(Received for publication, October 10, 1995)
From the
The 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
(A
AdoR) 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 A
AdoR. Confocal microscopy
corroborated the apical enrichment of the epitope-tagged
A
AdoR. 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
A
AdoR-expressing cells as monolayers was achieved only
following Transwell culture in the presence of A
AdoR
antagonists, which decreased cell growth, suggesting that
A
AdoR elicit MDCKII cell proliferation. The preferential
apical but detectable basolateral localization of A
AdoR
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.
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 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) .
The present
studies characterize the localization and delivery of another seven
transmembrane-spanning G-protein-coupled receptor, the A adenosine receptor (A
AdoR), 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 A
AdoR, 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 A
AdoR 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 A
AdoR. The
apical versus basolateral distribution of the
A
AdoR in polarized renal epithelial cells was determined
biochemically and morphologically, and the general mechanism by which
this predominantly apical localization is achieved was explored.
For polarity experiments, MDCKII and LLC-PKI cells
were seeded at a density of 1 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.
For immunostaining of cells
grown in Transwell culture, cultures were grown for 4-6 days and
assessed for [H]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.
To determine the steady-state
localization of the AAdoR, we employed surface
biotinylation and subsequent photoaffinity labeling. The
A
AdoR extracted from membrane preparations and eluted from
streptavidin-agarose was evaluated using SDS-PAGE. The
A
AdoR 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
A
AdoR was enriched on the apical surface in cell lines
expressing both the wild-type (65-68%) and the
TAG-A
AdoR (75-83%) structures (Fig. 1A). These data indicate that the presence of the
HA-epitope did not significantly alter the apical enrichment of the
A
AdoR at steady state.
Figure 1:
The wild-type and epitope-tagged
AAdoR are enriched on the apical membrane of MDCKII cells
at steady state. A, the MDCKII clonal cell lines expressing
either wild-type A
AdoR (A
AdoR#10) or
epitope-tagged A
AdoR (TAG-A
AdoR#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 A
AdoR 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-A
AdoR#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 A
AdoR antagonists (100
µM DPSPX and 60 µM theophylline) to permit
cell growth as monolayers (see
``Results'').
Figure 5:
Comparison of the AAdoR
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 A
AdoR 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).
Figure 2:
The wild-type AAdoR is
enriched on the apical membrane of LLC-PKI cells at steady-state. An
LLC-PKI clonal cell line expressing the A
AdoR
(A
AdoR#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.
Figure 6:
The AAdoR 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-A
AdoR#17 in MDCKII cells grown on
Transwell filters in the presence of A
AdoR 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-A
AdoR#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).
Figure 3:
Introduction of AAdoR, 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.
Figure 4:
Growth in the presence of adenosine
receptor antagonists fosters MDCKII cell growth as a monolayer in
AAdoR-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 A
AdoR localization (clonal
cell line TA
AdoR#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.
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 AAdoR
reported in preliminary studies from our laboratory(18) . We
thus compared the relative apical localization of A
AdoR 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
A
AdoR 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
A
AdoR.
In an attempt to determine if some of the ``haze''
seen in the z scans of the TAG-AAdoR 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-A
AdoR-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 A
AdoR 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.
Figure 7:
The AAdoR 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 A
AdoR
(TAG-A
AdoR#17) grown in Transwell culture in the presence
of A
AdoR 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 A
AdoR
(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 A
AdoR was
included in the experiment to demonstrate the migration of the
A
AdoR for comparison with the metabolically labeled,
epitope-tagged protein isolated by sequential protein A and
streptavidin-agarose chromatography. In all gels where
TAG-A
AdoR 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.
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
AAdoR (TA
AdoR#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
A
AdoR on the apical compared to the basolateral surface,
but appears to result from preferential delivery to the apical surface.
Figure 8:
The AAdoR has a comparable
surface half-life on both the apical and basolateral surface of MDCKII
cells. MDCKII cells expressing TAG-A
AdoR#17 and grown in
Transwell culture in the presence of A
AdoR 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.
A
AdoR 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
A
AdoR 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.
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 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
A
AdoR 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. A
AdoR-accelerated
phosphatidylinositol turnover and inhibition of forskolin-stimulated
cAMP production following basolateral but not apical administration of
adenosine(3) . Similarly, basolaterally localized
A
AdoR 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) .
Our studies are the first to characterize the
localization of the AAdoR 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 A
AdoR is preferentially
localized to the apical surface of MDCKII and LLC-PKI cells in multiple
independent clonal cell lines. The apical enrichment of the
A
AdoR at steady state is mirrored by the preferential
delivery of A
AdoR to the apical versus basolateral
surface. Since the A
AdoR has a similar half-life on the
apical and the basolateral surfaces after its delivery, our findings
are consistent with the interpretation that the A
AdoR
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 A
AdoR (Fig. 3) are consistent
with this interpretation; both the A
AdoR and the
AR elicit responses via
G
/G
-coupled GTP-binding proteins, but only
introduction of the A
AdoR 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 A
AdoR has not been determined. Nevertheless,
inclusion of A
AdoR antagonists was necessary to obtain a
monolayer of cells required for performing polarization studies.
The
finding that the AAdoR 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 A
AdoR
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 A
AdoR
and the
AR might therefore permit identification of
structural regions in G-protein-coupled receptors that confer
basolateral versus apical targeting.