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(Received for publication, October 10, 1995) From the
The
It is important to understand the molecular properties that
govern polarized expression of epithelial cell proteins, as this
polarity is an intrinsic part of the vectorial functioning of these
cells. The coordinated cellular functions mediated by endogenous and
exogenous ligands depend on the availability of appropriate receptors
at the particular surface domains to which the ligand has access. Our
laboratory is interested in elucidating the mechanisms and structural
regions within G-protein-coupled receptors responsible for polarized
expression of these regulatory molecules in renal epithelial cells. We
have demonstrated previously that the The present
studies characterize the localization and delivery of another seven
transmembrane-spanning G-protein-coupled receptor, the A
For polarity experiments, MDCKII and LLC-PKI cells
were seeded at a density of 1
For immunostaining of cells
grown in Transwell culture, cultures were grown for 4-6 days and
assessed for [
To determine the steady-state
localization of the A
Figure 1:
The wild-type and epitope-tagged
A
Figure 5:
Comparison of the A
Figure 2:
The wild-type A
Figure 6:
The A
Figure 3:
Introduction of A
Figure 4:
Growth in the presence of adenosine
receptor antagonists fosters MDCKII cell growth as a monolayer in
A
Because characterization of receptor polarization by biochemical
means requires a non-permeable monolayer, we examined whether the
proliferation of cells expressing adenosine receptors grown in the
absence of adenosine receptor antagonists might have contributed to the
variable (65-85%) apical localization of A
In an attempt to determine if some of the ``haze''
seen in the z scans of the TAG-A
Figure 7:
The A
Since
the steady-state localization of a receptor is influenced by both the
initial delivery to a particular surface and the eventual retention to
that specific surface, we examined the half-life of the
A
Figure 8:
The A
Endogenous compounds or drugs must first be recognized by the
appropriate receptor or, in the case of synthetic compounds, receptor
subtype to exert their desired effect. Multiple mechanisms likely
contribute to specificity in signal transduction by endogenous
agonists: the existence of numerous receptor subtypes that couple to
distinct signal transduction pathways; coupling of receptors to
distinct effector systems in various tissues; and receptor localization
to discrete microdomains on the cell surface, restricting the
G-proteins, effectors, and other molecules with which the receptor
interacts. The importance of receptor localization in signal
transduction is inferred by the number of pathophysiologic states that
result from mislocalized receptors (22; 23). For example, one form of
retinitis pigmentosa results from intracellular trapping of the
G-protein-coupled receptor for light, rhodopsin. It therefore is
essential to understand the mechanisms that govern the trafficking of
receptors and signal transducing proteins in order to both gain
insights into their role in receptor-mediated signal transduction
events under physiologic conditions and to probe for potential culprits
in pathophysiological states. We previously have demonstrated that
the Our studies are the first to characterize the
localization of the A The
finding that the A
Volume 271,
Number 2,
Issue of January 12, 1996 pp. 995-1002
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
A
ADENOSINE RECEPTORS ACHIEVE APICAL AND 
ADRENERGIC RECEPTORS ACHIEVE BASOLATERAL LOCALIZATION (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
adrenergic receptor
(
AR) previously was shown to be directly delivered to
and retained on the lateral subdomain of renal epithelial cells. The
present studies demonstrate that, in contrast, wild-type and
epitope-tagged canine A
adenosine receptors
(AAdoR) are apically enriched (65-83%) in Madin-Darby
canine kidney (MDCKII) and porcine renal epithelial (LLC-PKI) cells,
based on surface biotinylation strategies detecting
photoaffinity-labeled AAdoR. Confocal microscopy
corroborated the apical enrichment of the epitope-tagged
AAdoR. Metabolic labeling studies revealed that this
steady-state polarization is achieved by direct delivery to both the
apical (60-75%) and basolateral surface. Growth of
AAdoR-expressing cells as monolayers was achieved only
following Transwell culture in the presence of AAdoR
antagonists, which decreased cell growth, suggesting that
AAdoR elicit MDCKII cell proliferation. The preferential
apical but detectable basolateral localization of AAdoR
provides a molecular understanding of published reports that functional
responses can be elicited following apical as well as basolateral
delivery of adenosine agonists in varying renal preparations. These
findings also suggest that receptor chimeras derived from the
G
/G
-protein-coupled AR and
A
AdoR will be informative in revealing structural features
critical for basolateral versus apical targeting.
AR is
predominantly localized (>85%) to the basolateral surface of
Madin-Darby canine kidney (MDCKII) (
)cells, a polarized
model system for renal epithelia that accurately reflects
AR localization in vivo(1) .
Immunolocalization studies revealed that the
AR is
enriched in the lateral subdomain of the basolateral surface, and
metabolic labeling studies indicated that the
AR is
directly targeted to the basolateral domain(1) .
adenosine receptor (AAdoR), in polarized renal
epithelial cells. Interestingly, this receptor is enriched on the
apical surface in both MDCKII and porcine renal LLC-PKI cells. In the
kidney, adenosine, present in both blood and urine(2) , acts as
a paracrine regulator of renal blood pressure, renin secretion, and
renal excretion. The AAdoR, like the AR,
regulates cellular processes by interacting with G-proteins in the
G
/G family. Although some have explored the
localization of the adenosine receptors by means of functional studies (3, 4, 5, 6, 7) , there is
no evidence of the localization of these receptors using direct
biochemical and morphological strategies. In an attempt to study the
localization of the AAdoR in renal epithelial cells, we
have created permanent clonal cell lines of MDCKII and LLC-PKI cells
expressing either a wild-type or epitope-tagged AAdoR. The
apical versus basolateral distribution of the
AAdoR in polarized renal epithelial cells was determined
biochemically and morphologically, and the general mechanism by which
this predominantly apical localization is achieved was explored.
Materials
8-Cyclopentyl-1,3-di-(2,3[
H])propylxanthine
([H]DPCPX, 109 Ci/mmol),
ExpreS
S protein labeling mixture (1200
Ci/mmol), [
H]methoxyinulin (125.6 mCi/g), and
[-S]dATP (1389 Ci/mmol) were purchased from
DuPont NEN. The A
AdoR antagonist,
1,3-dipropyl-8-(4-sulfophenyl)xanthine or DPSPX, was prepared according
to Daly et al..(8) Biotin hydrazide and streptavidin-agarose
were purchased from Pierce. The protein A-purified 12CA5 monoclonal
antibody was purchased from the Berkley Antibody Co. The gp135 and EGF
receptor monoclonal antibodies were generously donated by Dr. Peter
Dempsey (Vanderbilt University). Cy-3-conjugated donkey anti-mouse IgG
was purchased from Jackson Immunochemicals.Cell Culture and Functional Confirmation of Intact
Monolayers
Madin-Darby canine kidney (MDCKII) cells were
obtained from Enrique Rodriquez-Boulan (Cornell University, New York,
NY) and maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum (Sigma), 100 units/ml
penicillin, and 100 µg/ml streptomycin (complete Dulbecco's
modified Eagle's medium) at 37 °C, 5% CO
. LLC-PKI
clone 4 cells were obtained from Anne E. Rubega-Male in the laboratory
of Carolyn Slayman (Yale University, New Haven, CT). The parental cells
and the permanent cell lines were maintained in minimum essential
medium supplemented with 10% heat-inactivated fetal bovine serum
(Sigma), 100 units/ml penicillin G, and 100 µg/ml streptomycin
( minimum essential complete medium) in a 5% CO, 37
°C incubator.
10
cells/24.5-mm
polycarbonate membrane filter (Transwell chambers, 0.4-µm pore
size, Costar, Cambridge, MA), and cultured for 5-8 days with
medium changes every day. Where stated, cells were grown in the
presence of 60 µM theophylline and 100 µM DPSPX. Prior to each experiment, the integrity of the monolayer
was assessed by adding [H]methoxyinulin to the
apical medium, and monitoring the leak of
[H]methoxyinulin from the apical compartment to
the basolateral compartment by sampling and counting the basolateral
medium in a 
-counter (Packard Tricarb) after a 1-h incubation at
37 °C. Chambers with leaks greater than 3%/h were discarded.Construction of the A
RDC7 (canine thyroid adenosine A and TAG-A Adenosine Receptor receptor cDNA) cloned into the pBluescript SK+ phagemid at
the EcoRI site was obtained from the laboratory of J. E.
Dumont and G. Vassart, Universite Libre de Bruxelles, Brussels. A
2.028-kilobase pair KpnI-XbaI fragment containing the
AAdoR full-length coding region, plus 53 bases of
5`-noncoding and 997 bases of 3`-noncoding sequence, was excised and
cloned into the KpnI-XbaI sites of the mammalian
expression vector pCMV4. To create the epitope-tagged
AAdoR, oligonucleotide-directed mutagenesis was utilized to
add 13 amino acids (YPYDVPDYALVPR) to the extracellular amino terminus
of the canine AAdoR immediately after the initiator
methionine(9) . The first 9 amino acids constitute a
hemagglutinin (HA) epitope (10) recognized by the commercially
available monoclonal antibody 12CA5 (Berkley Antibody Co.), while the
last 4 amino acids were added as a spacer arm. Positive mutations were
verified using dideoxy-DNA sequencing (Sequenase kit, U. S. Biochemical
Corp.) utilizing T7 DNA polymerase with
[-S]dATP. Once verified, the insert
encoding the epitope tag was subcloned into the pCMV4-A
AdoR
plasmid(11) , and reverified with sequencing. All the resulting
pCMV4- AAdoR and pCMV4-TAG-AAdoR constructs
were transiently transfected into COSM6 cells, and membranes from these
transfectants were assayed for binding of the AAdoR
antagonist, [H]DPCPX (see below). Binding of
[H]DPCPX to the epitope-tagged AAdoR
was indistinguishable from binding to the wild-type AAdoR.Development of Permanent Transformants of LLC-PKI and
MDCKII Cells
The pCMV4-AAdoR (20 µg) or
pCMV4-TAG-AAdoR adenosine receptor (20 µg) was
co-transfected with 2 µg of pRSVneo into 1 10 MDCKII cells using a modification of a standard
Ca(PO
) precipitation method(12) ,
except that 2 days following transfection, 500 µg of active G418/ml
was added either directly to the original plate of transfected cells or
to plates containing different dilutions of the transfected cells.
G418-resistant colonies were screened for AAdoR expression
by assaying the binding of the AAdoR antagonist
[H]DPCPX(13) . Specific AAdoR
binding was defined as that binding not competed for by 1 mM theophylline. Untransfected MDCKII cells displayed no detectable
specific binding of [H]DPCPX.A
MDCKII and LLC-PKI cell membrane particulate preparations
were prepared essentially as described (11) with the following
modifications. The lysis buffer was 15 mM Tris-HCl, 5 mM EGTA, 5 mM EDTA, pH 8.0 containing 0.5 µg/ml
leupeptin, 0.7 µg/ml pepstatin, 100 µg/ml soybean trypsin
inhibitor, and 1 mM phenylmethylsulfonyl fluoride. The final
pellet was resuspended in 900 µl of membrane binding buffer (50
mM Tris-HCl, 10 mM MgCl Adenosine Receptor Binding
Assay
6HO, pH 7.4). The assay of
[H]DPCPX binding was performed in 12
75-mm polypropylene tubes containing 3 nM [H]DPCPX (diluted in 1% ethanol in 50 mM Tris-HCl, 10 mM MgCl, pH 7.4) in the absence
(total binding) or presence (nonspecific binding) of 1 mM theophylline. Incubations were for 45 min at 25 °C. Reactions
were terminated by the addition of 3.5-ml ice-cold membrane binding
buffer and filtration through Whatman GF/B glass microfiber filters,
which were pretreated with 0.3% polyethylenimine in membrane binding
buffer at 4 °C for 30 min. Upon termination of the binding assay,
the filters were dried at 60 °C for 20 min and were counted in 5 ml
of NEN-963 liquid scintillation fluid.Steady-state Localization of the A
The method for quantitating the apical versus basolateral distribution of the wild-type and epitope-tagged
A- and
TAG-A Adenosine Receptors: Biotin Surface Labeling
StrategyAdoR in polarized MDCKII cells was biotinylation of the
apical versus basolateral surface of cells grown in Transwell
culture, photoaffinity labeling of the functional AAdoR in
harvested membranes, detergent extraction, and isolation of
biotinylated receptors via streptavidin-agarose chromatography. The
studies were performed essentially as described previously(1) .
Briefly, MDCKII or LLC-PKI cells grown in Transwell culture were washed
three times for 5 min in PBS containing 0.5 mM CaCl and 1 mM MgCl (PBS/CM) and 1 mM theophylline. Following sequential treatment with sodium periodate
and biotin-LC-hydrazide, cells were harvested into PBS containing 1
mM EDTA and 0.1 mM phenylmethylsulfonyl fluoride and
membranes were prepared as described above. A small sample of the
membrane preparation was removed and assayed for
[H]DPCPX binding. To photoaffinity label the
AAdoR in these membranes, aliquots containing
25,000-50,000 cpm of specific [H]DPCPX
binding were incubated with 2 nM AAdoR antagonist, I-azido-BW-A844U(13) , for 45 min in membrane
binding buffer at room temperature in the dark. Nonspecific
photoaffinity labeling was assessed in a separate incubation by
inclusion of 1 mM theophylline, added 15 min before
I-azido-BW-A844U. Photoincorporation was initiated by
irradiation of the membrane preparations with ultraviolet light at 254
nm (Rayonet miniphotochemical reactor) for 3 min. The
photoaffinity-labeled membranes, derived from Transwell cultures that
had been biotinylated on either the apical or the basolateral side,
were collected by centrifugation, resuspended in RIPA buffer (150
mM NaCl, 50 mM Tris, pH 8.0, 5 mM EDTA, 1%
Nonidet P-40, 0.5% deoxycholate, 0.1% SDS) and prepared for
streptavidin-agarose chromatography as described
previously(1) . The eluates were then subjected to SDS-PAGE and
autoradiography, and the area of the gel corresponding to the
A
AdoR signal was excised and counted on a Beckman 5000
-counter to quantify the amount of
I-azido-BW-A844U-labeled A
AdoR on the apical versus basolateral surface.Metabolic Labeling/Biotinylation Strategy for Determining
Surface Delivery of the TAG-A
The
amount of newly synthesized A Adenosine ReceptorAdoR delivered to the apical versus basolateral surface was quantified by biotinylating one
surface or the other of metabolically labeled MDCKII cells in Transwell
culture and subsequently isolating radiolabeled receptor on the
biotinylated surface through sequential immunoprecipitation and
streptavidin-agarose chromatography. These procedures were performed
essentially as described(1) , except that membranes were
prepared in adenosine receptor membrane binding buffer, as described
above.Immunolocalization of A
Primary and secondary immunostaining protocols were
optimized using cells plated on coverslips before evaluating cells
polarized by culture in Transwells. For immunostaining of MDCKII cells
on coverslips, cells were plated at confluence on glass coverslips and
cultured for 2-3 days prior to staining. Cells were fixed and
stained with a 1:50 dilution of 12CA5 primary antibody as
described(1) . Upon rinsing the cells after the incubation of
the primary antibody, a 1:200 dilution of the secondary Cy3-conjugated
donkey anti-mouse IgG was added to the cells in PBS containing 2%
bovine serum albumin and incubated for 1 h at room temperature in the
dark. The cells then were mounted on glass slides with Aqua-Poly/Mount
(Polysciences Inc., Warrington, PA). Adenosine
ReceptorH]methoxyinulin leak; wells with
leaks greater than 3% were discarded. The cells on the polycarbonate
filter were fixed and stained via the same protocol as those grown on
the coverslips, except that the filter was excised from the Transwell
support prior to the first antibody incubation. Samples were visualized
by confocal microscopy on a Zeiss Axiovert 135 Micro Systems LSM
(Germany). The samples were first visualized in the xy plane,
and then in the xz plane; the images were downloaded onto a
Silicon Graphics Iris Indigo workstation for analysis using Showcase
software.
The Wild-type and the Epitope-tagged A
To examine the trafficking of the A Adenosine Receptor Are Enriched on the Apical Membrane Domain of
MDCKII Cells-
and TAG-AAdoR in canine renal epithelial cells, clones of
MDCKII cells permanently expressing wild-type and epitope-tagged
receptors were made. The cell lines characterized in most detail in
this study expressed 17.2 pmol of wild-type AAdoR/mg
protein (clonal line AAdoR#10) and 36.9 pmol of
TAG-AAdoR/mg of total membrane protein (clonal line
TAG-AAdoR#17). Two other independent untagged cell lines of
lower densities also were evaluated; the findings with all cell lines
evaluated were comparable.AdoR, we employed surface
biotinylation and subsequent photoaffinity labeling. The
AAdoR extracted from membrane preparations and eluted from
streptavidin-agarose was evaluated using SDS-PAGE. The
AAdoR migrated on SDS-PAGE with an apparent mass of
43-45 kDa; this photolabeled species was not detected when the
incubation with I-azido-BW-A844U was carried out in the
presence of the A
AdoR antagonist, theophylline. The
AAdoR was enriched on the apical surface in cell lines
expressing both the wild-type (65-68%) and the
TAG-AAdoR (75-83%) structures (Fig. 1A). These data indicate that the presence of the
HA-epitope did not significantly alter the apical enrichment of the
AAdoR at steady state.
AdoR are enriched on the apical membrane of MDCKII cells
at steady state. A, the MDCKII clonal cell lines expressing
either wild-type AAdoR (AAdoR#10) or
epitope-tagged AAdoR (TAG-AAdoR#17) were grown
in Transwell culture and treated with biotin hydrazide to selectively
label either the apical or the basolateral membrane surface. In the
studies shown here, three Transwell cultures were pooled for each
surface determination. The AAdoR was identified with
photoaffinity labeling using I-azido-BW-A844U as
described under ``Experimental Procedures''; non-receptor
labeling was assessed in the presence of the adenosine receptor
antagonist, theophylline. The autoradiogram was exposed for 7 days to
Kodak X-Omat film between two Quanta III screens at -70 °C.
Gel slices corresponding to the position of the A
AdoR
signal on autoradiograms were excised and counted in a -counter.
The findings shown are representative of two separate experiments. B, confocal microscopy images of the MDCKII clonal cell line
A
AdoR#10 or TAG-AAdoR#17 cultured on Transwell
filters, fixed, and incubated, in the presence of 0.1% Triton X-100,
with a 1/50 dilution of 12CA5 monoclonal antibody directed against the
hemagglutinin epitope as described under ``Experimental
Procedures.'' The x-y scan shows the cells oriented with
the apical surface facing up toward the eye-piece. The yellow line represents the section of cells through which the z scan
was made. The z scan at the top of the photograph represents a
longitudinal cut through the cells, where the horizontal signal
represents the apical (top) surface of the cells. (See Fig. 5, B and D, for comparison with pattern of endogenous
protein expression characteristic of apical versus basolateral
localization.) Experiments in both A and B were
performed on cells grown in AAdoR antagonists (100
µM DPSPX and 60 µM theophylline) to permit
cell growth as monolayers (see
``Results'').
AdoR
localization with the basolateral AR and endogenous
protein markers of apical and lateral surfaces. The confocal image of
the TAG-A
AdoR#17 in MDCKII cells grown on Transwell filters
in the presence of AAdoR antagonists (A) reveals
apical staining over the entire cell surface in the x-y scan
and the horizontal line of red in the z scan. This is
indistinguishable from the confocal image of the endogenous protein,
gp135, which serves as a marker for the apical surface in MDCKII cells (B). The confocal image of the TAG-AR (also
tagged (1) with the same HA epitope used to identify the
A
AdoR) in MDCKII cells grown on Transwell filters (C) shows a lateral staining pattern indistinguishable from
the image of the known, endogenous EGF receptor, which serves as a
marker for the basolateral surface in MDCKII cells (D).
A Similar Apical Localization Is Detected for the A
Because there are
examples where polarization of a particular cell surface protein
differs in different cultured renal epithelial cell backgrounds, ( Adenosine Receptor in LLC-PKI Cells)we examined the localization of the wild-type
AAdoR after transfection into porcine renal epithelial
LLC-PKI cells. As seen with MDCKII cells, the AAdoR was
enriched on the apical surface (80-85%) of LLC-PKI cells (Fig. 2). In the LLC-PKI cells, the AAdoR migrated
consistently between 38 and 42 kDa. We presume that the difference in
migration of specific AAdoR photolabeling in MDCKII cells
(estimated at 43-45 kDa) versus LLC-PKI cells is due to
different glycosylation in the two cellular backgrounds, since
treatment of the AAdoR in LLC-PKI cells with
endoglycosidase F reduces the apparent molecular mass of the
photolabeled AAdoR to 36 kDa (data not shown), a value
consistent with the mass of the amino acids encoded by the
A
AdoR cDNA and that previously
reported(14, 15) . The finding that the
AAdoR is similarly enriched on the apical surface following
expression in MDCKII and LLC-PKI cells is consistent with the
interpretation that the polarization observed is a consequence of the
AAdoR structure and its interaction with shared renal
epithelial trafficking mechanisms. The apical enrichment does not seem
to be a function of receptor density as there seems to be little
difference between clones 50, 17, and 10, expressing 57, 37, and 17
pmol/mg protein, respectively. Thus, apical enrichment does not seem to
be driven by increased receptor density. Because MDCKII cells
frequently are used for the study of cell surface protein polarization
and because we have studied a basolaterally targeted protein, the
AR, extensively in these cells, the further
characterization of A
AdoR localization reported here was
evaluated in MDCKII cells.
AdoR is
enriched on the apical membrane of LLC-PKI cells at steady-state. An
LLC-PKI clonal cell line expressing the AAdoR
(AAdoR#50) was grown in Transwell culture and evaluated for
polarization as in Fig. 1. The calculated apical polarization
was 80-85%. The data shown are from one experiment performed
three times with indistinguishable
outcomes.
Immunolocalization of A
Because introduction of the HA-epitope did not
appear to alter the localization of the A Adenosine Receptor in
MDCKII CellsAdoR in MDCKII
cells based on the findings from surface biotinylation studies, we
performed immunocytochemical studies to independently determine the
localization of the AAdoR in MDCKII cells using the
introduced epitope as the receptor marker. For these studies, clones of
MDCKII cells permanently expressing the hemagglutinin epitope-tagged
version of the AAdoR (TAG-AAdoR#17 and
TAG-AAdoR#1) were evaluated. (The receptor density of clone
1 was about half that of clone 17 based on binding assays.)
Immunocytochemical studies revealed a predominantly apical localization
consistent with the biochemical data obtained using
photoaffinity-labeled receptor (Fig. 1B). The x-y scan revealed staining over the top surface of the cells, and the z scan reinforced this interpretation by depicting a distinct
horizontal band on the top of the cells. Lateral staining, although
detectable, was significantly less than apical staining, even though
staining of the lateral subdomain reflects the signal emerging from two
cell surfaces juxtaposed to each other. No staining was detected in
MDCKII cells expressing wild-type AAdoR, indicating that
the immunofluorescent signal detected is indeed due to the
epitope-tagged amino terminus of the TAG-AAdoR. The
specificity of identification of the HA epitope with the 12CA5 antibody
is further revealed by comparison of localization of the
TAG-AAdoR with the basolaterally targeted
TAG-AR structures in subsequent figures (cf.Fig. 5and Fig. 6).
AdoR is expressed
primarily on the surface of MDCKII cells as revealed by
immunolocalization in the absence and presence of Triton X-100. The
confocal image of the TAG-AAdoR#17 in MDCKII cells grown on
Transwell filters in the presence of AAdoR antagonists and
stained in the presence of Triton X-100 shows apical staining, as
evidenced by the strong red signal of the Cy3 chromophore present in
both the x-y scan and the z scan (A). There
is some red ``haze'' present below the apical signal
in the z scan. The confocal image of the
TAG-AAdoR#17 stained in the absence of Triton X-100 shows a
weaker apical signal, with an attenuated red ``haze'' (B). The confocal image of the epitope-tagged
AR subtype, known to be expressed on the surface as
well as in the interior of MDCKII cells (see Footnote 3), shows a
lateral staining pattern with detectable intracellular staining in the
presence of Triton X-100 (C) and the absence of detectable
intracellular signal in the absence of Triton X-100 (D).
Growth of A
Upon growing the AAdoR-expressing MDCKII Cells as a
Polarized Monolayer Requires the Presence of Adenosine Receptor
AntagonistsAdoR-expressing clones
in culture, we observed that the growth pattern of the adenosine
receptor-transfected MDCKII cells looked uncharacteristic of other
MDCKII cell clones, in particular, growth in patches and in
multilayers. We noted that when cells were plated and grown in the
presence of two adenosine receptor antagonists, DPSPX (a hydrophilic,
highly selective A receptor antagonist) and theophylline (a
non-subtype selective A/A receptor antagonist),
the cells were prevented from forming multilayers in culture. To
evaluate more directly the effect of AAdoR antagonists on
MDCKII cell growth, subconfluent AAdoR-transfected and
untransfected MDCKII cells were grown in the absence and presence of 60
µM theophylline, a nonspecific adenosine receptor
antagonist, for 2 days, at which point the cells were harvested and
counted. Theophylline significantly inhibited the growth rate of the
AAdoR-transfected cells, but not untransfected MDCKII cells (Fig. 3A). Furthermore, cell number achieved over time
in the AAdoR-transfected cells was greater compared to the
untransfected cells when both were plated at the same density and grown
in the absence of theophylline (2.77 ± 0.24
AAdoR-transfected cells/dish versus 1.92 ±
0.29 untransfected cells/dish; p = 0.058, where n = 5 for each group). These findings suggest that
AAdoR, either via theophylline-sensitive,
agonist-independent receptor activation of MDCKII cells or in response
to ambient adenosine, a proliferative agent in some
cells(16, 17) , evokes cell growth. To assess whether
or not stimulation of growth of MDCKII cells is a characteristic
response to other G/G-coupled receptors in
these cells, we examined the effect of the AR agonist,
UK-14304, on cells in which the AR had been
introduced by transfection(1) ; the
AR
agonist had no significant effect on cell growth (Fig. 3B). These findings indicate that introduction of
the A
AdoR, but not of the AR, changes the
growth properties of the MDCKII cells, suggesting (albeit not to the
exclusion of other explanations) that the A
AdoR and the
AR are localized in distinct compartments containing
different G
/G-coupled effector systems.
Although the precise mechanisms by which adenosine receptors mediate
proliferation of MDCKII cells was not explored in these studies, our
studies did establish that growth of Transwell cultures in the presence
of adenosine receptor antagonists prevented multilayer cellular
accumulation. (This interpretation is corroborated by the
immunocytochemical data in Fig. 4B, described below.)
AdoR, but not
the AR, into MDCKII cells causes proliferation of
MDCKII cells. A, introduction of A
AdoR into MDCKII
cells caused an increase in cell growth and multilayer morphology (see Fig. 4) that was suppressed by the inclusion of 60 µM theophylline, a non-subtype selective adenosine receptor
antagonist, in the culture medium. B, the effect of an
exogenous AR agonist on MDCKII cell growth was
examined by looking at the change in cell number for
AR-transfected MDCKII cells grown in the absence or
presence 10 µM of the
AR agonist,
UK-14,304. For both A and B, cells were plated at 5
10
cells/dish (subconfluent) in 60-mm dishes, at
which point drug or buffer was added. Two days later, the cells were
harvested and counted using a Coulter
counter.
AdoR-expressing cell lines. Cells were grown either in the
absence or presence of the adenosine receptor antagonists, theophylline
(60 µM) and DPSPX (100 µM). A,
biochemical characterization of AAdoR localization (clonal
cell line TAAdoR#17) was determined as in A. The
data obtained after exposure of the autoradiogram for 7 days to Kodak
X-Omat film between two Quanta III screens at -70 °C; the
findings are from one experiment performed twice with indistinguishable
results. B, confocal microscopy images of the MDCKII clonal
cell line TA1AdoR#17 cultured on Transwell filters, fixed, and stained
as described under ``Experimental Procedures.'' Both the x-y and z scan reveal that cells are piled on top of
each other in the absence of the antagonists, whereas growth in the
presence of antagonists results in a cell monolayer detected in both
the x-y and z scans.
AdoR
reported in preliminary studies from our laboratory(18) . We
thus compared the relative apical localization of AAdoR in
Transwell cultures grown in the absence and presence of the combined
adenosine receptor antagonists, theophylline (60 µM) and
DPSPX (100 µM) using both biotinylation (Fig. 4A) and immunocytochemical (Fig. 4B) strategies. It was apparent from the
immunolocalization experiments that in most clonal cell lines, the
cells grew in a multilayer in the absence of adenosine receptor
antagonists (Fig. 4B). In some cases, as in Fig. 4A, the percent apical polarization estimated from
streptavidin-agarose recovery of biotinylated and photolabeled
AAdoR increased notably when growth in Transwell culture
occurred in the presence of antagonists, and in this example was 56% in
the absence and 75% in the presence of antagonists. Since cell growth
in multiple layers would confound analysis of receptor localization by
biochemical strategies, these two receptor antagonists were included in
all subsequent experiments of all cell lines expressing the
AAdoR.Immunolocalization of Epitope-tagged A
To confirm the apical localization of the epitope-tagged
AAdoR,
AR, and
AR in MDCKII
Cells
AdoR, we compared the localization of the
AAdoR with endogenous MDCKII markers for the apical and
basolateral surfaces, and with the AR, already
characterized in terms of its lateral targeting following expression in
MDCKII cells(1) . The apical staining observed for
TAG-A
AdoR#17 (Fig. 5A) was consistent with
the immunostaining pattern of an endogenous MDCKII cell protein,
gp135(19) , that serves as a marker for apical staining in
MDCKII cells (Fig. 5B). In contrast, the epitope-tagged
AR revealed a predominantly lateral staining profile (Fig. 5C) and was consistent with the immunostaining
pattern of the endogenous EGF receptor (Fig. 5D), which
is localized laterally in MDCKII cells(20) . The appearance of
areas with no cells in the x-y scan of the
A
AdoR-stained cells is attributed to the various heights of
the cells, such that the apical signal comes into focus at different
levels.AdoR could be
attributable to a genuine signal arising from one or more intracellular
compartments, the cells were stained in the presence and absence of the
detergent Triton X-100 as a permeabilizing agent. The relative
distribution of surface to intracellular fluorescence was not
significantly altered when the 12CA5 primary antibody directed against
the HA epitope was incubated with fixed,
TAG-AAdoR-expressing cells in the presence (Fig. 6A) or absence (Fig. 6B) of
Triton X-100. The apical staining is somewhat fainter when the
procedure is performed in the absence of Triton X-100, perhaps because
the detergent facilitates access of the 12CA5 antibody to the
amino-terminal epitope on the AAdoR even on the
extracellular surface. To test our ability to detect the HA epitope
when expressed intracellularly, we also examined MDCKII cells
expressing the AR receptor subtype, which previously
has been demonstrated to be localized on the surface as well as in an
intracellular compartment in MDCKII cells, (
)as well as COS
and human embryonic kidney cells(21) . The ability to readily
identify the intracellular compartment containing the
TAG-AR structure required incubation of the fixed
Transwells with primary antibody in the presence of Triton X-100 (Fig. 6C), as the detection of this intracellular
compartment was not clearly visible in the absence of Triton X-100 (Fig. 6D). These control studies indicate that if a
substantial fraction of the TAG-A
AdoR were enriched in an
intracellular compartment, incubation with the 12CA5 primary antibody
in the presence of Triton X-100 should have detected it.The A
To investigate the mechanism by which apical enrichment is
achieved, the delivery of the A Adenosine Receptor Is Delivered
Directly to the Apical and Basolateral Surfaces of MDCKII
CellsAdoR to the apical versus the basolateral surface was determined using metabolic
labeling in combination with surface biotinylation (Fig. 7).
Newly synthesized, metabolically labeled AAdoR was first
detectable after 30 min of pulse labeling. At this time, metabolically
labeled receptor was detectable on both the apical and basolateral
surfaces, but appeared to be preferentially delivered to the apical
surface (63% apical, 37% basolateral). Over all the time points
examined (30, 45, 60, 90, and 120 min), the apical to basolateral ratio
remained relatively constant, consistent with the interpretation that
the amount of S-labeled A
AdoR detected at each
surface is due to direct delivery to these surfaces (Fig. 7A). We are confident that the S-labeled protein, migrating slightly above the 43-kDa
molecular size marker, is the A
AdoR, since it comigrates
with the photoaffinity-labeled AAdoR and because no
radioactive band is visible at this molecular weight when MDCKII cells
expressing wild-type AAdoR (AAdoR#10) are
metabolically labeled for 60 min and processed exactly as MDCKII cells
expressing TAG-AAdoR (Fig. 7B).
AdoR is preferentially
delivered to the apical membrane domain of MDCKII cells, consistent
with its steady-state enrichment on that surface. A, MDCKII
cells expressing epitope-tagged AAdoR
(TAG-AAdoR#17) grown in Transwell culture in the presence
of AAdoR antagonists were metabolically labeled with 1
µCi/µl
[S]Met/[
S]Cys protein
labeling mix (150 µl) for the indicated times and then harvested
and processed using sequential immunoprecipitation and
streptavidin-agarose chromotography as described under
``Experimental Procedures.'' The autoradiogram shown is for
an SDS-PAGE gel exposed for 7 days to Kodak X-Omat film between two
Quanta III screens at -70 °C. Gel slices corresponding to the
position of the A
AdoR signal on autoradiograms were excised
and counted in a 10 ml of NEN-963 scintillation fluid in a
-scintillation counter. Localization on the apical membrane was
not significantly different over the range of time points examined; the
findings from multiple individual experiments evaluating varying time
points (in percent apical enrichment) are as follows: 30 min (n = 2) mean = 61, range = 59-63; 45 min (n = 3) mean = 67.3, range = 62-76;
60 min (n = 6) mean = 62.3, range =
56-66; 90 min (n = 2) mean = 57.5 range
= 55-60; 120 min (n = 1) is 62% apical
enrichment. B, cells expressing wild-type AAdoR
(clone 10) were metabolically labeled for 60 min and then treated as in panel A. The autoradiogram for metabolically labeled wild-type
receptor was exposed for 7 days with Kodak X-Omat film at -70
°C. Migration of the photoaffinity-labeled AAdoR was
included in the experiment to demonstrate the migration of the
AAdoR for comparison with the metabolically labeled,
epitope-tagged protein isolated by sequential protein A and
streptavidin-agarose chromatography. In all gels where
TAG-AAdoR were analyzed, there were visible
[S]Met/Cys-labeled bands migrating at about 55,
67, 80, and 94 kDa; these presumably correspond to receptor aggregates,
since they also were seen when the A
AdoR was identified by
photoaffinity labeling. No [S]Met/Cys signal was
detected at any molecular weight when cells expressing the wild-type
receptor were analyzed.
AdoR (TAAdoR#17) on both the apical and
basolateral surface. The kinetics of the removal of the receptor from
the apical plasma membrane were not significantly different from those
of the basolateral membrane (Fig. 8). The calculated half-life
on the apical surface was about 9-13 h and on the basolateral
surface about 11-12 h. Thus, the apical enrichment observed at
steady state cannot be attributed to differential retention of the
AAdoR on the apical compared to the basolateral surface,
but appears to result from preferential delivery to the apical surface.
AdoR has a comparable
surface half-life on both the apical and basolateral surface of MDCKII
cells. MDCKII cells expressing TAG-AAdoR#17 and grown in
Transwell culture in the presence of AAdoR antagonists were
metabolically labeled as in Fig. 7A with 1
µCi/µl
[S]Met/[
S]Cys for a
60-min ``pulse'' and rapidly rinsed in PBS before the
``chase'' phase was initiated by addition of complete
Dulbecco's modified Eagle's medium supplemented with
additional 1 mM methionine, 1 mM cysteine, and
A
AdoR antagonists. For monitoring of half-life, the time
= 0 corresponds to a 6-h chase period, since after this time
point no increase in metabolic labeling signal occurs at the surface.
AAdoR present on the apical surface of labeled cells at
each time point was calculated by surface biotinylation, followed by
sequential immunoisolation and streptavidin-agarose chromatography of
cell extracts, as described under ``Experimental
Procedures.'' Each data point represents the streptavidin-agarose
eluate derived from three Transwells per time point per surface
evaluated. Gel slices corresponding to the position of the
AAdoR signal on autoradiograms were excised and counted in
10 ml of NEN-963 scintillation fluid and counted in a Packard-Tricarb
liquid scintillation counter on the -channel. The data also were
analyzed by scanning the gel on a phosphorimager, where the results
were not significantly different from those obtained from cutting and
counting the gel slices. The data (in h) are as follows: the calculated
half-life ranged from 9-13 h (n = 2) on the
apical surface and 11-12 h (n = 3) on the
basolateral surface.
AR is localized basolaterally in renal epithelial
cells(1) . One strategy that could reveal the structural
regions of the
AR, and perhaps all G-protein-coupled
receptors, necessary for conferring basolateral localization is to make
receptor chimeras with other seven-transmembrane-spanning,
G-protein-coupled receptors that achieve opposite localization in
polarized cells. We chose the A
AdoR as a potential
candidate since adenosine is available in sufficient concentrations (2) to activate adenosine receptors on the apical surface of
renal cells in vivo due to the existence of caveolar
5`-nucleotidase in that domain(24) . Also, in in vivo studies, Franco et al. (25) found that an
AAdoR agonist, when administered lumenally, participated in
the tubuloglomerular feedback mechanism. However, it should be noted
that responses to basolaterally introduced adenosine receptor agonists
also have been demonstrated in renal epithelial cells in
vitro, e.g. AAdoR-accelerated
phosphatidylinositol turnover and inhibition of forskolin-stimulated
cAMP production following basolateral but not apical administration of
adenosine(3) . Similarly, basolaterally localized
AAdoR have been suggested by addition of A analogues to the basolateral compartment (7) or to the
basolateral surface of perfused inner medullary collecting duct
tubules(5) .AdoR based on its structure, using
biochemical and morphological detection strategies, rather than relying
on receptor-mediated functions to infer receptor localization. Our
studies demonstrate that the AAdoR is preferentially
localized to the apical surface of MDCKII and LLC-PKI cells in multiple
independent clonal cell lines. The apical enrichment of the
AAdoR at steady state is mirrored by the preferential
delivery of AAdoR to the apical versus basolateral
surface. Since the AAdoR has a similar half-life on the
apical and the basolateral surfaces after its delivery, our findings
are consistent with the interpretation that the AAdoR
localization is achieved by direct delivery to the apical versus basolateral surfaces. It may be that both the apically and
basolaterally localized receptor populations elicit activities
important for polarized function, albeit by coupling to different
effector mechanisms. Our own observations on proliferation of MDCKII
cells expressing the AAdoR (Fig. 3) are consistent
with this interpretation; both the AAdoR and the
AR elicit responses via
G
/G-coupled GTP-binding proteins, but only
introduction of the AAdoR led to an increase in cell number
in polarized MDCKII cells. Whether or not the effects of theophylline
to slow cell doubling are due to agonist-independent effects of the
receptor or result from endogenously produced adenosine acting at the
heterologous AAdoR has not been determined. Nevertheless,
inclusion of AAdoR antagonists was necessary to obtain a
monolayer of cells required for performing polarization studies.AdoR is delivered to both the apical and
basolateral surfaces, but preferentially to the apical surface,
distinguishes this receptor from other G-protein-coupled receptors
characterized to date. The AR is delivered
exclusively to the basolateral surface(1) ; in contrast, the
AR achieves steady-state basolateral localization in
MDCKII cells via random delivery and selective retention on the
basolateral surface.
In studies not characterized as
rigorously as those presented here, we observed that the M2 and M3
muscarinic receptors are localized to both the apical and basolateral
surfaces of MDCKII cells as revealed by confocal microscopy, but
preferentially on the basolateral surface. ()Thus, of all
the G-protein-coupled receptors studied to date, the AAdoR
is the only structure preferentially delivered to the apical surface
that appears to be retained at that surface without redistribution to
the lateral subdomain. Chimeric structures between AAdoR
and the AR might therefore permit identification of
structural regions in G-protein-coupled receptors that confer
basolateral versus apical targeting.
)AR, adrenergic receptor; AAdoR, A adenosine
receptor; HA, hemagglutinin; WT, wild-type; PBS, phosphate-buffered
saline; PAGE, polyacrylamide gel electrophoresis;
[H]DPCPX,
8-cyclopentyl-1,3-di-(2,3[H])propylxanthine;
DPSPX, 1,3-dipropyl-8-(4-sulfophenyl)xanthine.)))
We thank Carol Ann Bonner for technical assistance in
the maintenance of all cell lines and development of several of the
AAdoR-expressing cell lines evaluated. We are grateful to
Dr. Magdalena Wozniak for providing the
TAG-AR-expressing MDCKII clonal cell line used in the
immunolocalization comparison with TAG-
AR in Fig. 6. We thank Dr. Peter Dempsey for the gift of the gp135 and
EGF receptor monoclonal antibodies. We are also grateful to Dr. Tom
Jetton for constant and patient assistance with the use of the confocal
microscope and discussions concerning immunocytochemical techniques. J.
R. K. acknowledges Dr. Michael Welch (University of Iowa) for his
suggestion to test the A
AdoR for possible apical
localization in MDCKII cells. We are very appreciative to the members
of the Limbird laboratory for helpful discussions and assistance during
these experiments. Finally, we thank Dr. Enrique Rodriguez-Boulan for
providing our laboratory with the parental MDCKII cells.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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