From the Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232
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ABSTRACT |
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Previous studies have shown that
The Receptor localization and stabilization on the cell surface of target
cells are two critical contributors to the sensitivity and extent of
signaling by G protein-coupled receptors. There is a growing body of
evidence that discrete localization of G protein-coupled receptors may
play a role in specificity of signaling by these receptors (5-8). A
precedent already exists for the microcompartmentation of signaling
molecules such as protein kinase C (9), cAMP-dependent
protein kinase (10), Ca2+/calmodulin-dependent protein kinase
II (11), kinases involved in the yeast mating response (12), and NO
synthase (13, 14) by interaction of these effector molecules with
signaling "scaffold" proteins.
In polarized cells, receptor localization is essential for vectorial
information transfer, as occurs for Examination of molecular regions of the three Materials--
[ Cell Culture--
Madin-Darby canine kidney cells (MDCKII) were
obtained from Enrique Rodriguez-Boulan (Cornell Univ. New York, NY) and
maintained in Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum (Sigma), 100 units/ml penicillin, and 100 mg/ml
streptomycin (referred to as complete Dulbecco's modified Eagle's
medium) at 37 °C, 5% CO2. For polarity experiments,
MDCK II cells were seeded at a density of 1 × 106
cells/24.5- mm polycarbonate membrane filter (Transwell chambers, 0.4-µm pore size, Costar, Cambridge, MA) and cultured for 5-8 days
with a change of medium every 1-2 days. Before each experiment, the
integrity of the monolayer was assessed by adding
[3H]methoxyinulin to the apical medium and monitoring its
leak after a 1-h incubation at 37 °C from the apical to the
basolateral compartment by sampling and counting the basolateral medium
in a scintillation counter (Packard Tricarb). Chambers with greater
than 5% leak/h were not evaluated.
Construction of Mutant
Oligo-directed mutagenesis in M13 phage was utilized to create
incremental deletions of the predicted third intracellular loop of the
The nonsense loop was designed by taking advantage of the method used
for making the original
All mutations were verified using dideoxy-DNA sequencing (Sequenase
kit, U. S. Biochemical Corp.) of the single-stranded DNA utilizing T7
DNA polymerase with Determination of the Half-life of Wild-type or Mutant
Analysis of Ligand Binding to Adrenergic Receptors--
The
density of
To assess the effects of agonist on Determining the Triton X-100 Extractability of Wild-type and
To determine the Triton X-100 extractability of No Small Region in the
Five ~20 amino acids deletions were made within the
The surface stability for each of the Direct Cytoskeletal Interactions Do Not Appear to Account for
Stabilization of the
As shown in Fig. 2A, both the
wild-type Specific Amino Acid Sequences Within the Third Intracellular Loop
Do Not Appear to Be Required for Stabilization of the
These findings are consistent with a mechanism where the size of the 3i
loop structure plays an important role in stability of the
Sustained Agonist Exposure in MDCKII Cells Does Not Decrease
Receptor Density for Either Wild-type or The present findings suggest that the retention of the
2A-adrenergic receptor (
2A-AR)
retention at the basolateral surface of polarized MDCKII cells involves
its third intracellular (3i loop). The present studies examining mutant
2A-ARs possessing short deletions of the 3i loop
indicate that no single region can completely account for the
accelerated surface turnover of the
3i
2A-AR,
suggesting that the entire 3i loop is involved in basolateral
retention. Both wild-type and
3i loop
2A-ARs are
extracted from polarized Madin-Darby canine kidney (MDCK) cells with
0.2% Triton X-100 and with a similar concentration/response profile,
suggesting that Triton X-100-resistant interactions of the
2A-AR with cytoskeletal proteins are not involved in
receptor retention on the basolateral surface. The indistinguishable
basolateral t1/2 for either the wild-type or
nonsense 3i loop
2A-AR suggests that the stabilizing
properties of the
2A-AR 3i loop are not uniquely dependent on a specific sequence of amino acids. The accelerated turnover of
3i
2A-AR cannot be attributed to
alteration in agonist-elicited
2A-AR redistribution,
because
2A-ARs are not down-regulated in response to
agonist. Taken together, the present studies show that stabilization of
the
2A-AR on the basolateral surface of MDCKII cells
involves multiple mechanisms, with the third intracellular loop playing
a central role in regulating these processes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES
2-adrenergic receptor
(
2-AR)1 is a
member of a large family of G protein-coupled receptors that are
predicted to have seven transmembrane-spanning regions (1, 2). Three
subtypes of
2-ARs exist and couple to members of
the Gi and Go class of G-proteins to
mediate a variety of physiological responses (3, 4).
2-AR regulation of
Na+ and H2O transport in renal (15) and
intestinal (16, 17) epithelial cells. Madin-Darby canine kidney
(MDCKII) cells cultured in Transwell culture dishes have provided an
excellent model system for polarized renal epithelial cells. The
localization of the
2A-AR subtype on the basolateral
surface of these cells (18) recapitulates the basolateral localization
of this receptor in vivo, based on physiological (19) and
pharmacological (20) data.
2-AR
subtypes in polarized MDCKII cells indicates that basolateral targeting of these receptors involves sequences in or near the membrane bilayer
(18, 21, 22). In contrast, the large third intracellular loop of the
receptor appears to play a role in stabilizing the
2A-AR
on the plasma membrane, because mutant
2A-ARs that lack 119 amino acids from the large third intracellular loop (
3i loop
2A-AR) have a cell surface half-life
(t1/2) of 4.5 h compared with a
t1/2 of 10 h for the wild-type
2A-AR (21). The present studies have explored the
structural features of the
2A-AR 3i loop responsible for
stabilizing the
2A-AR on the plasma membrane of MDCKII cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES
-32P]ATP (6000 Ci/mmol),
[3H]yohimbine (70-90 Ci/mmol),
p-[125I]iodoclonidine (2200 Ci/mmol),
35S-Express protein labeling mix (1200 Ci/mmol),
[3H]methoxyinulin (Ci/mmol), and
[
-35S]dATP (1389Ci/mmol) were purchased from NEN Life
Science Products. Phentolamine was kindly provided by CIBA
Pharmaceutical Co. 125I-Rau-AzPEC
(17-hydroxyl-20-yohimban-16-(N-4-azido-3-[125I]iodophenyl)car-boxamide)
was synthesized in our laboratory by the method of Lanier et
al. (23). Biotin hydrazide, Sulfo-NHS-biotin, and
streptavidin-agarose were purchased from Pierce. The protein A-purified
12CA5 monoclonal antibody was purchased from the Berkeley Antibody Co.
Epinephrine was obtained from Sigma, and ascorbic acid was purchased
from Fisher.
2A-AR Permanently
Expressing Cell Lines--
Generation and characterization of
3i
loop
2A-AR (
aa240-359) has been previously
described(21, 24). Briefly, site-directed mutagenesis was used to
create a novel NotI restriction enzyme site in the region of
the
2A-AR cDNA encoding the C-terminal end of the
putative third intracellular (3i) loop. Cleavage of this mutant
2A-AR cDNA with NotI restriction enzymes
removes the DNA fragment encoding amino acids 240-359. This
3i loop
mutant receptor has 36 amino acids linking transmembrane domains 5 and 6 of the
2A-AR as shown in Fig. 1A.
2A-AR. Single oligos were designed against sequences flanking those encoding the amino acids selected for each deletion. The
deletion mutations were confirmed by dideoxynucleotide sequencing and
then subcloned into the pCMV4 mammalian expression vector. Deletions
corresponding to DNA encoding the following amino acids were made in
this manner:
aa252-267,
aa268-285,
aa286-303,
aa315-326, and
aa327-340. Fig. 1 provides a schematic diagram of the regions encoded by these deleted amino acids.
aa240-359 (
3i loop
2A-AR). Because two NotI enzyme sites were
used to remove the 3i loop, it was possible to subclone this segment of
the gene back into the receptor in two orientations. The correct
orientation produced a receptor that corresponded to the wild-type
2A-AR sequence except for two point mutations at the
site of the engineered NotI site (K359A, S360A). The
opposite orientation also produces an open reading frame with a 3i loop
of the same amino acid length but with very little sequence homology to
the wild-type receptor (See Fig. 4). We refer to this mutant
2A-AR as the nonsense 3i loop
2A-AR.
-35S-dATP. Once verified, the mutant
inserts were subcloned from M13 into the pCMV4-TAG-
2A-AR
expression vector (18) containing an N-terminal hemagglutinin epitope
(YPYDVPDYA) to which antibodies are available commercially (Berkeley
Antibody Co.). These plasmid constructs were verified by
double-stranded DNA sequencing through the region of the mutation. COS
M6 cells were transiently transfected with the plasmid DNA encoding the
wild-type and mutant
2A-ARs, and membranes from the
transient transfectants were assayed for [3H]yohimbine
binding before developing permanent MDCK cell lines expressing these
mutant
2A-AR. MDCK cell lines permanently expressing the
wild-type or mutant
2A-AR were created as described
previously (18) (Table I).
Wild-type 2A-AR and mutant
2A-AR-expressing MDCK
cell lines utilized in polarization and stability studies
2A-AR on the Basolateral Membrane--
To determine
2A-AR half-life on the basolateral surface, a metabolic
labeling strategy was employed. MDCK cells expressing wild-type or
mutant
2A-AR were incubated with
[35S]Cys/Met ("pulse") and then incubated for various
periods of time ("chase") before isolation of basolateral
2A-AR using sequential biotinylation, extraction,
immunoisolation, and streptavidin-agarose chromatography. The
procedures utilized have been previously described (21) except for the
modifications outlined as follows. Specifically, MDCK cells grown in
Transwell culture were metabolically labeled with 1 µCi/µl
35S-Express protein labeling mix for 60 min in Cys/Met-free
Dulbecco's modified Eagle's medium (18). After labeling, the cells
were washed once with Dulbecco's phosphate-buffered saline (dPBS) and incubated for various periods of time (generally 0, 3, 6, and 18 h) at 37 °C, 5% CO2 in chase medium (complete
Dulbecco's modified Eagle's medium supplemented with 1 mM
cysteine and 1 mM methionine). At the conclusion of each
chase period,
2A-ARs residing on the basolateral surface
of these cells were isolated by biotinylating the basolateral cell
surface with biotin hydrazide or sulfo-NHS biotin and subjecting
detergent extracts of membranes prepared from these cells to sequential
immunoprecipitation with the 12CA5 anti-hemagglutinin epitope antibody
followed by streptavidin-agarose chromatography (21). The
streptavidin-agarose eluates were separated by SDS-polyacrylamide gel
electrophoresis, and the amount of radiolabeled
2A-AR
remaining on the basolateral surface after various durations of chase
was determined. The gels were exposed to film, and the gel area
corresponding to the radiolabeled
2A-AR was removed and
counted (in 10 ml of scintillation Fluor) in a Packard beta counter.
Similar-sized gel slices that did not correspond to any radiolabeled
protein band were excised and counted to quantify the background
35S signal in each lane; these background counts were
subtracted from the counts in the
2A-AR slices to
determine the specific
2A-AR cpm at each time point.
2A-ARs in membrane preparations of MDCK cells
was determined via saturation binding using the
2A-AR
antagonist [3H]yohimbine as the radioligand. Membranes
were prepared by hypotonic lysis and were resuspended in membrane
binding buffer (25 mM glycylglycine, 20 mM
HEPES, 100 mM NaCl, and 5 mM EDTA, pH 8.0) such
that the specific binding at 7.5 nM
[3H]yohimbine in a 250-µl aliquot (12-400 µg of
protein/ml) represented approximately 10,000 cpm of specific binding.
The density of [3H]yohimbine binding sites was then
assessed in a subsequent assay using increasing concentrations of
[3H]yohimbine (0.5-15 nM) in a total volume
of 250 µl. Binding was performed at 25 °C for 1.5 h and was
terminated by the addition of 4.5 ml of ice-cold 25 mM
glycylglycine, pH 7.6, followed by vacuum filtration through GF/B glass
fiber filters and two washes with 4.5 ml of ice-cold buffer.
Nonspecific binding was defined as that binding detectable in the
presence of 10 µM phentolamine. The
Bmax and KD for
[3H]yohimbine binding to the
2A-AR was
determined by analysis of saturation binding data using GraphPad Prism
software (GraphPad, San Diego, CA).
2A-AR
redistribution, MDCKII cells expressing either wild-type or
3i loop
2A-AR were treated for 24 h with 100 µM epinephrine and 100 µM ascorbic acid to
prevent oxidation of the epinephrine, as described previously (25).
Control cells were treated with 100 µM ascorbic acid
alone. Receptor density was determined by [3H]yohimbine
saturation binding.
3i Loop
2A-AR--
MDCK cells permanently
transfected with either wild-type or
3i loop
2A-AR
were grown on Transwells for 7 days. Transepithelial leak of
[3H]methoxyinulin was determined as described earlier for
confirmation of functional polarization. To compare the Triton X-100
extractability of wild-type and
3i loop
2A-AR, the
2A-ARs in intact cells were covalently modified via a
radioiodinated photoaffinity label and then extracted with increasing
concentrations of Triton X-100 (%v/v). Briefly, cells were washed with
dPBS supplemented with 0.5 mM CaCl2 and 1.0 mM MgCl2 (dPBS-CM), and the Transwells were inverted and incubated 1 h at 22 °C in the dark with 150 µl
of dPBS-CM containing 0.2 µCi/well (0.9 nM)
125I-Rau-Az-Pec. After 1 h, the wells were washed with
dPBS containing 1 mM glutathione, suspended in a Rayonet UV
photoilluminator, and photolysed for 3 min with 300-nm light. The
2A-ARs expressed on the basolateral surface were
identified by biotinylation with Sulfo-NHS-biotin in triethanolamine
buffer for 2 20-min incubations as described above.
2A-ARs
in polarized cells, Transwells with the photolabeled
2A-AR were washed with dPBS and extracted with
increasing concentrations of Triton X-100 using a modification of a
previously described protocol (26). The polycarbonate filters were
first removed from the Transwell support and placed into a 12-well dish
with each 24-mm well containing 190 µl of a Triton X-100 extraction
buffer (15 mM Tris, pH 8, 120 mM NaCl, 25 mM KCl, 0.1 mM EGTA, 0.5 mM EDTA) with no Triton X-100. The cells on polycarbonate filters were rocked
gently for 5 min at 4 °C. The filters were then transferred to a
well with extraction buffer containing 0.05% Triton X-100 and rocked 5 min. This procedure was repeated with increasing concentrations of
Triton X-100 (0.1, 0.2, 0.5, and 1.0%). After exposure to 1% Triton
X-100, the residual cellular material, operationally defined as
"Triton shells," was scraped into 200 µl of RIPA buffer. A set of
control Transwells were subjected to the same procedure, except that
each successive buffer contained no Triton X-100. The final RIPA
extraction buffer from each well was transferred to a 0.6-ml Eppendorf
tube, and the wells were washed with 200 µl of RIPA buffer. All
extracts were brought up to a final volume of 500 µl with RIPA
buffer, and biotinylated proteins were isolated using
streptavidin-agarose chromatography. After an overnight incubation, the
streptavidin beads were eluted with 1× SDS sample buffer at 90 °C
for 30 min. This elution was repeated, and the combined eluates were
loaded onto a 10% SDS-polyacrylamide gel. The dried gels were exposed
to preflashed Kodak film for 3-5 days. The receptor was identified as
a radioactive band migrating at a position characteristic of the
2A-AR and whose photoaffinity-labeling was blocked in
the presence of 10 µM phentolamine in separate control studies.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES
2A-AR Third Intracellular
Loop Contains All of the Necessary Information for Stabilization of the
Receptor on the Cell Surface--
We observed previously that deletion
of 119 amino acids from the 3i loop of the
2A-AR (amino
acids 240-359) generates a structure (
3i
2A-AR) that
has a basolateral t1/2 of ~4.5 h compared with
10-12 h for the wild-type
2A-AR in polarized MDCKII
cells. By analogy with the ability of a 21-amino acid insert into the
short (D2S) dopamine receptor to create the long dopamine receptor
isoform (D2L) and dramatically slow the rate of sequestration (27), the
3i loop of the
2A-AR was examined to determine whether a
single small amino acid sequence could account for the stabilization of
the receptor.
2A-AR 3i loop, as shown schematically in Fig.
1A. Demarcation of the regions
selected for individual deletions was based on secondary structural
predictions of Chou and Fasman analysis (52); for example,
aa286-303 and
aa315-326 are predicted by this analysis to form
amphipathic
helices. In addition, the
aa286-303 removes the
LEESSSS sequence recognized for phosphorylation by G protein-coupled receptor kinases (28, 29). This was of interest because G protein-coupled receptor kinase-mediated phosphorylation of these receptors promotes association with arrestins that have been shown to
act as adaptors and recruit some G protein-coupled receptors into
clathrin-coated pits (30-32).
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Fig. 1.
No small region of the
2A-AR third intracellular loop can
account for the stabilizing properties of the entire 3i loop.
A, a schematic diagram of the different deletions evaluated
in this study. The
aa240-359 structure, termed
3i loop
2A-AR (21), and incremental deletions of the predicted
third intracellular loop of the
2A-AR (
aa252-267,
aa268-285,
aa286-303,
aa315-326, and
aa327-340) were
prepared as described under "Experimental Procedures".
B, the residence time of each
2A-AR structure
on the cell surface was determined by metabolically labeling MDCK cells
expressing wild-type (WT) or mutant
2A-AR
(pulse) and then incubating those labeled cells for various periods of
time (chase) before isolating basolateral
2A-AR using
sequential biotinylation, extraction, immunoisolation, and
streptavidin-agarose chromatography, as described under "Experimental
Procedures." The data shown are from one experiment representative of
at least four separate experiments for wild-type,
aa286-303, and
3i loop
2A-AR. C, multiple incremental
deletions of the
2A-AR 3i loop were compared for the
amount of receptor detected on the basolateral surface following a 1-h
pulse (100%) versus a 1-h pulse followed by a 6-h chase.
The results shown are the mean +S.E. of multiple individual experiments
as follows: wild type (n = 9),
3i loop
(n = 7),
aa286-303 (n = 6),
aa327-340 (n = 4),
aa252-267 (n = 5), and
aa315-326 (n = 4).
2A-AR structures
examined (Fig. 1A) was determined by pulse/chase metabolic
labeling strategies and isolation of
2A-AR on the
basolateral surface by sequential biotinylation and
streptavidin-agarose isolation of detergent-solubilized receptor (see
"Experimental Procedures"). As shown in Fig. 1B,
aa286-303
2A-AR does not mimic the accelerated turnover characteristic of the
3i loop
2A-AR. Similar
studies were performed for the other deletions shown in Fig.
1A and are summarized in Fig. 1C. Although three
of the deletions may affect
2A-AR residence time on the
cell surface somewhat, none of the smaller deletions within the 3i loop
appear to mimic the accelerated turnover characteristic of the
3i
loop
2A-AR. These data suggest that multiple
noncontiguous sequences or bulk size of the 3i loop determine the
residence time of
2A-AR on the cell surface.
2A-AR via Its 3i Loop--
One
mechanism that might account for stabilization of the
2A-AR on the basolateral surface of polarized renal
epithelial cells could be direct interaction of the receptor with the
underlying cytoskeleton. In this case, accelerated turnover of the
3i loop
2A-AR could result from loss of these direct
cytoskeletal interactions. We utilized differential sensitivity to
extraction by Triton X-100 as an indicator of direct and stable
association with the cytoskeleton (33-35). This approach has been
informative in revealing the association of the polytopic
Na+-K+-ATPase (26, 36) and of the single
transmembrane-spanning CD44 protein (37) with the
cadherin-dependent ankyrin-fodrin matrix underlying the
basolateral surface of polarized MDCK cells.
2A-AR and the
3i
2A-AR are
released from polarized MDCKII cells when exposed to 0.2% Triton X-100
in the presence of 2 mM EDTA and 2 mM EGTA (0 mM [Ca2+]o and 0 mM
[Mg2+]o). For comparison, proteins directly
associated with the cytoskeleton, such as the
Na+-K+-ATPase, are not completely extracted by
0.5% Triton X-100 under similar, but slightly more stringent, divalent
cation-free extracellular conditions, whereas the
2A-AR
is completely extracted (26, 36),2 suggesting that the
2A-AR does not interact directly or stably with the
cytoskeleton. When Triton X-100 extractions were performed in the
presence of 0.5 mM Ca2+ and 1 mM
Mg2+, the amount of Triton X-100 required for extraction of
60% of the photoaffinity-labeled surface receptors was increased
from 0.2% (Fig. 2A) to 0.5% (Fig. 2B) but with
no difference in extraction efficiency between wild-type and mutant
2A-AR. These data suggest that
2A-AR
stability on the basolateral surface is influenced by protein-protein
interactions that involve a Ca2+ (likely
cadherin)-organized substratum, but that these interactions cannot
explain the difference in cell surface stability of the wild-type and
3i loop structures, as they are extracted in a comparable manner
even in the presence of Ca2+.
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Fig. 2.
Neither the
2A-AR nor the
3i loop
2A-AR
remains associated with Triton X-100-resistant structures in polarized
MDCKII cells. Polarized MDCK cells grown for 1 week on Transwell
filters were radiolabeled in intact cells using 0.2 µCi/well (0.9 nM) 125I-Rau-AzPEC, an
2A-AR-selective photoaffinity label. Proteins present on
the basolateral surface, including the
2A-AR, were
covalently modified with Sulfo-NHS-biotin as described under
"Experimental Procedures." Wild-type or
3i loop
2A-AR were then extracted with increasing concentrations
of Triton X-100 by rocking each Transwell in extraction buffers
containing varying concentrations of Triton X-100 as indicated. All
buffers contained either 2 mM EDTA, 2 mM EGTA
(denoted [0 mM Ca2+]o and [0
mM Mg2+]o) (A) or 0.5 mM [Ca2+]o, 1.0 mM
[Mg2+]o (B). After exposure to 1%
Triton X-100, the residual cellular material, defined as Triton shells,
was solubilized with RIPA buffer (Non Ext.). Biotinylated
proteins were isolated using streptavidin-agarose chromatography. The
2A-AR in the eluates was resolved on a 10%
SDS-polyacrylamide gel. Control experiments indicated that the
radioactive band shown corresponds to 125I-Rau-AzPec
photoaffinity-labeled
2A-AR, based on its relative
migration on 10% gels and the blockade of its labeling by
2A-AR antagonists. The results shown compare wild-type
2A-AR (Tag3 clone at 25 pmol/mg of protein) and
3i
loop
2A-AR (T3 at 3.4 pmol/mg of protein (A)
or T66B at 2.8 pmol/mg of protein (B)). These data are
representative of at least three separate experiments. This extraction
profile is not dependent on receptor density because two cell lines
with nearly 10-fold different levels of wild-type
2A-AR
expression (Tag3 clone at 25 pmol/mg of protein versus T24
clone at 3.4 pmol/mg of protein) were examined with the same
results.
2A-AR on the Cell Surface--
If bulk size of the 3i
loop is sufficient for stabilization of the
2A-AR, then
a loop containing the same number of amino acids as the wild-type
2A-AR should manifest the same surface t1/2 regardless of the primary sequence within the
3i loop. To test this hypothesis, we constructed a receptor that
contains a nonsense 3i loop corresponding to the exact number of amino
acids as in the wild-type
2A-AR 3i loop but with very
little sequence homology. The sequences of the wild-type and nonsense
2A-AR 3i loops are compared in Fig.
3. In four experiments using two
different clonal cell lines expressing the
2A-AR 3i nonsense loop, the half-life of this structure was indistinguishable from that characteristic of the wild-type receptor as shown in Fig.
3.
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Fig. 3.
Specific amino acid sequences within third
intracellular loop do not appear to be required for stabilization of
the 2A-AR on the basolateral
surface of polarized MDCKII cells. The nonsense 3i loop
(NS)
2A-AR was designed as described under
"Experimental Procedures," and the residence time of this structure
on the basolateral surface was compared with that of wild-type
2A-AR. This mutant
2A-AR contains a 3i
loop of the same exact amino acid length but with very little sequence
homology to the wild-type receptor; the calculated net charge of the
wild-type
2A-AR 3i loop is +1, whereas the calculated
net charge of the nonsense loop is +11 because of a decreased number of
acidic amino acids. The surface half-life of the wild-type and nonsense
3i loop
2A-AR was determined as in Fig. 1B
and described in detail under "Experimental Procedures."
2A-AR on the cell surface. There are examples of
membrane proteins localized in surface microdomains by virtue of
so-called "corrals," often established by the cytoskeletal proteins
underlying the cell surface (38-40). Consequently,
2A-AR surface stability might arise by steric
principles, dictated by the size of the 3i loop (Fig. 3). If corrals
partitioned the
2A-AR, and lack of corralling were
responsible for accelerated turnover of the
3i loop
2A-AR, then we should expect a more rapid lateral
diffusion coefficient and a significantly greater mobile fraction for
the
3i loop
2A-AR. However, Uhlén et
al. (41) have previously reported that the difference in surface
half-life between
2A-AR and
3i loop
2A-AR is not paralleled by a difference in lateral diffusion coefficients for each receptor structure, estimated at
~2.2 × 10
10 cm2/s using fluorescence
recovery after photobleaching. Thus, the mechanistic significance of
the retention of the
2A-AR 3i nonsense loop on the
basolateral surface for a duration comparable with the wild-type
receptor is unexplained at present.
3i loop
2A-AR--
One mechanism that might account for
accelerated surface turnover of the
3i loop
2A-AR
would be enhanced agonist-elicited redistribution and subsequent
down-regulation of this mutant receptor compared with the wild-type
receptor. Consequently, we examined the effect of prolonged agonist
exposure on steady-state
2A-AR density in MDCKII cells
expressing wild-type or
3i loop
2A-AR. As shown in
Fig. 4, treatment of MDCKII cells with
100 µM epinephrine for 24 h results in no detectable
down-regulation of either the wild-type or the
3i loop
2A-AR. In fact, there is even a slight increase in
receptor density following agonist incubation, perhaps because of
ligand-dependent receptor stabilization
(42).3 These findings are
consistent with previous reports that the
2A-AR subtype
does not undergo agonist-induced down-regulation in MDCKII cells (43)
and Chinese hamster fibroblast cells (28), although this subtype has
been reported to down-regulate in Chinese hamster ovary cells (25, 44,
45). In addition, the
2A-AR subtype, in contrast to the
2B-AR subtype, does not manifest agonist-elicited
redistribution (46),4 nor is
there any evidence for intracellular localization of the
3i loop
2A-AR in MDCKII cells either by immunocytochemistry (48)
or cell surface
biotinylation.5 In contrast
to our findings and the lack of effect of removal of the 3i loop on
agonist-elicited
2A-AR redistribution, several muscarinic receptor subtypes have been shown to undergo
agonist-elicited sequestration and down-regulation in a manner
influenced by the 3i loop; mutations within the 3i loop reduce
sequestration (47, 49-51) and deletion of the 3i loop slows the rate
of down-regulation for the m2 receptor (47).
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Fig. 4.
Sustained agonist exposure in MDCKII cells
does not alter either wild-type (WT) or
3i loop
2A-AR
density. MDCKII cells expressing either wild-type or
3i loop
2A-AR were treated for 24 h with 100 µM epinephrine (+100 µM ascorbic acid to
prevent epinephrine oxidation). Control cells were treated with 100 µM ascorbic acid alone. Receptor density was determined
by [3H]yohimbine binding as described under
"Experimental Procedures." Results are the mean ±S.E. from six
separate experiments. Results shown are from MDCKII cells grown to
confluence on 60-mm cell culture dishes. However, analysis of MDCKII
cells polarized in Transwells gave the same results. No decrease in
receptor density was observed; in fact, an increase was noted by
analogy with findings in other cultured cell systems3
suggesting that ligand occupancy stabilizes receptor density and
affirming that epinephrine remains viable during the course of the
incubation.
CONCLUSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES
2A-AR on the basolateral surface of polarized renal
epithelial cells involves the 3i loop in its entirety, because smaller
deletions within the 3i loop cannot mimic the accelerated turnover
characteristic of the mutant
3i loop
2A-AR.
Stabilization of the
2A-AR on the basolateral surface
does not appear to involve interaction with cytoskeletal proteins,
because concentrations of Triton X-100 that do not destabilize direct
membrane protein interaction with the cytoskeleton nonetheless extract
the wild-type
2A-AR and
3i loop
2A-AR
with a similar concentration-response. Deletion of the 3i loop does not
accelerate agonist-elicited redistribution and down-regulation of the
2A-AR, suggesting that the 3i loop likely does not
prevent
2A-AR interactions with proteins involved in
receptor internalization. A mutant
2A-AR containing a 3i
loop of similar length but disparate sequence from wild-type
2A-AR (nonsense loop) has a surface half-life in
polarized MDCKII cells indistinguishable from wild-type receptors,
indicating that the specific amino acid sequences are not necessarily
required for basolateral retention and suggesting that the bulk of the
3i loop may be sufficient to stabilize the
2A-AR on the
basolateral surface.
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ACKNOWLEDGEMENTS |
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We thank Jeffrey R. Keefer (Department of
Pediatrics, Johns Hopkins University Medical Center, Baltimore, MD) for
the design and creation of cDNA constructs with incremental
deletions within the 2A-AR 3i loop and for help in
producing the MDCKII cell lines expressing these mutant
2A-ARs. We also thank Carol Ann Bonner for assistance
with transfection, screening, and maintenance of all cell lines used in
these experiments and for synthesis of the 125I-Rau-AzPEC
photoaffinity label. The precurser for the 125I-Rau-AzPEC
photoaffinity label was a gift from Steve Lanier (Medical University of
South Carolina, SC). We appreciate the advice from W. James Nelson
(Stanford University, CA) especially regarding the Triton X-100
extraction experiments, and we are grateful to all members of the
Limbird Lab for helpful discussions during these experiments.
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FOOTNOTES |
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* This work was funded by National Institutes of Health Grants DK43879 and HL25182 (to L. E. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by Pharmacological Sciences Training Grant GM07628 and
Molecular Biophysics Training Grant GM08320.
§ To whom correspondence should be addressed: Dept. of Pharmacology, Vanderbilt University Medical Center, MRBI 464, Nashville, TN 37232-6600. Tel.: 615-343-3538; Fax: 615-343-1084; E-mail: lee.limbird{at}mcmail.vanderbilt.edu.
2 S. W. Edwards and W. J. Nelson, unpublished observations.
3 M. H. Wilson and L. E. Limbird, submitted for publication.
4 N. L. Schramm and L. E. Limbird, submitted for publication.
5 J. R. Keefer, S. W. Edwards, and L. E. Limbird, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
2A-AR,
2A-adrenergic receptor;
MDCKII, Madin-Darby canine kidney II;
3i loop, third intracellular loop;
3i
loop
2A-AR,
aa240-359
2A-AR;
125I-Rau-AzPEC, 17-hydroxyl-20-yohimban-16-(N-4-azido-3-[125I]iodophenyl)carboxamide;
dPBS, Dulbecco's modified phosphate-buffered saline;
RIPA, radioimmune
precipitation buffer.
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