by Sandra Gendler and
Christian Lytle, respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression of ClC-3A and ClC-3B.
A, sequence alignment of ClC-3A and ClC-3B. ClC-3A and
ClC-3B share identical sequences with the exception of the last 47 amino acids. ClC-3B has at its C-terminal end a PDZ-binding motif
highlighted in green. -Helices crossing the membrane
partially or completely are underlined (B
to R), and residues important for chloride selectivity are
shown in red. These predictions were derived from an
alignment with prokaryotic ClC channels, whose structures have been
determined (16). Two potential N-glycosylation sites are
shown in blue. A shorter form of ClC-3A has been described
(37). The N-terminal end of the short form is indicated with an
arrow. B, heterologous expression of ClC-3A
and ClC-3B in BHK-21 and HEK293T cells. Cells were transiently or
stably transfected as described under "Experimental Procedures."
ClC-3A was detected with antibody 42.4, which recognizes the N-terminal
MRP1 tag (BHK-21 transient) or with antibody 34.1, which recognizes the
C-terminal tail (BHK-21 stable). ClC-3B was detected with antibody
9E10, which recognizes the N-terminal c-Myc-tag or with antibody 69.16, which recognizes the C-terminal tail (BHK-21 stable and HEK293T
stable). In the first lane of each panel, the same amount of
protein was loaded from cells not expressing ClC-3A or ClC-3B
(control). C, endoglycosidase digestion of
ClC-3A and ClC-3B. Membranes were prepared from BHK cells stably
expressing ClC-3A or ClC-3B, treated with N-glycosidase F or
endoglycosidase H, separated by SDS-PAGE, and ClC-3A or ClC-3B were
detected by immunoblotting.
-adaptin. In distinct contrast ClC-3B
did co-localize with these markers, especially giantin, GM130, and
-adaptin (Fig. 2B). Of the endosomal markers there was
some co-localization only with EEA-1, suggesting that some of both
isoforms of the ClC-3 channel may occupy the early endosome
compartment, possibly even as heterodimers (see below). The primarily
distinct localization of the two channels; however, is emphasized when
each is compared with the same marker. With giantin, for example, there
is nearly complete correspondence with ClC-3B but very little with
ClC-3A, even though some of it is situated contiguous with that
compartment. This is not surprising of course since there is extensive
traffic between the trans-Golgi network and endosomes. The
Golgi localization of ClC-3B is emphatically confirmed in Fig.
2C where its staining in HEK293 cells coincides with that of
three Golgi proteins. Thus while Ogura et al. (6) showed
that most heterologously expressed ClC-3B was intracellular, our
results further resolve this primarily to the Golgi indicating that the
two ClC-3 isoforms reside in virtually continuous but clearly distinct
organelles.
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Fig. 2.
Localization of ClC-3 proteins relative to
organelle markers. A, ClC-3A heterologously expressed
in BHK-21 cells. Cells were transiently transfected with ClC-3A. ClC-3A
was detected by immunofluorescence as described under "Experimental
Procedures" using antibody 42.4. B, ClC-3B
heterologously expressed in BHK-21 cells. Cells were transiently
(rows 2-6) or stably (Giantin, first row)
transfected with ClC-3B and ClC-3B was detected by immunofluorescence
using antibody 69.16 or 9E10. C, ClC-3B heterologously
expressed in HEK293T cells. Cells were transiently transfected with
ClC-3B. ClC-3B was detected by immunofluorescence as described under
"Experimental Procedures" using antibody 69.16.
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Fig. 3.
ClC-3A and ClC-3B interact with each other.
A, co-immunoprecipitation of co-expressed ClC-3A and
ClC-3B. Membranes from BHK-21 cells transiently expressing ClC-3A and
ClC-3B were subjected to immunoprecipitation of ClC-3A or ClC-3B using
antibodies 42.4 or 9E10, respectively. Co-immunoprecipitated ClC-3A and
ClC-3B were detected by Western blotting using the same antibodies.
B, influence of ClC-3A and ClC-3B co-expression on the
localization of each. ClC-3A and ClC-3B were transiently co-expressed
in BHK-21 cells. ClC-3A was visualized with antibody 42.4 conjugated to
Alexa 568, and ClC-3B was detected using antibody 69.16 conjugated to
Alexa 488.
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Fig. 4.
Endogenous expression of ClC-3B in epithelial
cells. A, ClC-3B in human colonic and bronchial
epithelia. ClC-3B was immunoprecipitated from lysates of human colonic
and bronchial epithelia using antibody 69.16 and detected with the same
antibody by Western blotting. B, ClC-3B and CFTR in
epithelial cell lines. Membranes were prepared from epithelial cell
lines and separated by 6% SDS-PAGE. ClC-3B was detected using antibody
69.16, and CFTR was detected using antibody 596. C,
localization of ClC-3B in polarized Calu-3 cells. Calu-3 cells were
grown at an air-liquid interface and stained for immunofluorescence
microscopy as described under "Experimental Procedures." MUC1,
EBP50, and CFTR localize to the apical membrane. Immunofluorescence of
sodium, potassium ATPase (NaK-ATPase), sodium potassium
chloride cotransporter 1 (NKCC), and Zonula occludens
protein 1 (ZO-1) demonstrate polarization of the cells.
Nuclei were stained with propidium iodide and are shown in
blue in the CFTR panel.
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Fig. 5.
Abilities of GST fusions of PDZ domains of
EBP50 (A), PDZK1 (B), and GOPC
(C) to interact with ClC-3B and CFTR. GST fusions
with PDZ domains of EBP50, PDZK1, and GOPC were bound to
glutathione-Sepharose beads and incubated with lysates from BHK-21
cells expressing ClC-3B or CFTR. The beads were washed, and bound
proteins eluted. Lysates from cells expressing ClC-3B or CFTR were
loaded as a positive control. Individual or combined PDZ domains
employed are indicated above each lane. The high molecular
weight ClC-3B bands near the top of the gels reflect strong aggregation
in non-ionic detergent.
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Fig. 6.
Effect of EBP50, PDZK1, and GOPC on the
localization of ClC3B and CFTR. ClC-3B or CFTR were transiently
co-expressed with EBP50, PDZK1, or GOPC, and immunofluorescence
microscopy was performed as described under "Experimental
Procedures." Individual ClC-3B and CFTR panels at top
indicate localization of proteins expressed alone; below are
their localization on co-expression with the PDZ protein
indicated.
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Fig. 7.
Influence of overexpression of PDZ proteins
on the amount of ClC-3B and CFTR. A, co-expression of
GOPC with ClC-3B or CFTR. ClC-3B or CFTR were transiently overexpressed
in BHK-21 cells without or with GOPC as described under "Experimental
Procedures." Cell lysates were analyzed by Western blotting using the
antibodies 69.16 and 596 for ClC-3B and CFTR, respectively.
B, co-expression of EBP50, GOPC, or PDZK1 with ClC-3B.
EBP50, GOPC, or PDZK1 were transiently cotransfected with ClC-3B as
described under "Experimental Procedures." ClC-3B was visualized by
Western blotting using antibody 69.16.
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Fig. 8.
Interaction of ClC-3B and CFTR.
A, co-immunoprecipitation of PDZK1 and ClC-3B or CFTR.
PDZK1 was immunoprecipitated from solubilized membranes prepared from
BHK-21 cells expressing ClC-3B or CFTR alone or with PDZK1. ClC-3B or
CFTR were detected by immunoblotting. B, influence of
overexpression of PDZK1, GOPC, and EBP50 on the association of ClC-3B
with the C-terminal tail of CFTR. A fusion of the last 40 amino acids
of CFTR (1440-1480) and GST was bound to glutathione-Sepharose beads
and incubated with lysates or solubilized membranes from BHK-21 cells
expressing ClC-3B with or without PDZK1 or EBP50. To avoid the
down-regulation of ClC-3B in the presence of co-expressed GOPC two
different membrane preparations from cells overexpressing ClC-3B or
GOPC were combined and solubilized to test the influence of GOPC on the
association of ClC-3B with the C terminus of CFTR.
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Fig. 9.
Co-localization of a small pool of CFTR with
ClC-3B. CFTR, ClC-3B, and EBP50, PDZK1, or GOPC and were
transiently expressed in BHK-21 cells. CFTR and ClC-3B were visualized
by immunofluorescence as described under "Experimental
Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 glutamate ion channel, and its
C-terminal coiled-coil domain with Beclin1, a factor promoting
autophagy where cellular constituents are trafficked to lysosomes and
degraded (28). Hence, GOPC appears capable of more generally shifting
the balance of PDZ-binding membrane proteins in the degradative
direction. The other important clue to GOPC function comes from the
knock-out of its gene in mice which results in failure of acrosome
formation, a Golgi-dependent function, during
spermatogenesis without detectable effect on other tissues (29).
-subunit as is the case with ClC-Ka, where
barttin plays this role (35) or interference with recognition of a PY
internalization and degradation motif, as is the case with ClC-5 (36).
However, our findings do not support the idea that interactions with
subplasma membrane PDZ proteins such as EBP50 (or PDZK1) bring about
either cell surface localization or association with CFTR at the plasma membrane.
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ACKNOWLEDGEMENTS |
---|
We thank Ian Macara for providing the pKH3-PIST plasmid and Olivier Kocher for a PDZK1 cDNA; Sandra Gendler and Christian Lytle for supplying antibodies against MUC1 and NKCC, respectively; and Richard Boucher and Marcus Mall for bronchial and colonic epithelial scrapings. We thank Sharon Fleck and Marv Ruona for preparation of the article and figures, respectively. We further thank Bradley Bone and Tammy Brehm-Gibson for antibody production and purification.
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FOOTNOTES |
---|
* This work was supported by grants from the NIDDK, National Institutes of Health.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.
To whom correspondence should be addressed: Mayo Clinic
Scottsdale, S. C. Johnson Medical Research Center, 13400 E. Shea
Blvd., Scottsdale, AZ 85259. Tel.: 480-301-6206; Fax: 480-301-7017;
E-mail: riordan@mayo.edu.
Published, JBC Papers in Press, December 5, 2002, DOI 10.1074/jbc.M211050200
2 GOPC or Golgi-associated PDZ coiled-coil protein was discovered and named by Yao et al. (27). Homologous cDNAs were independently isolated by Neudauer et al. (11) and termed PIST for PDZ domain protein interacting specifically with TC10; by Charest et al. (20) and termed FIG for Fused In Glioblastoma; and by Cheng et al. (19) and termed CAL for CFTR-associated ligand. We have used the term GOPC, which seems most descriptive and does not derive from one of the proteins with which it interacts.
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ABBREVIATIONS |
---|
The abbreviations used are: PDZ, PSD-95/discs large/ZO-1; EBP50, ERM-binding phosphoprotein 50; CFTR, cystic fibrosis transmembrane conductance regulator; GIPC, GAIP-interacting protein, C terminus; GOPC, Golgi-associated PDZ and coiled-coil motif-containing protein; GRASP, Golgi reassembly stacking protein; GST, glutathione S-transferase; Mint, Munc18-interacting protein; NKCC, sodium potassium chloride cotransporter 1; HA, hemagglutinin; mAb, monoclonal antibody; SNARE, soluble NSF attachment protein receptors.
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