(Received for publication, November 7, 1995; and in revised form, December 13, 1995)
From the
The present studies examined the localization of the
- and
-adrenergic receptor (AR)
subtypes in polarized Madin-Darby canine kidney cells (MDCK II) and the
mechanisms by which this is achieved. Previously we demonstrated that
the
AR subtype is directly delivered to lateral
subdomain of MDCK II cells. Surface biotinylation strategies
demonstrated that the
AR, like the
AR, achieves 85-90% basolateral localization at
steady-state. However, in contrast to the
AR, this
polarization occurs after initial random insertion of the
AR into both apical and basolateral surfaces followed
by selective retention on the lateral subdomain (t
on the apical surface is 15-30 min; t
on the basolateral surface is 8-10 h). The
AR also is enriched on the basolateral surface at
steady-state and, like the
AR, is directly delivered
there. Morphological evaluation of the epitope-tagged
AR,
AR, and
AR
subtypes by laser confocal microscopy not only corroborated the
biochemically-defined basolateral localization of all three
AR subtypes but also revealed that the
AR uniquely exists in an intracellular compartment(s)
as well. Immunofluorescence due to intracellular
AR
partially overlaps that due to calnexin, a marker for endoplasmic
reticulum, as well as that due to mannosidase II, a marker for the
trans-Golgi network. Taken together, the present findings demonstrate
that the
AR,
AR, and
AR subtypes, which possess highly homologous
structures and ultimately achieve similar polarization to the lateral
surface of MDCK II cells, nonetheless manifest distinct trafficking
itineraries.
-Adrenergic receptors (
ARs) (
)belong to a superfamily of G-protein-coupled receptors
that have seven predicted transmembrane spanning regions. The three
AR subtypes, called
,
, and
AR, all couple to the
G-proteins of the G
and G
class and mediate a variety of physiological responses via
pertussis toxin-sensitive signal transduction pathways, including
inhibition of adenylyl cyclase, activation of receptor-operated
K
channels, and suppression of voltage-gated
Ca
channels(1) .
The AR
has been demonstrated in the kidney of several mammalian
species(2, 3) . Although these receptors are
concentrated in the proximal tubular segment of the nephron, they also
are found in the glomerulus, thin descending limb of Henle's loop
and cortical collecting duct. The physiological function of the
AR in the proximal tubule is to increase Na
reabsorption and proton secretion via the modulation of
Na
/H
exchange(2, 4) . The precise physiological role
for each of the
AR subtypes is not yet defined.
The
expression of multiple AR subtypes in the kidney (5) and the effects of adrenergic agents on renal physiology
encouraged us to explore one determinant of receptor-mediated function,
namely the precise localization of the receptor molecules in the plasma
membrane of renal epithelial cells. Renal epithelial cells are
polarized both morphologically and functionally into at least two
distinct compartments: apical and basolateral. Both radioligand binding
studies and renal microperfusion experiments are consistent with the
interpretation that
ARs are present primarily on the
basolateral membrane domain of renal epithelial cells in
vivo(6, 7) . How this localization is achieved
for the
AR, or for any G-protein-coupled receptor, is
only beginning to be revealed.
Recent studies from our laboratory
have examined the targeting and retention of wild-type and
epitope-tagged AR in cultured Madin-Darby canine
kidney (MDCK II) cells, a model system that achieves morphological and
functional polarity following culture on Transwell filters(8) .
Surface biotinylation strategies demonstrated that 85-90% of the
wild-type
AR population is localized on the
basolateral surface of MDCK II cells, in a manner analogous to the
localization of this receptor in vivo. Furthermore,
immunochemical detection of the
AR, based on the
recognition of an artificial epitope tag introduced by mutagenesis into
the amino terminus of the receptor, has revealed that the
AR is not randomly distributed in the basolateral
surface but is highly enriched on the lateral subdomain of polarized
MDCK II cells. This localization is achieved by direct delivery to this
surface, based on findings from metabolic labeling studies. Extensive
mutational analysis of the
AR to reveal structural
regions that confer direct
AR delivery to the
basolateral surface suggests that basolateral targeting of
AR does not rely on amino-terminal glycosylation,
carboxyl-terminal acylation, nor amino acid sequences within the third
cytoplasmic loop and the carboxyl-terminal tail(9) . These
findings are consistent with the interpretation that sequences in or
near the lipid bilayer are involved in the delivery of the
AR to the basolateral surface. In contrast, the
deletion of the predicted third cytoplasmic loop of the
AR significantly decreases its half-life on the
epithelial cell surface, suggesting that this structural region of
AR may participate in mechanisms that stabilize the
receptor on the lateral subdomain.
The existence of three subtypes
of AR that possess regions of structural similarity
and diversity provides natural reagents to further understand the
structural regions of G-protein-coupled receptors that confer
information for targeting and stabilization in polarized cells. The
three
AR subtypes (
AR,
AR, and
AR) can be distinguished
not only by differences in pharmacological specificity but also by
differences in their primary amino acid sequences. Although sequences
in the seven transmembrane-spanning domains are highly conserved among
the
AR subtypes, the large third cytoplasmic loop and
the amino and carboxyl termini of the
AR subtypes
demonstrate significant sequence
dissimilarity(10, 11, 12, 13) . In
addition, the post-translational modification of these three receptors
is quite different. For example, the
AR has sequences (10, 11) which confer amino-terminal glycosylation (14) and carboxyl-terminal acylation(15) . In contrast,
the
AR is not
glycosylated(13, 14, 16) , but its carboxyl
terminus does contain the sequences appropriate for acylation, whereas
the
AR does not contain acylation signals but does
possess glycosylation signals in the amino terminus (12) .
The structural differences among the AR subtypes
led us to explore the possibility of a different targeting mechanism
for each subtype, possibly resulting in differential receptor subtype
localization. The present findings demonstrate that although the
,
, and
AR
subtypes ultimately achieve basolateral localization at steady-state,
they do so by different molecular mechanisms. Thus, the
AR shares the direct basolateral delivery pathway
characteristic of the
AR, but is not exclusively
localized on the cell surface at steady-state. The
AR, which achieves almost complete basolateral
localization at steady-state, does so following apparent random
delivery to both surfaces and prolonged retention on the basolateral
surface.
Introduction of the epitope tag into the
carboxyl termini of the and
ARs
(termed
-TAG-AR and
-TAG-AR) was
achieved using polymerase chain reaction (PCR)-based mutagenesis. The
AR 5` oligonucleotide (24-mer) was
5`-GGCTACTGCAACAGCTCTTTGAAC. The
AR 3`
oligonucleotide containing the hemagglutinin TAG sequence, the stop
codon, and an EcoRV restriction site (82-mer with 24 bases
annealing) was
5`-CTAGGATATCTCAACGAGGAGCTAGCGCGTAGTCAGGAACGTCGTAAGGATAGCTAGCCCAGCCAGTCTGGGTCCACGGCCG.
The
AR 5` oligonucleotide (24-mer) was
5`-CGCATCTACCGCGTGGCCAAGCTG. The
AR 3`
oligonucleotide containing the hemagglutinin TAG sequence, the stop
codon, and a SmaI restriction site (82-mer with 24 bases
annealing) was
5`-CTAGCCCGGGTCAACGAGGAGCTAGCGCGTAGTCAGGAACGTCGTAAGGATAGCTAGCCTGCCTGAAGCCCCTTCTCCTCCT.
The 82-mer oligonucleotides were purified on a 9% PAGE, the appropriate
bands were excised, eluted, and passed over C18 Sep-Pak columns. The
24-mer oligonucleotides were purified on C18 Sep-Pak columns. The PCR
reactions were done at high denaturing temperature (98 °C) in the
presence of 5% dimethyl sulfoxide following a ``hot start''
in the absence of DNA polymerase at 90 °C for 30 min. Thermal
stable VENT
DNA polymerase was required for these PCR
reactions due to a high GC content of the DNA sequences encoding the
predicted third cytoplasmic loop of the
AR subtypes.
In case of each
AR subtype, double-stranded DNA
template (5 ng) was used with the respective oligonucleotides at a
final concentration of 0.5 µM. The incubation also
contained 0.25 mM dNTPs, 1
VENT
DNA
polymerase buffer, and 1 unit of VENT
DNA polymerase
in a final volume of 100 µl with a top layer of mineral oil. The
PCR conditions were as follows: denaturing at 98 °C for 1 min,
annealing at 68 °C for 1 min, extension at 72 °C for 2 min for
35 cycles, followed by a 10-min extension at 72 °C and storage at 4
°C. Obtained PCR products were purified on agarose gels and
digested with the appropriate restriction enzymes;
-TAG-AR PCR product (167 bp) was cut with EcoRV and BstBI, and
-TAG-AR PCR
product (744 bp) was digested with SmaI and MluI.
Obtained
-TAG-AR (99 bp) and
-TAG-AR (740 bp) fragments were purified on agarose
gels and subcloned into the pCMV4-
AR plasmid (in
place of the EcoRV-BstBI fragment encoding wild type
sequence) and pCMV4-
AR expression plasmid (instead of
the wild type SmaI-MluI fragment), respectively.
The sequence of all of these mutant constructs was confirmed by
double-stranded dideoxy-DNA sequencing using T7 DNA polymerase and
[-
S]dATP. Transient transfection of COS M6
cells (100-mm dish plated at 1
10
cells/dish) with
10 µg of each expression plasmid utilizing the DEAE-dextran method (9) was employed to assess the level of receptor expression
using [
H]yohimbine binding as well as to examine
the detectability of the epitope TAG by immunocytochemistry prior to
utilization of the DNA to develop permanent transformants.
Immunological co-localization studies were done essentially as
described above, although expanded by the additional application of
another primary and secondary antibody. Incubation with 1:400 dilution
of an anti-calnexin rabbit polyclonal antibody (used as a marker for
the endoplasmic reticulum(18) ), or a 1:1000 dilution of
anti-mannosidase II rabbit polyclonal antibody (used to detect
trans-Golgi network(17) ) in 1% BSA, 0.2% Triton X-100, 0.04%
sodium azide followed the initial detection of the AR
with 12CA5 antibody and FITC (1:60 dilution) or Cy3 (1:100
dilution)-conjugated donkey anti-mouse IgG. The anti-calnexin or
anti-mannosidase II primary antibodies were incubated with the sample
for 1 h at room temperature, followed by four 15-min washes with 0.2%
Triton X-100 in PBS and a 1-h incubation with a secondary donkey
anti-rabbit FITC (1:60 dilution) or Cy3 (1:200 dilution)-conjugated IgG
in 1% BSA, 0.2% Triton X-100, 0.04% sodium azide in PBS. The cells were
then washed four times for 15 min with PBS, 0.2% Triton X-100, mounted,
and analyzed as described above.
Since the initial immunoanalysis of
AR localization in MDCK II cells revealed a
significantly lower level of fluorescent signal as compared to the
AR, we generated additional
AR and
AR DNA constructs and clonal cell lines. We were
concerned that the epitope tag of the TAG-
AR was not
easily accessible to 12CA5 antibody because this subtype has an
unusually high alanine content (17 alanine residues) in its
amino-terminal region, potentially leading to the formation of helices.
Consequently, we deleted the 3`-untranslated regions (280 bp in
TAG-
AR and approximately 500 bp in
TAG-
AR) in order to increase receptor expression (20) and simultaneously inserted the hemagglutinin epitope tag
into carboxyl termini of both subtypes to make the epitope more
accessible to the antibody and also to ascertain that the localization
was independent of the epitope-insertion site.
In the present studies, we examined whether the three
subtypes of AR are differentially localized in MDCK II
cells and whether the modes of receptor delivery to the cell surface or
their half-lives on the membrane after surface delivery differ among
the three subtypes.
Figure 1:
Basolateral localization of all three
AR subtypes in MDCK II cells at steady-state.
Autoradiograms presented in this figure were obtained using the
following permanent MDCK II clonal cell lines expressing either the
wild-type or the epitope-tagged
AR subtypes:
WT-
AR 12 and TAG-
AR 3 (both shown
in panel A); WT-
AR 67 and
TAG-
AR 122 (both shown in panel B);
WT-
AR 90 and TAG-
AR 11 (both shown
in panel C). Polarized MDCK II cells from three 24-mm
Transwell filters were biotinylated on either the apical or the
basolateral surface and the
ARs were photoaffinity
labeled with [
I]Rau-AzPEC in the absence or
presence of the
-AR antagonist phentolamine. We have noted that
significantly less total receptor in the preparation of
AR was required to obtain a sufficient signal
comparable to that of
AR and
AR
subtypes. This observation would suggest that the
AR
has a greater affinity for the iodinated photolabel than the other
AR subtypes. Following detergent extraction of the
photolabeled preparations and subsequent streptavidin-agarose
chromatography, the eluates from the streptavidin-agarose were analyzed
on 7-20% gradient SDS-PAGE and subsequently subjected to
autoradiography. Autoradiograms shown represent one of at least five
independent experiments for each receptor subtype, where comparable
findings were obtained. To quantitate the fraction of the
AR subtype localized apically or basolaterally, the
gel regions corresponding to migration of the
AR
subtypes were cut and counted in a
-counter, as described under
``Experimental Procedures.''
Since surface biotinylation
strategies only reveal the fraction of the receptor population at
either the apical or basolateral surface, we also employed
immunocytochemical methods to evaluate the surface as well as possible
intracellular localization of each of the AR subtypes
within MDCK II cells. MDCK II clones were grown in Transwell culture
and immunostained using the Affi-Gel-purified 12CA5 antibody directed
against the epitope tag (see ``Experimental Procedures'').
The surface staining pattern of the three
AR subtypes
corroborates the basolateral localization revealed using surface
biotinylation strategies. For the
AR and
AR, the immunofluorescence was detected exclusively
on the lateral subdomain of MDCK II cells (Fig. 2). The
AR, however, is localized not only on the lateral
subdomain, but also in intracellular compartments. Our findings in MDCK
II cells resemble previous reports of both surface and intracellular
localization of the
AR when expressed in COS-7 and
HEK-293 renal cell lines(21) . This bimodal surface and
intracellular localization of the
AR, unique among
the
AR subtypes, must represent a property of the
AR structure per se, since the same 12CA5
antibody preparation was used to immunologically localize all three
subtypes of the
AR. Furthermore, as mentioned above,
introduction of the epitope tag into the amino- versus the
carboxyl-terminal domains of the
AR did not alter its
steady-state distribution, assessed using either biochemical or
morphological strategies. Finally, we also examined several independent
clonal cell lines (see Table 1), and found that the relative
distribution of
AR for all subtypes was unmodified
over the range of 2 to 25 pmol of
AR/mg of protein.
Thus, we are confident that the receptor distribution shown in Fig. 2represents properties of the structures of the varying
AR subtypes.
Figure 2:
Immunological localization of the three
AR subtypes in polarized MDCK II cells using 12CA5
antibody. MDCK II clones expressing the epitope-tagged
AR subtypes (TAG-
AR 3,
TAG-
AR 122, and TAG-
AR 11 clonal
cell lines) were polarized on the Transwell system, fixed, and
immunostained as described under ``Experimental Procedures.''
The localization of the hemagglutinin epitope tag determined by
immunochemical strategies described in detail under ``Experimental
Procedures'' was analyzed on a Zeiss laser confocal microscope.
Localization of the epitope-tagged
AR,
AR, and
AR in the XY plane is
presented in the lower panel of each image set. Z scans shown
in the upper panel of each image set show a laser-sectioned
side view of MDCK II cells expressing each of the three receptor
subtypes. The yellow line across each XY plane represents the
exact site where the cells were sectioned from top to bottom with the
laser beam to create the Z scan.
Figure 3:
Immunological co-localization of the
AR subtype with markers for endoplasmic reticulum
(calnexin) and trans-Golgi network (mannosidase II). MDCK II clones
permanently expressing the HA epitope-tagged
AR
subtype (TAG-
AR 11 and
-TAG-AR 77)
were polarized on Transwells, fixed, and immunostained as described
under ``Experimental Procedures.'' The 12CA5 primary mouse
monoclonal antibody was detected by a secondary donkey anti-mouse FITC (panels A and D) or Cy3 (G)-conjugated IgG. The
calnexin or mannosidase II rabbit polyclonal antibodies were detected
with a secondary donkey anti-rabbit Cy3 (panels B and E) or FITC (panel H)-conjugated IgG. Panel I shows a co-localization of 12CA5 and anti-calnexin antibody
staining, while panels C and F show a co-localization
of 12CA5 and anti-mannosidase II antibody staining. In panels C, F, and I, the 12CA5 incubation was followed by a secondary
donkey anti-mouse FITC (panels C and F) or Cy3 (panel I)-conjugated IgG; subsequently a second primary
antibody (anti-Calnexin, in panel I, or anti-mannosidase II in panels C and F) was added for 1 h, followed by a
secondary donkey anti-rabbit FITC (panel I) or Cy3 (panels
C and F)-conjugated IgG. The yellow line across
the XY scan indicates the plane of sectioning of the Z scan, as
described in the legend to Fig. 2.
Metabolic labeling studies require the
epitope-tagged receptor as a means to identify and isolate the
AR among the other radiolabeled proteins. Thus, for
these studies we were restricted to using clonal cell lines permanently
expressing the epitope-tagged versions of
AR subtypes
to study receptor delivery to the cell surface. Fortunately, all of our
steady-state data comparing the localization of photoaffinity-labeled
AR indicate that introduction of the epitope tag does
not influence the localization of any of the
AR
subtypes (cf. Fig. 1). It was of interest, however,
that we were able to immunoisolate the
AR subtype
only when the epitope tag was inserted into the carboxyl terminus;
perhaps the alanine-rich sequence in the amino terminus of the
AR rendered the tag inaccessible to the 12CA5
antibody when inserted in the amino terminus.
Fig. 4examines
the delivery of all three AR subtypes in MDCK II
cells. In all cases, we are confident that the radiolabeled band
identified as
,
, or
AR represents the particular subtype under study,
because of its comigration with photoaffinity-labeled receptor and its
absence when comparable metabolic labeling studies are performed in
parental MDCK II cells lacking expression of any of these subtypes. The
band migrating just above the 66-kDa molecular mass marker on SDS-PAGE
is interpreted to represent the
AR (Fig. 4A) and
AR (Fig. 4C) subtype. Newly synthesized
ARs (Fig. 4C), like the
AR (Fig. 4A), appear virtually
exclusively on the basolateral surface after each labeling time point
tested. These data are consistent with the interpretation that both the
AR and
AR subtypes are delivered
directly to the basolateral surface. In contrast, the
AR shows a fundamentally different targeting pattern
when compared to the
AR and
AR
subtypes. As shown in Fig. 4B, the 45-kDa
AR is initially randomly inserted into both apical
and basolateral surfaces, where it is first detectable 30 min after
initiation of metabolic labeling. This random expression on both the
apical and basolateral surfaces also is repeatedly observed 45 min
after pulse labeling of this receptor subtype. However, 60 min after
the initiation of metabolic labeling, the majority of the
AR is found on the basolateral membrane. The data
shown in Fig. 4B are from one experiment repeated 6
times with comparable outcome. The
AR subtype
delivery was assessed using receptor structures where the epitope tag
was introduced either into the amino or carboxyl terminus, and the
indistinguishable results obtained for either epitope-tagged structure
suggest that position of epitope tag does not influence the apparent
random delivery of the
AR in MDCK II cells.
Figure 4:
Direct versus random cell surface
delivery of different subtypes of the AR in MDCK II
cells. The following MDCK II clones permanently expressing the
epitope-tagged
AR subtypes were used to obtain the
shown autoradiograms: TAG-
AR 3 (A),
TAG-
AR 122 (B), and
-TAG-AR 28 (C). Polarized MDCK II clonal
cell lines grown on three or four 24-mm Transwell filters (depending on
the receptor expression level) for 7 days were metabolically labeled
with 150 µCi of [
S]cysteine/methionine in
150 µl of medium at 37 °C, 5% CO
for the indicated
periods of time and then biotinylated on either the apical or the
basolateral surface. The
AR delivered to the
biotinylated surface were isolated via sequential Protein A and
streptavidin-agarose chromatography, as described under
``Experimental Procedures.'' Eluates from the
streptavidin-agarose resin were analyzed by 7-20% gradient
SDS-PAGE and subsequent autoradiography. Shown here are representative
autoradiograms of at least three independent experiments for each
receptor subtype (for the
AR, this experiment was
repeated 6 times), which yielded comparable
findings.
Figure 5:
Differential retention of the
AR subtype on the apical versus the
basolateral surface of MDCK II cells. MDCK II cells permanently
expressing the
-TAG-AR clone 42 (A) or
TAG-
AR clone 122 (B) were metabolically
labeled with 150 µCi of
[
S]cysteine/methionine in 150 µl of medium
at 37 °C, 5% CO
for 30 (A) or 60 (B)
min, then chased in medium supplemented with 1 mM methionine
and 1 mM cysteine for the indicated time periods. After
completion of the chase period, cells were biotinylated on either the
apical (A) or the basolateral (B) surface with
NHS-biotin. Subsequently, the
AR was isolated via
sequential protein A-agarose and streptavidin-agarose chromatography,
and resolved on SDS-PAGE. The upper panels of A and B show representative autoradiograms. Upon excision and
counting gel bands that correspond to the
AR, the
percentage of radioactivity present in each receptor band was plotted
as a function of time (radioactivity present in t
(time = 0) band was ascribed as 100%). The means ±
S.E. of radioactivity present in
AR corresponding gel
bands averaged from n = 7 (A) and n = 3 (B) are shown in the lower panels of A and B. The calculated half-life of the
AR on the apical surface was 15-30 min, whereas
the calculated half-life of the
AR on the basolateral
surface was 8-10 h.
Fig. 6demonstrates that the AR,
AR, and
AR have comparable
half-lives on the basolateral surface. Previous data from our
laboratory suggest, at least for the
AR, that
receptor retention involves protein-protein interactions involving
endofacial domains of the receptor, since deletion of the third
cytoplasmic loop of the
AR measurably accelerates
receptor turnover on the basolateral surface(9) . The similar
half-life of the
AR,
AR, and
AR subtypes on the basolateral surface suggests that
similar tethering mechanisms may exist for all three structures despite
the fact that the endofacial third loop sequences of these three
AR subtypes are quite distinct. Perhaps these
AR subtypes, when they achieve their three-dimensional
structure, project similar surfaces to endofacial proteins that
stabilize
ARs on the surface. More rapid turnover of
the
AR on the apical domain suggests that if such
tethering proteins do exist, they may be absent or exist in a reduced
density underneath the apical surface, resulting in a more rapid
AR turnover on the apical, when compared with the
basolateral, domain.
Figure 6:
Retention of the three AR
subtypes on the basolateral surface. MDCK II permanent clonal cell
lines expressing the epitope-tagged
AR subtypes used
in representative autoradiograms shown above included
-TAG-AR 52 (A), TAG-
AR 122 (B), and
-TAG-AR 28 (C). Polarized
MDCK II cells grown on three or four 24-mm Transwell filters (depending
on the clonal receptor expression) for 7 days were metabolically
labeled (pulsed) with 150 µCi of
[
S]cysteine/methionine in 150 µl of medium
for 60 min at 37 °C, 5% CO
, and then incubated in
medium supplemented with 1 mM methionine, 1 mM cysteine for various periods of time (``chase period'')
at 37 °C. The 2-h chase period was designated as t
(time = 0) based on our previous observations of time
required for the nascent
AR to arrive at the
basolateral surface. In addition to the t
,
ARs were also isolated following 6- and 24-h chase
periods, using biotin surface labeling strategy coupled with receptor
immunoisolation and streptavidin-agarose chromatography, as described
under ``Experimental Procedures.'' The upper panel of A, B, and C provides autoradiograms of
7-20% gradient SDS-PAGE from a representative experiment; the lower panels show plots of the means ± S.E. of
radioactivity in each band averaged from three experiments. Receptor
bands were excised from autoradiograms according to the position of
each
AR subtype and counted in a
-counter with 10
ml of scintillation liquid. The percentage of radioactivity present in
each receptor band was plotted on a semi-log scale as a function of
time defining radioactivity at t
as
100%.
Fig. 7provides a schematic diagram of the different
trafficking itineraries observed for the three AR
subtypes in these studies. As described previously (8) , the
AR subtype is delivered directly to the basolateral
membrane and at steady-state virtually all
AR is
present on the surface. The
AR also is directly
delivered to the basolateral surface, but at steady-state is
distributed between cell surface and one or more intracellular
compartments. Finally, the
AR subtype is initially
delivered randomly to both cell surfaces but then is preferentially
retained on the basolateral membrane. Although all three
AR subtypes have been detected in renal epithelia of
varying species(2, 3, 5) , the functional
consequences of these differing receptor itineraries in renal
epithelial cells revealed by the present studies or the implications
for targeting and retention of these
AR subtypes in
other polarized cells, such as neurons, remain to be established.
Figure 7:
Schematic representation of differential
targeting mechanisms that lead to the basolateral localization of all
three AR subtypes in MDCK II cells. This schematic
diagram compares the targeting mechanism of the
AR,
AR, and
AR subtypes, and summarizes
the differing trafficking or steady-state localization observed for
each subtype. The
AR is delivered directly to the
basolateral membrane (present data, (8) ). The present findings
have revealed that the
AR is inserted randomly into
both cell surface domains and is retained preferentially on the
basolateral surface. At steady-state, the
AR and
AR are detected exclusively on the lateral surface of
MDCK II cells, whereas the
AR is distributed between
cell surface and intracellular compartments. However, the fraction of
the
AR that is localized to the basolateral surface
is delivered there directly.