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J Biol Chem, Vol. 273, Issue 45, 29381-29388, November 6, 1998
From the Department of Cell Biology, University of Geneva, 1211 Geneva, Switzerland
In Madin-Darby canine kidney cells, newly
synthesized apical and basolateral membrane proteins are generally
transported directly to their respective cell surface domain due to
targeting determinants that mediate sorting in the Golgi complex. In
several basolateral membrane proteins, these targeting determinants
reside in the cytoplasmic domains. We show here that basolateral
expression of the human The plasma membrane of polarized epithelial cells is divided into
two morphologically, functionally, and biochemically distinct cell
surface domains. The maintenance of cell surface polarity requires the
continuous sorting of newly synthesized and internalized membrane
components. Once arrived at the trans-side of the Golgi apparatus, newly synthesized membrane proteins are sorted into cell
surface transport pathways in a manner that can vary from one
epithelial tissue to another and from one protein to another (1-4).
With one exception, all studied apical and basolateral membrane
proteins are directly sorted to their respective cell surface domain in
the Golgi apparatus in MDCK1
cells. Apical sorting can be mediated by different types of
determinants including transmembrane domains and
glycosylphosphatidylinositol anchors as well as luminal
N-linked and perhaps O-linked carbohydrates (5-10). Basolateral targeting has been associated with cytoplasmic domain determinants that mediate direct basolateral transport in the
Golgi apparatus as well as in endosomes (2, 3). Membrane proteins
lacking apical and basolateral targeting determinants accumulate in the
Golgi apparatus, indicating that efficient cell surface transport does
not occur by default (10).
In one clone of MDCK cells, polarization of
Na+K+-ATPase is mediated by selective
basolateral stabilization after delivery to both cell surface domains
(11). The apical pool of Na+K+-ATPase does not
become transcytosed but is degraded, as seen during the repolarization
of MDCK cells in calcium switch experiments (12). Although nonpolarized
cell surface transport occurs only in a mutant MDCK cell line deficient
in glycolipid sorting, the steady-state polarity of
Na+K+-ATPase seems to be enhanced by selective
basolateral stabilization also in wild-type MDCK cells (13, 14).
Integrins are functionally important basolateral membrane proteins
composed of an We studied basolateral sorting of the human
Cell Culture, Mutagenesis, and Transfection--
MDCK strain II
cells were grown in Dulbecco's modified Eagle's medium (4.5 g/liter
glucose) as described previously (21). For polarity experiments, the
cells were cultured for 5-8 days on tissue culture-treated
polycarbonate filters (Costar Transwells) with a pore size of 0.4 µm
and a diameter of 24 mm (for biochemical assays) or 12 mm (for immunofluorescence).
The cDNAs coding for human
MDCK cells were transfected and selected using G418 (pCB6) or
hygromycin (pCB7), respectively (21). For double transfections, stable
cell lines were first generated expressing either wild-type or
truncated human Immunofluorescence and Confocal Microscopy--
Transfected
cells were cultured on filters for 5-8 days and then fixed with or
without an overnight preincubation with 10 mM sodium
butyrate. To label chimeric Fc receptors with a polyclonal antibody
(22) and human Cell Surface Appearance of Newly Synthesized Proteins and
Differential Stability--
Filter-grown MDCK cells were metabolically
labeled for 20 min with
[35S]methionine/35S-cysteine, chased for
different periods of time, and selectively biotinylated (25). The
biotinylated cells were solubilized with extraction buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.2% SDS, 1% Empigen BB, 10 mM glycine, and 40 µg/ml phenylmethylsulfonyl fluoride; 1 ml/filter). The extracts were left on ice for 30 min and then spun in a
microfuge for 15 min at 4 °C. The human integrin subunits were
immunoprecipitated with the monoclonal antibodies linked to protein
A-Sepharose via a rabbit anti-mouse secondary antibody, and the
chimeras were immunopurified with the rabbit anti-Fc receptor antibody
(21). After washing, the immunocomplexes were dissociated, and each precipitate was divided into three samples: (a) one sample
was fractionated on a SDS gel, and radioactively labeled protein was visualized by fluorography; (b) another sample was diluted,
reprecipitated with streptavidin-agarose, and then analyzed by
electrophoresis and fluorography; and (c) the third sample
was fractionated on a SDS gel, transferred to nitrocellulose, and then
probed with streptavidin-HRP.
For the determination of the half-lives of proteins in the apical and
the basolateral cell surface domains, cells were cooled on ice, and one
of the two cell surface domains was biotinylated as described above.
After incubating the cells for different periods of time at 37 °C in
normal tissue culture medium, the samples were extracted, and the
transfected protein(s) was immunoprecipitated. The precipitates were
fractionated on SDS gels and transferred to nitrocellulose, and the
presence of biotinylated protein was tested using streptavidin-HRP and ECL.
Steady-State Distribution of Chimeric Fc Receptors--
The
steady-state distribution of Fc receptors was determined using a
modified antibody-binding assay (21). Filter-grown MDCK cells
expressing chimeras were incubated with the polyclonal antibody against
the Fc receptor (diluted in phosphate-buffered saline containing 0.5%
bovine serum albumin and 0.5 mM CaCl2) from
either the apical or the basolateral side on ice for 2 h (the
opposite side contained buffer alone). The cells were then washed and
incubated for another 2 h in the same way with a HRP-conjugated goat anti-rabbit antibody. After washing, the filters were excised from
the holders and transferred to phosphate-buffered saline containing 1%
Triton X-100, and HRP was assayed colorimetrically (26).
Human
Fig. 1A shows confocal
sections obtained from monolayers preincubated overnight with sodium
butyrate and then labeled with the two integrin antibodies. In both
cases, very little apical staining could be detected, but the lateral
and the basal plasma membranes were efficiently labeled. When
nontransfected MDCK cells were labeled with the two antibodies under
the same conditions, no staining could be detected (the
anti- Both the
We next co-expressed mutant Both Cytoplasmic Domains Are Independently Sufficient to Mediate
Basolateral Expression--
Because integrin affinity to the ligand
can be affected by mutations in the cytoplasmic domains, it could be
that the deletions of the cytoplasmic domains caused a redistribution
due to the effects on substrate binding. The effects of mutations that
we observed on the polarized cell surface distribution, however, do not
correlate with the known effects on affinity (27). To test a direct
involvement of the integrin cytoplasmic domains in basolateral sorting,
we linked the entire cytoplasmic tail of
Fig. 3 shows confocal sections taken from
such samples derived from cells treated with (A and
B) or without (C) sodium butyrate. The chimera
containing the cytoplasmic domain of
The apical expression of the
We next introduced different deletions into the cytoplasmic domains of
the
We then expressed a set of Newly Synthesized
Fig. 4A shows that
Fig. 5 shows the result of such an
experiment in which samples from the same cell extracts were
immunoprecipitated with either anti- Our experiments indicate that basolateral polarization of
transfected human Newly synthesized Basolateral polarization aided by selective stabilization has also been
observed for Na+K+-ATPase (11, 14). In this
case, significant apical transport of newly synthesized protein was
only observed in MDCK cells deficient in apical glycolipid sorting (13,
14). Importantly, our MDCK strain II cells efficiently sort many apical
and basolateral membrane proteins including
Na+K+-ATPase and apically expressed
glycosylphosphatidylinositol-anchored proteins and are hence able to
recognize all known types of plasma membrane sorting signals ranging
from cytoplasmic basolateral targeting determinants over
glycosylphosphatidylinositol anchors to extracytoplasmic apically
sorting carbohydrates (5, 10, 21, 25, 28, 36).
Although the Stabilization of the basolateral pool of integrins could occur either
by selective recycling of basolaterally internalized molecules or by
interactions with the submembrane cytoskeleton at the cell surface.
Cytoplasmic determinants that mediate basolateral recycling are similar
or even identical to determinants that mediate basolateral sorting in
the Golgi apparatus (25, 39); hence, it is unlikely that the
Of all the proteins known to interact with integrin cytoplasmic
domains, only calreticulin is known to bind to The cytoplasmic domain of In MDCK cells, We thank Dr. E. Ruoslahti for the cDNAs
coding for the human *
This research was supported by the Swiss National Science
Foundation and the Canton de Genève.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.
§
Recipient of a fellowship from the Swiss Talents in Academic
Research and Teaching program of the Swiss National Science Foundation. To whom correspondence should be addressed: Dept. of Cell Biology, University of Geneva, Sciences III, 30, Quai Ernest-Ansermet, 1211 Genève-4, Switzerland. Tel.: 41-22-702-6729; Fax: 41-22-781-1747; E-mail: Matter{at}cellbio.unige.ch.
The abbreviations used are:
MDCK, Madin-Darby
canine kidney; HRP, horseradish peroxidase; PAGE, polyacrylamide
gel electrophoresis.
The Cytoplasmic Domains of a 
1 Integrin Mediate
Polarization in Madin-Darby Canine Kidney Cells by Selective
Basolateral Stabilization*
,, and
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
5
1 integrin
in stably transfected Madin-Darby canine kidney cells is also mediated
by the cytoplasmic domains. Distinct regions in both cytoplasmic
domains were found to be sufficient to mediate basolateral expression
independently from one another. Unexpectedly, newly synthesized
wild-type 51 and basolaterally expressed
chimeras containing the cytoplasmic domain of either 5
or 1 were integrated into both cell surface domains,
preferentially apically, during biosynthesis. The apical pools of
wild-type integrin and chimeric subunits were found to become quickly
degraded, whereas the basolateral pools were stabilized. Thus, the
cytoplasmic domains of the 51 integrin
are independently sufficient to mediate sorting by selective
basolateral stabilization.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
subunit and a subunit (15, 16). MDCK cells
express a variety of different and subunits (17, 18).
1 seems to be the predominant subunit and is
involved in the formation of functional receptors for different
extracellular matrix components as well as in the determination of the
spatial orientation of polarized epithelial cells (18-20).
51 integrin in stably transfected MDCK
cells, which was basolaterally expressed in the steady state. The
cytoplasmic domains of both subunits were found to be able to mediate
the basolateral sorting of dimeric integrins as well as that of a
chimeric reporter protein independently from one another but exhibited
different capacities. Unlike other basolateral sorting signals, the
presence of the cytoplasmic domains of 5 and/or
1 did not result in direct basolateral targeting, but in
stabilization of the basolateral pool after transport to both cell
surface domains.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
5 and 1
(generously provided by Dr. E. Ruoslahti, La Jolla Cancer Research
Foundation, La Jolla, CA) were cloned into Bluescript SK+
and into the eukaryotic expression vectors pCB7 (5;
selectable with hygromycin) or pCB6 (1; selectable with
G418). 5CT9 and 1CT10 were constructed
using the Bluescript constructs and PCR-based mutagenesis to introduce
premature stop codons. Fc receptor chimeras were constructed as
described previously using a modified FcRII-B2 cDNA coding for the
apically expressed tail-minus receptor with a AflII site
just before the stop codon (21). cDNA fragments corresponding to
the cytoplasmic domain of 5 (amino acids 3-28, resulting in FcR-5:3-28) or 1 (amino
acids 11-47, resulting in FcR-1:11-47) with a
AflII and a XbaI site were synthesized and cloned
into the Fc receptor cDNA in pCB6. These two cDNAs were then
used to construct chimeras with modified cytoplasmic domains using
primers with 5' XbaI sites for cloning. All constructs were
sequenced over the entire subcloned PCR-derived fragment by dideoxy sequencing.
1. These cell lines were then used to
co-express 5 cDNAs. Single clones were picked and
analyzed for expression by immunofluorescence. At least three clones
and an uncloned cell line (derived from a pool of the nonpicked clones)
were grown and analyzed for all transfections.
5 with monoclonal antibody P1D6 (Life Technologies, Inc.), the cells were fixed for 20 min; to label human
1 with monoclonal antibody P4C10 (Life Technologies,
Inc.), the cells were fixed for 60 min with 3% paraformaldehyde. The long fixation for the 1 staining eliminated background
staining due to endogenous 1 if the primary antibody was
sufficiently diluted (1:8000; each batch was newly titrated). The
samples were then blocked and permeabilized with phosphate-buffered
saline containing 0.1% saponin, 10 mM glycine, 0.5%
bovine serum albumin, and 0.05% sodium azide (23). The primary and
secondary antibodies were diluted in the same solutions, and the
incubations were performed for 60 min at room temperature. In some
experiments, cell surface chimeras were labeled by incubating the
unfixed cells with primary antibody diluted in phosphate-buffered
saline containing 0.5% bovine serum albumin and 0.5 mM
CaCl2 added from both sides of the filter culture for
2 h on ice (24). The samples were analyzed by confocal microscopy
as described previously (23).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
51 Integrin Is Expressed at the
Basolateral Cell Surface in Stably Transfected MDCK Cells--
MDCK
cells endogenously express several different integrins, but
1 seems to be the most prominent subunit (17, 18). We therefore decided to use human 1 and 5
for the targeting studies; these two subunits can dimerize together,
forming a receptor for fibronectin, and the human 5
subunit can easily be specifically detected because MDCK cells do not
seem to express significant amounts of the 5 integrin
subunit (17). To reconstitute this human integrin in MDCK cells, we
first generated stable cell lines expressing human 1.
This resulted in several clones that all expressed the human integrin
subunit in the basolateral membrane (data not shown). We chose two
homogeneously expressing clones into which we transfected a cDNA
coding for human 5. Cell lines expressing the two human
integrin subunits were then cultured on filters for 5 days to allow
full polarization, and the polarity of expression was then studied by
immunofluorescence combined with confocal microscopy. Because both the
anti-5 and anti-1 antibodies are derived
from mouse, we stained separate cultures for the two subunits.
1 antibody showed some staining in cells that were
gently fixed for 15 min, but not if the cells were fixed as described
here for 60 min). Basolateral expression of the two subunits was also
obtained when they were separately transfected (data not shown). The
cell surface expression in such single transfections was lower, and the
1 subunit could be detected accumulating in the
endoplasmic reticulum, suggesting that the expression of only one
subunit did not result in efficient cell surface transport due to
insufficient amounts of partners for dimerization. Although this
suggests that the two transfected subunits dimerized together, it could
also be that at least a fraction of the transfected subunits was
transported to the cell surface in association with an endogenous
integrin subunit. As shown below, however, co-expressed subunits can
determine the polarity of each other's cell surface distribution,
indicating that in the steady state, the vast majorities of
co-transfected human integrin subunits must be dimerized with one
another.
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Fig. 1.
Polarized expression of wild-type and
C-terminally truncated human 51 integrin
in MDCK cells. Stably transfected MDCK cells co-expressing the
human 5 and 1 integrin subunits
(A), 5CT9 and 1CT10
(B), wild-type 5 and 1CT10
(C), or 5CT9 and wild-type 1
(D) were plated on filters; after 1 week of culture and an
overnight incubation with sodium butyrate, they were fixed and
processed for immunofluorescence using either an anti-5
(top) or anti-1 (bottom)
monoclonal antibody. The samples were analyzed with a confocal
microscope. For each staining, an optical section derived from the
apical region of the monolayer (apical), one from
approximately the middle of the monolayer (lateral), and one
taken close to the filter (basal) are shown. At least two
different clones and an uncloned cell line were analyzed for each
combination of the transfected subunits. Bars, 10 µm.
5 and the 1 Cytoplasmic
Domain Contain Basolateral Sorting Information--
Because
basolateral sorting of several membrane proteins has been shown to
depend on distinct cytoplasmic targeting determinants, we constructed
C-terminally truncated 5 and 1 integrin
subunits lacking most of the cytoplasmic domains (Fig.
2: 5CT9 and
1CT10) and generated stable cell lines co-expressing the
two mutant subunits. Filter-grown cells preincubated overnight with
sodium butyrate were fluorescence labeled using either the
anti-5 or the anti-1 monoclonal antibody
and analyzed by confocal microscopy. Fig. 1B shows that both
antibodies efficiently stained the apical membrane of MDCK cells
expressing human 5 and 1 integrin
subunits lacking their cytoplasmic domains (5CT9 and
1CT10), and only a little staining of the basolateral
cell surface was detected. Thus, one or both of the cytoplasmic domains
are required for basolateral expression of human
51 integrin in MDCK cells. Furthermore, the absence of cytoplasmic basolateral sorting information results in
the preferential apical expression of the integrin, as in the case of
many monomeric membrane proteins (2).
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Fig. 2.
Amino acid sequences of the cytoplasmic
domains of wild-type, mutant, and chimeric human 5 and
1 integrin subunits. The cytoplasmic domain amino
acid sequences of wild-type and mutant 5 and
1 (A) and of chimeras consisting of the ecto-
and transmembrane domains of the mouse Fc receptor for IgG
(FcRII) and 5 or 1 cytoplasmic
domain sequences (B) are shown in the single-letter code. In
A, the arrows indicate the positions of the
C-terminal truncations (the number after CT indicates the
number of residual cytosolic amino acids). In B, amino acids
derived from either 5 or 1, respectively,
are underlined, and the arrows indicate the
position of amino acid substitutions (labeled with the code of the
replaced and the introduced amino acids and the position in the
cytoplasmic domain). For the different chimeric constructs of
5 (FcR-5) and 1
(FcR-1), the stretch of amino acids derived
from 5 or 1, respectively, is indicated
with a double-headed arrow and the number of the
first and the last integrin-derived amino acid.
5 and 1
subunits with the corresponding wild-type subunit to test whether one
or both of the cytoplasmic domains are required for basolateral
expression; if only one was required, we tested to see which one. In
cells expressing wild-type 5 and 1CT10,
both antibodies resulted in efficient basolateral labeling with little
apical fluorescence (Fig. 1C), indicating that the
5 cytoplasmic domain is sufficient to mediate basolateral expression. Cells expressing wild-type 1 and
5CT9 were labeled by both antibodies on both cell
surface domains (Fig. 1D), suggesting that the cytoplasmic
domain of 1 also mediates basolateral expression, but at
an apparently lower efficiency than the cytoplasmic domain of
5. These experiments also demonstrated that one
transfected subunit could efficiently redirect the other one,
indicating that most of the integrin staining we observed must have
been due to the transfected human 5 and 1
dimerized with each other. This is also supported by the observation
that efficient apical expression was not observed when the cells were transfected with either one of the mutant subunits alone (data not
shown). It is important to note that expression of mutant integrin
subunits did not negatively affect MDCK cells. We could not detect
decreases in transepithelial electrical resistance, indicating that the
transfected cells were still forming electrically tight monolayers, and
the cells did not show any obvious morphological alterations when
observed by electron microscopy after embedding in Epon and thin
sectioning (data not shown).
5 (amino acids
1-28) or the region of 1 required for basolateral expression of the integrin (amino acids 11-47) to the transmembrane and extracytoplasmic domain of the mouse Fc receptor for IgG (Fig. 2), a protein that is apically expressed in the absence of a
basolateral sorting signal in its cytoplasmic domain (28, 21). We then generated stable MDCK cell lines expressing these chimeric constructs and analyzed polarized expression in filter-grown cells with an antibody recognizing the Fc receptor ectodomain.
5 (Fig.
3A) was basolaterally expressed, and only a little apical
staining could be detected. The cytoplasmic domain of human
5 is thus sufficient to mediate basolateral expression
of the reporter protein. When MDCK cells expressing the
1 Fc receptor chimera were analyzed, labeling of both
cell surface domains could be detected after the induction of high
expression levels by sodium butyrate (Fig. 3B). The staining
appeared heterogeneous: some cells were only basolaterally labeled,
whereas other cells were fluorescent on both cell surface domains.
Because random labeling seemed to coincide with more expression, we
repeated the same experiment without induction with sodium butyrate.
Indeed, primarily basolateral fluorescence was detected at low
expression levels (Fig. 3C). Thus, the cytoplasmic domain of
1 is also sufficient to mediate basolateral expression
of the Fc receptor but seems to do so at a lower efficiency than that
of 5. This is in agreement with the mixed cell surface
polarity observed in cells co-expressing wild-type 1 and
5CT9 (Fig. 1D).
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Fig. 3.
Polarized expression of Fc receptor-integrin
chimeras. Chimeras consisting of the ecto- and transmembrane
domains of the mouse IgG Fc receptor and the cytoplasmic domain of
either 5 (FcR-5:3-28, A) or
1 (FcR-1:11-47, B and
C) were stably expressed in MDCK cells. Mature filter
cultures of such cells, preincubated with (A and
B) or without (C) sodium butyrate to induce
higher expression levels, were then incubated with anti-Fc receptor
antibodies on ice to label only chimeras at the cell surface. After
washing and fixing, the bound antibody was visualized with a
fluorescent secondary antibody and confocal microscopy. Shown are three
sections derived from a set of serial sections representing the apical,
the middle (lateral), and the basal region of the monolayer.
Bar, 20 µm. In D and E, the cell
surface distribution of the FcR-5 (D) and
FcR-1 (E) chimeras was determined by binding
first anti-Fc receptor antibody followed by HRP-conjugated secondary
antibodies to filter-grown transfected and nontransfected cells. Bound
secondary antibody was then assayed by measuring HRP with a
colorimetric assay. The values obtained from nontransfected cells were
deducted as background from those from of transfected cells. The values
are given as a percentage of total activity (apical plus basolateral).
To avoid saturation of basolateral expression mediated by the
1 cytoplasmic domain, all determinations in B
were done without preincubation with sodium butyrate. The values for
each construct are derived from two clones and were determined in two
independent experiments performed in duplicates.
1 Fc receptor chimera at
high expression levels could also have been due to a dominant negative effect of the integrin chimera causing a general defect in cell adhesion, as described for fibroblasts expressing a chimera containing the entire cytoplasmic domain of 1 (29, 30, 31). We
could not detect negative effects on the monolayer morphology, the
polarized expression of another basolateral membrane protein
(Na+/K+-ATPase), or on the localization of the
tight junction protein ZO-1 in cells expressing
FcR-1:11-47; such effects could easily be observed when
a chimera containing the entire cytoplasmic domain of 1
including the first 10 amino acids was expressed.
5 and 1 Fc receptor chimeras to map
the regions in the cytoplasmic tails required for basolateral
expression. We assayed the polarity of expression by binding the
anti-Fc receptor antibody to either the apical or the basolateral cell
surface domain of filter-grown transfected cells on ice. After washing, bound primary antibody was visualized with a secondary antibody conjugated to HRP and a colorimetric reaction. Fig. 3D shows
that 80% of the chimera containing the entire cytoplasmic domain of 5 was detected basolaterally, confirming the confocal
images shown above. In contrast, C-terminal deletions of 19 or 11 amino acids, respectively, resulted in preferentially apically expressed chimeras (FcR-5:3-9 and FcR-5:3-17). In
contrast, removal of the membrane proximal domain, which is conserved
in all subunits (32), did not affect basolateral expression
(FcR-5:8-28). Thus, only the C-terminal part of the
5 cytoplasmic domain is required for basolateral
expression of the chimeras.
1 Fc receptor chimeras and
analyzed the polarity of expression without preincubation with sodium butyrate to avoid saturation. Fig. 3E shows that C-terminal
truncation also resulted in apical expression
(FcR-1:11-28 and FcR-1:11-38), indicating
that the C-terminal part of the 1 cytoplasmic domain is
important for basolateral expression. Interestingly, substitution of
tyrosine 44 by an alanine (FcR-1:11-47Y-A44) reduced the
polarity of expression (~60% basolateral); hence, the tyrosine
substitution resulted in only a partial inactivation of the basolateral
sorting activity of the 1 cytoplasmic domain because the
complete absence of basolateral sorting information results in
preferential apical expression of Fc receptors (28). Thus, the last
nine amino acids of the cytoplasmic domain of 1 are
important for basolateral expression, and tyrosine 44 is involved in
this process.
51 Integrin Is
Transported to Both Cell Surface Membranes--
Cytoplasmic
domain-mediated basolateral expression has thus far always been found
to be achieved by direct transport from the Golgi apparatus to the
basolateral cell surface, circumventing the apical plasma membrane
domain (2). To test whether the cytoplasmic domains of the human
51 integrin also mediate direct basolateral transport, we pulse-chase labeled transfected cells co-expressing wild-type 51 integrin and
then biotinylated either the apical or the basolateral plasma membrane
domain. After extraction, the human integrin was immunoprecipitated
with the anti-5 antibody. Each precipitate was then
split into three samples: (a) the first sample was directly
analyzed by SDS-PAGE and fluorography to see the total labeled and
immunoprecipitable integrin (i.e. newly synthesized
integrin); (b) the second sample was reprecipitated with
streptavidin-agarose to see the metabolically labeled biotinylated integrin (i.e. newly synthesized integrin at the cell
surface); and (c) the third sample was fractionated by
SDS-PAGE and transferred to nitrocellulose, and biotinylated integrin
was visualized with HRP-streptavidin and enhanced chemiluminescence to
see the total biotinylated integrin (i.e. the steady-state
cell surface distribution of human 51 integrin).
5 was synthesized as a higher molecular weight precursor
(heaviest band at 0 min) and then processed and also started to
co-immunoprecipitate subunits. The total immunoprecipitated 5 appeared to increase with time, suggesting that the
monoclonal antibody used in this experiment recognizes the mature forms
more efficiently than the early forms, a common behavior of monoclonal antibodies that is often used to assay conformational maturation of
proteins in the early secretory pathway (for examples, see Refs. 33 and
34). Newly synthesized integrin was detected on the cell surface after
60 min of chase (Fig. 4A, surface 35S proteins)
and started to appear at both cell surface domains in similar amounts.
At longer times of chase, the integrin started to disappear from the
apical cell surface, whereas it remained at the basolateral domain.
Total biotinylated integrin exhibited a clear basolateral expression in
all cultures, suggesting that the apical appearance was not due to
preferential apical biotinylation or an inability of the antibody (or
of the technique used) to immunoprecipitate basolateral integrin. If
the immunoprecipitation was done with anti-1 antibodies,
the apical cell surface appearance was even more pronounced (Fig.
4B). It is currently not clear what caused the difference in
the amounts of apically detected integrin in Fig. 4A and
B, but it could be that a fraction of 5
formed dimers with a relatively short half-life (and therefore did not
influence the steady-state distribution) with an endogenous subunit
that, in contrast to 1, is able to mediate direct
basolateral transport. Moreover, preferential apical cell surface
insertion was also obtained when the human 1 subunit was
expressed alone and thus could only dimerize with endogenous subunits (data not shown), excluding that apical transport was induced
by human 5. Because the monoclonal anti-1
antibodies that were at our disposition did not immunoprecipitate
canine 1, we could not directly test whether endogenous
1 is also transported to the apical membrane.
Nevertheless, endogenous 1 integrins have previously been reported on the apical cell surface of MDCK cells, excluding that
the apical expression is a transfection artifact due to, for instance,
higher expression levels (18-20). Because the apical integrin pool has
a short half-life (see below), the different polarity of cell surface
insertion observed by different investigators might be due to the
different times of chase used (35, 20). In agreement with the results
obtained with the dimeric integrin, the 5 and the
1 Fc receptor chimera appeared preferentially at the
apical cell surface but were detected basolaterally if the steady-state
distribution was analyzed (Fig. 4, C and D). Thus, newly synthesized human 51 integrin
and the chimeras are transported to both cell surface domains.
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Fig. 4.
Cell surface insertion of newly synthesized
human 51 integrin and of chimeric
constructs. Cell lines expressing either human
51 integrin (A and
B), FcR-5:3-28 (C), or
FcR-1:11-47 (D) were metabolically labeled
for 20 min with [35S]methionine/35S-cysteine
and chased for the indicated periods of time, followed by selective
biotinylation of either the apical (A) or basolateral
(B) cell surface. After extraction, immunoprecipitations
were performed using antibodies specific for 5
(A), 1 (B), or the Fc receptor
(C and D). Each immunoprecipitate was then split
into three samples of which one was directly analyzed by SDS-PAGE and
fluorography to see total newly synthesized and immunoprecipitated
protein (total IP), one was reprecipitated with
streptavidin-agarose before electrophoresis and fluorography to detect
newly synthesized protein at the cell surface (surface
35S), and the third was transferred to nitrocellulose after
electrophoresis to visualize the steady-state distribution with
streptavidin-HRP (surface total). Note that the same
fraction of human 1 was found to be apically inserted
when cells transfected with only the human 1 cDNA
were analyzed, indicating that direct apical transport of
1 also occurred when it was associated with endogenous
subunits. For each type of transfection, cell surface transport was
monitored in at least two independent experiments.
51 Integrin Is More Stable at the
Basolateral Cell Surface than at the Apical Cell
Surface--
Basolateral polarization of the transfected integrin
could be achieved by internalization from the apical membrane followed by either transcytosis to the basolateral cell surface or degradation. We found that the cytoplasmic domains of 5 and
1 were both able to mediate efficient internalization of
Fc receptor chimeras, a prerequisite for both mechanisms (data not
shown). Because we observed that large fractions of newly synthesized
51 integrin and chimeras were degraded
relatively quickly, we tested whether apical and basolateral
51 integrins have different half-lives. To do this, we biotinylated filter-grown cells either apically or
basolaterally and then incubated the cells for different periods of
time at 37 °C. The cells were then lysed, and the integrin was
immunoprecipitated with either anti-5 or
anti-1 antibodies. The immunoprecipitates were
fractionated by SDS-PAGE and transferred to nitrocellulose, and
biotinylated integrin was detected with streptavidin-HRP.
5 (A) or
anti-1 (B) antibodies. In both cases,
apically biotinylated integrin disappeared relatively quickly
(t1/2, ~1.5 h), whereas basolaterally
biotinylated integrin was very stable and became only slowly degraded
(t1/2 
16 h). Thus,
51 integrin is more stable at the
basolateral cell surface than at the apical cell surface. To test
whether the cytoplasmic domains of the 5 and
1 subunits are sufficient to mediate selective
stabilization in the basolateral membrane, we repeated the same
experiment with MDCK cells expressing either one of the two integrin/Fc
receptor chimeras. Fig. 6 shows that both
the 5 (A) and the 1
(B) chimera exhibited a similar behavior as the integrin;
apically biotinylated chimeras were more quickly degraded
(t1/2 < 60 min) than basolaterally labeled
chimeras (t1/2 180 min). Although
basolaterally expressed chimeras were also more stable than apical
ones, they were not as stable as basolateral
51 integrin. Because apical chimeras and
apical integrins exhibited comparable half-lives, this did not seem to
be due to quicker proteolysis of internalized chimeras but to more
efficient stabilization of the basolateral pool of dimeric integrin,
due to cooperation between the two cytosolic domains and/or
interactions occurring in the extracytoplasmic domains (e.g.
with the extracellular matrix). Nevertheless, the cytoplasmic domains
of 5 and 1 were independently sufficient to mediate stabilization of basolaterally expressed chimeras. Although
these experiments do not completely exclude that small fractions of
apical chimeras and apical 51 integrin
are transcytosed, the degradation of apically expressed integrin is too
fast for efficient transcytosis to occur and too complete for
transcytosis to be responsible for a significant amount of the finally
basolaterally expressed protein (i.e. the apically
biotinylated integrins do not exist anymore). Thus, basolateral
polarization of wild-type and chimeric integrins is achieved by
selective stabilization of the basolaterally expressed pool.
View larger version (36K):
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Fig. 5.
Stabilization of human
51 integrin on the basolateral cell
surface. Filter-grown and sodium butyrate-treated MDCK cells
co-expressing 51 were apically or
basolaterally biotinylated on ice. After incubation at 37 °C for the
indicated periods of time, the cells were extracted, and
immunoprecipitations were performed with either anti-5
(A) or anti-1 (B) antibodies.
After fractionation by SDS-PAGE and transfer to nitrocellulose of the
immunoprecipitates, the presence of biotinylated integrin was
visualized with streptavidin-HRP and chemiluminescence. The films were
then quantified by densitometric scanning, and the signals obtained
after no incubation at 37 °C were set to 100%.
View larger version (36K):
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Fig. 6.
Basolateral stabilization of chimeric Fc
receptors containing the cytoplasmic domain of 5 or
1. Cells expressing either
FcR-5:3-28 (A) or FcR-1:11-47
(B) were biotinylated and incubated at 37 °C as described
in the Fig. 5 legend. The chimeras were then immunoprecipitated with
the anti-Fc receptor antibody, and biotinylated immunoprecipitated
protein was again visualized after SDS-PAGE and blotting to
nitrocellulose with streptavidin-HRP and chemiluminescence. The films
were quantified as described in the Fig. 5 legend. Note that when the
cells were radioactively labeled before the experiment, and total
immunoprecipitated chimera was directly detected by SDS-PAGE and
fluorography, disappearance was paralleled by the appearance of a smear
of immunoprecipitated protein indicating the protein was degraded and
not sheared into the medium (data not shown).
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
51 integrin in MDCK
cells is mediated by distinct determinants in the cytoplasmic domains
of each subunit, similar to other basolateral membrane proteins. In
contrast to the basolateral sorting determinants described thus far,
the cytoplasmic domains of 5 and 1 make
use of an alternative sorting mechanism that does not involve direct
basolateral sorting in the Golgi complex but involves selective
stabilization at the basolateral cell surface after transport to both
cell surface domains.
51 integrin and
chimeras containing the cytoplasmic domain of either one of the two
subunits were transported to both cell surface domains. This was also
observed when MDCK cells were only transfected with 1
cDNA, indicating that the apical cell surface transport was not due
to the 5 subunit, which is normally not an abundant subunit in MDCK cells (17), but also occurred when the human
1 subunit was associated with endogenous subunits.
Because the existence of endogenous 1 integrins in the
apical membrane of MDCK cells has been reported previously, apical
transport of transfected human 1 is apparently not just caused by a species difference between human and dog 1
(18-20).
5 and the 1 cytoplasmic
domains were independently sufficient to mediate basolateral
polarization, basolateral stabilization of the wild-type integrin was
more pronounced than stabilization of the chimeras. One possible reason
for this is that two cytoplasmic domains may be better than one because
at least two interactions, perhaps occurring in a cooperative manner, can be used for stabilization. It could also be that the transmembrane and extracytoplasmic domains of the wild-type integrin participate in
basolateral stabilization (e.g. substrate binding).
Nevertheless, the chimeras clearly indicate that the cytoplasmic domain
sequences that were required for basolateral polarization of the
dimeric integrin were also sufficient to mediate basolateral
polarization of the truncated Fc receptor, which has been shown to be
expressed only basolaterally if its cytoplasmic domain contains sorting signals specific for the basolateral membrane (28, 21, 37, 38).
51 integrin possesses a determinant for
basolateral recycling, because it is not efficiently sorted to the
basolateral membrane during biosynthesis or after internalization from
the apical membrane. Nevertheless, the short half-lives of apically
expressed chimeras and integrin made it impossible to measure
differences in endocytic trafficking. Because basolaterally recycling
receptors like those for low density lipoprotein or transferrin exhibit
extensive endosomal localization in the steady state, the absence of
clear endosomal staining for integrins in our immunofluorescence
experiments suggests that stabilization occurs at the basolateral cell
surface. Although we were not able to detect significant Triton X-100
insolubility of basolaterally expressed constructs (data not shown), it
is likely that basolateral stabilization of the
51 integrin is mediated by interactions with cytoskeletal components. Integrins are connected to the
actin-based cytoskeleton by a linkage consisting of a large complex of
proteins that is also involved in the transmission of signals across
the plasma membrane. In fibroblasts, integrins, which normally become quickly degraded, have been shown to become stabilized on the plasma
membrane upon the addition of ligand (40). Importantly, the addition of
ligand stabilizes not only the interacting integrin but also other
integrins that cannot interact with the added ligand (40), indicating
that the stabilization of integrins at the plasma membrane can also be
mediated by their cytoplasmic domains in fibroblasts. Thus, in the
steady state, cytoplasmic domain-mediated stabilization in epithelial
cells occurs at the basolateral plasma membrane domain because it
contains established extracellular matrix contacts and therefore the
necessary organization of the submembrane cytoskeleton.
subunits by
interacting with a membrane-proximal conserved sequence (41); this
conserved sequence was neither sufficient for basolateral expression of
the dimeric integrin nor required for basolateral expression of the
5-Fc receptor chimera. In contrast, the last 11 amino
acids of the 5 subunit were found to be required for basolateral expression. Although this could be due at least in part to
reduced rates of endocytosis, the C-terminal 11 amino acids do not
contain a motif reminiscent of an endocytosis determinant, suggesting a
direct involvement of the C-terminal 11 amino acids in basolateral stabilization.
1 is known to interact with
many different proteins, and the binding sites cover essentially the entire cytoplasmic domain (15, 41, 42). Similar to 5,
the C-terminal amino acids of 1 were required for
basolateral expression. Although the last 9 amino acids contain a
NPXY motif, of which the tyrosine is apparently involved in
basolateral expression, previous studies indicate that the C-terminal
NPXY motif is not important for integrin internalization but
for the accumulation of 51 in focal
adhesions (43-46). Of all the proteins known to interact with the
cytoplasmic domain of 1, only one of the proposed binding sites for talin and one for focal adhesion kinase overlap with
the C-terminal domain required for basolateral expression (47).
Additionally, the tyrosine residue involved in basolateral expression
is also important for the interaction with the recently identified
protein ICAP-1 (48).
1 integrins participate in the
determination of the spatial orientation of the cells and can trigger a
reversal of polarity in response to matrix in contact with the apical
membrane (18, 19, 20, 49). The formation of tubulocysts upon collagen overlay or growth in a collagen gel was observed with various epithelial cells, indicating that this is a general phenomenon and
suggesting that apical expression of 1 integrins is not
restricted to MDCK cells (for a review, see Ref. 50). These
observations, together with our results, suggest a model in which
1 integrins are constitutively transported to both cell
surface domains and act as sensors that signal the presence of matrix
to the interior of the cells; hence, cells will be able to respond
relatively quickly to changes in their environment. Reorientation of
the cellular polarity is then a complex process requiring
reorganization of the cytoskeleton, remodeling of the cell surface, and
stabilization of integrins at the newly forming basolateral membrane.
ACKNOWLEDGEMENTS
51 integrin and Dr.
W. Hunziker for the pCB7 plasmid. We are grateful to Dr. A. Whitney for
critical reading of the manuscript and helpful suggestions.
FOOTNOTES
These authors contributed equally to this work.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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