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2-Adrenergic Receptors
Department of Molecular and Cellular Physiology, Stanford University, Stanford, California 94305 (D.A.D., C.H.), Institut für Pharmakologie, Universität Würzburg, 97078 Würzburg, Germany (L.H.), Department of Pharmacology and Clinical Pharmacology, MediCity Research Laboratory, University of Turku, FIN-20520 Turku, Finland (J.K.), Biological Sciences, Stanford University, Stanford, California 94305 (F.F.), and Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, and Division of Cardiovascular Medicine, Stanford University, Stanford, California 94305 (B.K.K.)
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Summary |
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Summary
Introduction
Procedures
Results
Discussion
References
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The three 
2-adrenergic receptor subtypes
(2a, 2b, and 2c) are
highly homologous G protein-coupled receptors. These receptors all
couple to pertussis toxin-sensitive G proteins and have relatively similar pharmacological properties. To further explore functional differences between these receptors, we used immunocytochemical techniques to compare the ability of the three
2-receptor subtypes to undergo agonist-mediated
internalization. The 2a-receptor does not internalize
after agonist treatment. In contrast, we observed that the
2b-receptor is able to undergo agonist-induced internalization and seems to follow the same endosomal pathway used by
the 
2-adrenergic receptor. Attempts to examine
internalization of the 2c-receptor were complicated by
the fact that the majority of the 2c-receptor resides in
the endoplasmic reticulum and cis/medial Golgi and there is relatively
little cell surface localization. Nevertheless, we were able to detect
some internalization of the 2c-receptor after prolonged
agonist treatment. However, we observed no significant movement of
2c-receptor from the intracellular pool to the plasma
membrane during a 4-hr treatment of cells with cycloheximide,
suggesting that these cells are unable to process 2c-receptors in the same way they process the
2a or 2b subtypes.
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Introduction |
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Summary
Introduction
Procedures
Results
Discussion
References
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The 2-AR family
consists of three highly homologous subtypes: 2a,
2b, and 2c (1, 2). They belong to the
superfamily of G protein-coupled receptors and mediate the
physiological actions of the endogenous catecholamines, epinephrine and
norepinephrine. These receptors are targets for therapeutic agents for
hypertension, anesthesia, and analgesia. Although the three
2-ARs are highly homologous (50-60% identity), several
differences in their coupling to G proteins subunits and their
desensitization have been reported. In reconstitution experiments.
Kurose et al. (3) have shown that the human
2a- and 2c-ARs couple to the same G
proteins, with only slight differences in efficiency. In contrast,
using transfected cell lines, others (4) have shown that the
2c subtype (the rat RG10 receptor) preferentially
couples to Go, whereas 2a- and
2b-ARs couple to Gi (5). Initial studies indicated that 2-ARs transduce their signal through a
pertussis toxin-sensitive G protein (Gi or Go);
however, several more recent studies have demonstrated that all three
2-AR subtypes can stimulate adenylyl cyclase in
pertussis toxin-treated cells (6). The EC50 value of
agonists for stimulation of adenylyl cyclase by 2-ARs,
presumably through Gs, is higher than the EC50
value for inhibition of adenylyl cyclase through Gi (6).
The three 2-AR subtypes seem to differ in their
efficiency of coupling to Gs (6). Moreover, the capacity
and efficacy of Gs coupling by the three
2-ARs are dependent on the agonist (7) and the cell line
used to express the 2-AR subtype (6, 8). Similarly, others have shown that depending on the cell line studied,
2-ARs can inhibit or increase cellular cAMP levels,
suggesting that these differences are due to coupling to different G
proteins or isoforms of adenylate cyclase (9). Cotecchia et
al. (10) demonstrated that 2a- and
2c-ARs can stimulate phospholipase C activity. The
2a-AR has also been shown to inhibit voltage-dependent calcium currents and increase inwardly rectifying potassium currents (11); however, the ability of 2b and 2c
to couple to these ion channels has not been reported. Finally,
differences in receptor regulation have been described for the three
2-ARs. Agonist-promoted desensitization has been
observed for all 2-AR subtypes; however, the extent of
desensitization is greater for the 2a subtype (12, 13).
It has also been shown that phosphorylation of the 2a-AR is required for desensitization (14, 15).
In addition to these functional differences in signal transduction and
regulation, recent studies have identified differences in the
intracellular trafficking of this receptor family. The 2a- and 2b-ARs have been observed to
reside primarily in the plasma membrane, whereas a large portion of the
2c-AR is found in an intracellular compartment (16, 17).
We previously reported that there is no detectable agonist-induced
internalization of the 2a-AR in cells in which rapid
agonist-induced internalization of the 2-AR is seen
(16). Previously, agonist-induced internalization of the
2b and 2c-AR subtypes have not been
thoroughly characterized using immunocytochemical techniques; however,
these receptors are structurally and functionally more similar to the
2a-AR than to the 2-AR and might be
predicted to have similar trafficking properties. In this study, we
examined the steady state distribution and agonist-induced subcellular
sorting of each of the three 2-AR subtypes in the same
cell line. We observed that each subtype has distinctive trafficking
properties. The 2a- and 2b-ARs are located on the plasma membrane at steady state, whereas most of the
2c-AR is found in the endoplasmic reticulum. The
2b- and 2c-AR subtypes are internalized
into endosomes after exposure to agonists, whereas the
2a subtype is not.
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Experimental Procedures |
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Summary
Introduction
Procedures
Results
Discussion
References
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Materials. [3H]Atipamezole (50 Ci/mmol) and dexmedetomidine were kindly provided by Orion-Farmos (Turku, Finland). All other ligands and chemicals were purchased from Sigma Chemical (St. Louis, MO) unless indicated.
Plasmid construction and receptor expression in tissue culture
cells.
All cells were grown in DMEM (University of California, San
Francisco Cell Culture Facility) supplemented with 10% fetal bovine serum (Gemini Bio-Products, Calabasas, CA) and 25 µg/ml gentamicin (Boehringer-Mannheim Biochemicals, Indianapolis, IN). The three mouse
2-ARs were cloned into pBC 12BI, pREP4, or pcDNA3
expression vectors (InVitrogen, San Diego, CA) and epitope-tagged as
previously described (16). Briefly, the 12CA5 epitope (sequence
MGYPYDVPDYA) was inserted at the amino terminus of the three mouse
2-ARs, using oligonucleotide linker-adapters into the
NcoI site located at the 5
-end of the receptor coding
sequence. The sequence of the recombinant 2-ARs was
confirmed by dideoxy sequencing. To monitor the trafficking pathway of
2-ARs between intracellular organelles and the cell
surface, a double epitope tag with a thrombin cleavage site between the
two epitopes was added to the amino termini of the 2a-
and 2c-ARs (see Fig. 6). For this purpose, part of the
extracellular amino-terminal sequence of a flag epitope-tagged thrombin
receptor (18, 19) was combined with the coding sequences of the murine
2a- and 2c-ARs. The thrombin epitope tag
consists of the following domains: prolactin signal sequence, flag
epitope (italic), amino acids 53-64 of the human thrombin receptor
containing a thrombin cleavage site (dash) and the hirudin domain
epitope (bold) (20) (amino acid sequence:
MDSKGSSLLCQGVVSDYKDDDDVDATLDPR-SFLLRNPNDKY EPFWEDEEKNESGLTEYRLVSINDS). The sequence of the recombinant 2-ARs was confirmed by dideoxy sequencing and subcloned
into the pREP4 plasmid for expression in eukaryotic cells. The thrombin epitope tag can be detected by immunostaining using the monoclonal M1
antibody (Kodak IBI, Rochester, NY) recognizing the flag epitope and a
polyclonal antiserum that was raised against a peptide containing the
hirudin epitope domain of the human thrombin receptor (19). Epitope-tagged receptors were transfected by either DEAE-Dextran, calcium-phosphate precipitation, or electroporation methods. Stable cell lines were selected under 250-500 µg/ml G418 (GIBCO, Grand Island, NY) or 200 IU/ml hygromycin (Boehringer-Mannheim) in the media.
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Radioligand binding assay. For binding studies, COS-7 cells were transiently transfected using the DEAE-Dextran method, and cell membranes were harvested 3 days after transfection as previously described (21). Binding experiments were performed in 500-µl volumes of binding buffer (75 mM Tris, 12.5 mM MgCl2, and 1 mM EDTA, pH 7.4) for 90 min at room temperature. The bound radioactivity was separated from free by filtration through GF/C filters and washed three times with 5 ml of ice-cold binding buffer using a Brandel cell harvester. Saturation isotherms were performed by incubating the membranes with varying concentrations of [3H]atipamezole. Nonspecific binding was determined by the addition of 100 µM yohimbine. Competition binding experiments were carried out by incubating membranes with varying concentrations of competing ligand and 2 nM [3H]atipamezole. Nonspecific binding was determined by the addition of 100 µM yohimbine. Equilibrium dissociation constants were determined from saturation isotherms and competition curves using GraphPAD software (San Diego, CA). All binding experiments were done in duplicate and repeated at least three times.
Production of antisera.
The antigenic epitopes selected for
the polyclonal antibodies were the carboxyl termini of each
2-AR. The antigenic peptides were synthesized and
MBS-coupled to thyroglobulin, purified, and injected into New Zealand
White rabbits. Polyclonal sera directed against the 2b
and 2c epitopes were affinity-purified over peptide coupled to epoxy-activated Sepharose columns and eluted with either 3 M KSCN, pH 7.4, or 20 mM glycine, pH 3.0. The
three polyclonal antisera are specific for their respective
2-AR subtype and do not cross-react with other
2-AR subtypes at the dilutions used (2a,
1:500; 2b and 2c affinity-purified
antisera, 1:100; data not shown). The previously described
2 antibody was kindly provided by Dr. M. Von Zastrow
(University of California, San Francisco).
Immunocytochemical studies and techniques. Receptor trafficking and subcellular distribution were examined using indirect immunocytochemical staining. Nonspecific binding was blocked with blotto (5% nonfat dry milk in 50 mM Tris, pH 7.6) for 45 min. For staining in permeabilized cells, nonionic detergent (Nonidet P-40, Sigma) was added to blotto incubations to a final concentration of 0.2%. Cells were stained using 12CA5 hemagglutinin antibody (Berkeley Antibody Co., Berkeley, CA) monoclonal antibody (at 1:500 in blotto). For nonpermeabilized studies, receptors present on the plasma membrane were stained by incubation of live cells grown on coverslips at 37° with 6% CO2 for 60 min with 12CA5 monoclonal antibody at 1:500 in DMEM supplemented with 10% fetal calf serum. After a 60-min incubation, cells were washed with cold PBS and fixed with 4% formaldehyde for 30 min and then rinsed three times in room-temperature PBS. Nonpermeabilized immunocytochemistry was also performed by growing cells on coverslips for 2 or 3 days and then fixing them for 30 min in 4% formaldehyde and deleting detergent from the blotto. Secondary antibodies were either fluorescein-conjugated (fluorescein isothiocyanate) or Texas red-conjugated directed against the species of primary antibody used (Jackson ImmunoResearch, West Grove, PA).
One day after transient transfections, cells were trypsinized off tissue culture dishes and replated onto sterilized cover slips. For steady state studies, cells were grown on coverslips for 2 days and then fixed in 4% formaldehyde or 4% paraformaldehyde for 45 min, followed by three washes with room-temperature PBS. For adrenergic agonist treatment studies, after 2 days' growth on coverslips, cells were incubated for 30 min at 37° with 6% CO2 in either serum-free DMEM (control) or agonist in serum-free DMEM. After incubations, the medium was aspirated, and cells were fixed with 4% formaldehyde for 45 min at room temperature and then washed three times with room-temperature PBS. Studies involving basal trafficking of2c thrombin epitope-tagged receptors were carried out in
the absence of agonist after a 30-min treatment with 5 nM
thrombin to cleave off the M1 epitope from receptors present on the
plasma membrane. The effect of agonist stimulation on 2c
thrombin epitope-tagged receptor trafficking was examined by exposing
cells to 10 µM epinephrine in DMEM with 40 mM
HEPES, pH 7.4, and 10 µM cycloheximide to inhibit
de novo protein synthesis for 30 min at 37°. Steady state
trafficking of the cytosolic 2c thrombin epitope-tagged
receptor pool was also studied in the absence of de novo
protein synthesis by treated cells with 10 µM
cycloheximide for 4 hr. During inhibition of protein synthesis, cells
were concomitantly treated with 5 nM thrombin to remove the
M1 epitope from all receptors that were translocated to the plasma
membrane.
For studies using thrombin epitope-tagged 2-ARs, after 2 days of growth on coverslips, cells were washed three times with PBS
and incubated at 37° with 6% CO2 in DMEM with 40 mM HEPES, pH 7.4, for 30-45 min. After various treatments
as outlined above, cells were rinsed three time with PBS and fixed
using cold methanol (
20°) for 5 min. After fixation, cells were
washed three times with PBS supplemented with
Ca2+/Mg2+ over a 5-min period. To block
nonspecific binding, 5% nonfat dry milk in PBS with
Ca2+/Mg2+ supplemented with 50 mM
HEPES, pH 7.4, was applied to cells for a period of 30-45 min.
Selective dual antibody labeling of cells was performed in the blocking
agent applied at room temperature for 1 hr with either the monoclonal
M1 antibody (Kodak-IBI) at 1:500 or a polyclonal antibody to the
hirudin epitope (Dr. S. Coughlin, University of California, San
Francisco, CA) at 1:1000.
Colocalization immunocytochemical studies were performed on either the
wild-type 2c or 12CA5 epitope-tagged
2c-AR and a variety of antibodies selective to various
intracellular compartments. Cells were costained with either the 12CA5
monoclonal antibody or affinity-purified polyclonal antibody directed
against the wild-type 2c-AR as described above and one
of the following: lgp120 (Dr. S. Green, University of Virginia,
Charlottesville, VA) at 1:1000; cation-independent M6PR (Dr. William
Brown, Cornell University, Ithaca, NY) antibody at 1:500; mannosidase
II (Berkeley Antibody, Berkeley, CA) at 1:500; calnexin (StressGen
Biotechnologies, Victoria, BC, Canada) at 1:750; or BiP (GRP-78;
Affinity Bioreagents, Nashanic Station, NJ) at 1:500. After primary
antibody labeling, the cells were rinsed three times with PBS
supplemented with Ca2+/Mg2+. Blocking agent was
again applied for 30 min, and the aforementioned conjugated secondary
antibodies were applied in blocking agent for 1 hr at room temperature.
Cell were subsequently rinsed three times with PBS supplemented with
Ca2+/Mg2+, and the specimens were mounted on
glass slides for viewing.
Conventional immunofluorescence microscopy was performed using a Zeiss
Axiophot microscope with a Zeiss Neofluar 63×/1.25 or Zeiss Neofluar
100×/1.3 objective. Confocal microscopy was performed using a Zeiss
Axioskop microscope and a Sarastro Phoibos 1000 instrument with either
a Zeiss Apochromat 40×/1.0 or Zeiss Apochromat 63×/NA 1.4 objective,
a 50-µM confocal pinhole, and optical filters set as
described in the manufacturer's instructions for fluorescein
isothiocyanate/Texas red imaging.
ELISA. For quantification of receptor internalization, ELISA assays were performed. Briefly, nonclonal stable HEK 293 cell lines were plated onto 24-well tissue culture dishes. To improve adhesion of this cell line to plastic, the wells were treated for 30 min with 0.0005% poly-L-lysine in PBS immediately before plating out ~10 × 104 cells/well. At ~48 hr later, when cells were ~75% confluent, they were incubated for 30 min at 37° with 6% CO2 in serum-free DMEM or 10 µM norepinephrine in serum-free DMEM. After incubations, the medium was aspirated, and cells were fixed with 4% paraformaldehyde for 5 min at room temperature. Wells were washed three times with room-temperature PBS. Nonspecific binding was blocked with 1% BSA for 45 min with occasional gentle shaking. The 12CA5 antibody was diluted to 1:1000 in 1% BSA for 60 min, followed by three gentle PBS washes. Cells were briefly reblocked with 1% BSA for 15 min and then incubated with anti-mouse conjugated alkaline phosphatase (BioRad, Richmond, CA) diluted 1:1000 in 1% BSA for 60 min with occasional gentle shaking. Wells were then washed three times with PBS, and a colorimetric alkaline phosphate substrate was added. Plates were continuously but gently shaken (40 rotations/min) until an adequate color change had occurred, at which time a 100-µl sample was taken for colorimetric readings. Nontransfected cells were studied concurrently to determine background. All experiments were done in triplicate.
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Results |
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Summary
Introduction
Procedures
Results
Discussion
References
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Immunocytochemical techniques were used to examine the
distribution of 2-AR subtypes expressed in COS 7, HEK
293, MDCK, NRK and Rat1 fibroblast cell lines. Subtype-specific rabbit
polyclonal antibodies were prepared against a carboxyl-terminal peptide
as described in Experimental Procedures. The specificity of these antibodies was verified by staining untransfected COS 7 cells or COS 7 cells transfected with each of the 2-AR subtypes (data not shown). In addition, we used receptors modified with an
amino-terminal 12CA5 epitope, which is recognized by a commercially
available monoclonal antibody. The amino-terminal tag permits
examination of cell surface receptor density in nonpermeabilized cells.
We previously observed that the use of this epitope does not alter the
trafficking of the 2-AR (16). Moreover, the distribution and trafficking of epitope tagged 2-ARs (examined with
either 12CA5 monoclonal antibody or polyclonal subtype-specific
antibody) are indistinguishable from those of nontagged receptors
(examined with polyclonal antibody; data not shown). Binding affinity
for the antagonist atipamezole, the endogenous catecholamines
epinephrine and norepinephrine, and the specific 2
agonist dexmedetomidine are also comparable between wild-type and
epitope-tagged 2-ARs (Table 1).
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Fig. 1 shows the distribution of the three
2-AR subtypes in HEK 293 cells at steady state and after
agonist treatment. It can be seen that 2a and
2b subtypes are predominantly localized in the plasma
membrane at steady state, whereas a large portion of
2c-AR is found in an intracellular compartment. After
agonist treatment (10 µM norepinephrine) for 30 min at
37°, the 2b-AR is internalized, but the
2a subtype remains in the plasma membrane. This analysis
does not allow us to determine whether the 2c-AR is
internalized because of the large background of 2-AR
already in an intracellular membrane compartment. This intracellular
pool of 2c-AR is be further characterized below.
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To further investigate differences in agonist-mediated internalization,
12CA5 epitope-tagged 2b-AR was coexpressed with a nontagged 2a-AR, allowing us to examine differences in
trafficking between these two subtypes in the same cell. The
2a distribution was monitored with the polyclonal
2a subtype-specific antibody, and the epitope-tagged
2b-AR distribution was monitored with a monoclonal
antibody to the 12CA5 epitope. At steady state, both receptors are
found in the plasma membrane of the same cell (Fig. 2, A
and B). After exposure to 10 µM norepinephrine for 30 min, only the 2b-AR is internalized (Fig. 2, C and D).
Similar treatment with 10 nM of the nonselective
2 agonist dexmedetomidine also selectively internalized
the 2b-AR in cotransfected cells (Fig. 2, E and F).
Additional agonist treatment paradigms with the 2a-AR (30-min incubations with 10 µM epinephrine or 100 µM norepinephrine or 
4-hr incubation with 10 µM norepinephrine) resulted in similar findings (data not
shown).
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The 2b- and 2-ARs internalize by a
similar mechanism.
The mechanism of agonist-mediated
internalization of the 2-AR has been well characterized.
We therefore coexpressed the epitope-tagged 2b-AR along
with the 2-AR and examined the distribution of the two
receptors after agonist treatment. At steady state, both receptors are
on the plasma membrane (Fig. 3, A and B). Only the
2-AR is internalized in cells exposed to the -AR
agonist isoproterenol (Fig. 3, C and D), and only the
2b-AR is internalized in cells exposed to the
2-AR agonist dexmedetomidine (Fig. 3, E and F). Using
confocal microscopy, we demonstrated that both receptors are
internalized by exposure to the common agonist norepinephrine and seem
to reside in the same endocytic vesicles (Fig. 3, G and H).
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2a-AR after agonist treatment; however, it may not be possible to detect internalized receptor by
fluorescence microscopy if it is not concentrated in endosomes. To
investigate this possibility, we used an ELISA to monitor the loss of
cell surface receptor rather than accumulation of intracellular receptor protein after agonist activation. Epitope-tagged
2a-and 2b-ARs were expressed in HEK 293 cells, and the density of receptors was monitored in nonpermeabilized
cells with the 12CA5 antibody. At steady state, the cell surface
expression of the 2a- and 2b-ARs was
comparable. After treatment with norepinephrine for 30 min, there was a
loss of 31 ± 2.4% (mean ± standard error) of
2b-ARs and a loss of 7 ± 1.4% of
2a-ARs. These results confirm the difference between
2a- and 2b-ARs; however, they suggest
that there is a small but significant agonist-induced internalization
of the 2a-AR, possibly by a nonendosomal mechanism.
The 2c-AR is found in the endoplasmic reticulum in
Rat1 fibroblast cells.
Although there is some plasma staining, the
2c-AR seems to reside primarily in an intracellular
compartment in HEK 293 cells (Fig. 1C). To further study this, we
expressed the 2a- and 2c-ARs in a variety
of cell lines. Similar to studies reported by Wozniak et
al., (17) when expressed in a MDCK II cell line, the abundance of
2c-AR was localized in an intracellular compartment,
whereas the 2a-AR was targeted to the basolateral plasma
membrane (Fig. 4, A and B). When the 12CA5
epitope-tagged 2c-AR was transiently cotransfected in an
HEK-293 cell line stably expressing wild-type 2a-AR, the
2c subtype was also localized to an intracellular compartment (Fig. 4, C and D). Coexpression of the 2c
did not alter the trafficking of the 2a-AR subtype. We
attempted to quantify the amount of 12CA5 epitope-tagged
2c-AR in the plasma membrane using the ELISA method
discussed above. In contrast to the 2a- and
2b-AR subtypes, the signal generated from
2c-AR transfected cells was not significantly different
from that of untransfected cells. Thus, although we were able to detect
a small amount of 12CA5 epitope-tagged 2c-AR in the
plasma membrane of nonpermeabilized cells by the sensitive technique of
immunofluorescence microscopy, the amount was too small to quantify by
ELISA.
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2c-ARs was difficult to assess in HEK 293 cells because
of their relatively small size. To further investigate the subcellular distribution of 2c-ARs, we studied Rat1 fibroblast and
NRK cells, which have relatively large cytoplasms. The distribution of
the receptor in Rat1 cells colocalizes with both BiP and mannosidase II
(Fig. 5, A-D), suggesting that the 2c-AR
is found primarily in the endoplasmic reticulum and cis/medial Golgi.
No overlap was observed with markers for trans-Golgi network (M6PR),
endosomes (M6PR), or lysosomes (lgp120) (Fig. 5, E-H). We observed a
similar distribution of the 2c-AR in NRK cells (data not
shown).
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2c-AR
resides in the endoplasmic reticulum suggests several possibilities. This receptor may be improperly processed, or the cell may lack an
important chaperone protein and therefore the receptor is retained in
the endoplasmic reticulum. It is also possible that the receptor is
processed slowly and undergoes a relatively rapid turnover at the
plasma membrane. However, similar half-lives have been reported for all
three 2-AR subtypes in MDCK cells (17). Finally, it is
possible that the receptor is cycling between the plasma membrane and
cytosolic pool. To examine these possibilities, we constructed a
modified form of the 2c-AR that would allow us to
identify receptor that has been delivered to the plasma membrane. Fig.
6 shows the amino-terminal sequence of the modified
2c-AR. An M1 flag epitope and a thrombin cleavage site
were added to the amino terminus. Cleavage of the receptor by thrombin
present in the media results in loss of the flag epitope (which can be recognized by the commercially available M1 monoclonal antibody), whereas the hirudin binding domain of the thrombin cleavage site remains and can be recognized by a polyclonal antibody. Therefore, receptors that are on the plasma membrane are susceptible to thrombin cleavage. These thrombin-cleaved receptors will stain only with the
polyclonal antibody to the hirudin epitope, not with the M1 flag
antibody.
Fig. 7 demonstrates that the amino-terminal thrombin
epitope does not effect the distribution of the 2c-AR.
In permeabilized cells, most of the 2c-AR is found in an
intracellular compartment (Fig. 7, E-H); however, cell surface
expression can be demonstrated in nonpermeabilized cells (Fig. 7,
A-D). Exposure of the cell to thrombin results in loss of cell surface
receptor staining by the M1 flag antibody (Fig. 7, C and G); however,
the receptor is still recognized by the antibody to the hirudin epitope
(Fig. 7, D and H). To investigate the turnover of 2c-AR,
cells were exposed to cycloheximide to block protein synthesis and
incubated in the presence of epinephrine and thrombin for 4 hr (Fig.
8, A-F). Movement of receptor from the intracellular
pool to the cell surface would result in cleavage by thrombin and a
loss of staining by M1 flag antibody. If these receptors were then
internalized and recycled through the endoplasmic reticulum or
distributed to lysosomes, they would be recognized by staining with the
polyclonal antibody to the hirudin epitope but not the M1 flag
antibody. As seen in Fig. 8, there is no significant loss of
intracellular staining by the M1 antibody, indicating that the
intracellular pool of receptor is stable. Moreover, using the cleavable
thrombin/flag epitope, we were able to determine that a small amount of
2c-AR was internalized from the plasma membrane with
agonist treatment (Fig. 8, E and F). This internalized receptor stains
with the hirudin antibody but not the M1 antibody (Fig. 8, E and F,
arrows).
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2a-AR was similarly
tagged with the same thrombin-cleavable epitope (Fig.
9). At steady state, most of the receptor is susceptible
to cleavage with thrombin, indicating that the majority of
2a-AR is localized in the plasma membrane (Fig. 9, C and
D). A small amount of intracellular receptor (M1 flag antibody
staining) is observed before but not after exposure to cycloheximide
and thrombin for 4 hr (Fig. 9, E and F). This indicates that within 4 hr, all of the 2a-AR in the biosynthetic pathway has
been processed and inserted in the plasma membrane.
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2c-AR in endosomes may
not be as pronounced as that observed for the 2b-AR
because of the large amount of 2c-AR in the
intracellular compartment at steady state and the comparatively small
amount of 2c-AR in the plasma membrane (Fig. 8F). A more
sensitive technique for identifying agonist-induced receptor
internalization is shown in Fig. 10. Living, nonpermeabilized cells expressing M1 flag-tagged 2c-AR
are first labeled with M1 flag monoclonal antibody and then washed and
incubated in the presence or absence of agonist for 30 min. Cells were
then fixed, permeabilized, and stained with secondary antibody. This approach allowed us to selectively examine only 2c-ARs
that are in the plasma membrane. No internalization was detected in
control cells (Fig. 10A); however, after agonist treatment, receptor
was observed in an intracellular compartment (Fig. 10C, arrow). The intracellular receptor surrounds the nucleus, creating a nuclear shadow
in agonist-treated cells (Fig. 10C), whereas no nuclear shadow is
observed in control cells (Fig. 10A). The internalized 2c-AR colocalized with the M6PR that is present in the
trans-Golgi network and endosomes (Fig. 10D).
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| |
Discussion |
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Summary
Introduction
Procedures
Results
Discussion
References
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In this study, we report differences in steady state targeting and
agonist-induced internalization of the three 2-AR
subtypes. The 2b-AR behaves more like the
2-AR than the 2a-AR. This is particularly
interesting in light of the high degree of identity shared by
2a and 2b (55%, 2a versus
2b; 21%, 2 versus 2b). Moreover, we extend previous observations regarding the intracellular distribution of the 2c-AR subtype. The roles of
intracellular targeting and trafficking in signal transduction are not
well understood; however, agonist-induced internalization has been shown to play a role in receptor regulation. Studies have implicated agonist-induced internalization in the process of resensitization. Blocking internalization by selective receptor mutations or treatment of cells with concanavalin A or sucrose prevents dephosphorylation of
the receptor (22). Recent studies have suggested that binding of
-arrestin to the 2-AR after G protein-coupled
receptor kinase phosphorylation is necessary for internalization (23).
Of interest, the 2a-AR has been shown to undergo
phosphorylation by G protein-coupled receptor kinase (14, 15). The
inefficient agonist-induced internalization in the 2a-AR
may suggest that this receptor does not bind to -arrestin. These
results may also suggest that the 2a-AR is tethered to
the plasma membrane and is prevented from undergoing internalization
even when phosphorylated and bound to -arrestin. This may lead to a
difference in the rate of resensitization and therefore account for the
more extensive desensitization observed for the 2a-AR.
We observed a small amount of agonist-induced internalization of
2a using a sensitive ELISA technique for quantitative
changes in cell surface density of receptor antigen. However, there was no significant accumulation of receptor in endosomes that could be
detected by immunocytochemistry. This suggests that the small amount of
2a internalization may be occurring by a different mechanism than that used for internalization of 2b- and
2-ARs. The inefficient agonist-induced internalization
of 2a relative to 2b is in contrast to
previous reports (12, 24); Eason et al. found that a 30-min
exposure to agonist resulted in 35% sequestration of the
2b-AR and 26% sequestration in the
2a-AR. In those studies, CHO cells were used, and
internalization was assayed by ligand binding techniques that used
hydrophilic agonists to distinguish between cell surface and
intracellular receptor. Several possibilities might account for these
different results. First, CHO cells may express other proteins that are
critical for agonist-induced sequestration in the 2a-AR.
Although 2b- and 2-ARs undergo
agonist-promoted receptor internalization in HEK 293 cells, it is
possible that this cell line lacks a component necessary for
sequestration of 2a-ARs that is present in CHO cells.
Another possible explanation is that different methods were used to
assess sequestration in the two studies. In the current study, we used
immunocytochemical methods to examine receptor internalization. These
techniques are not influenced by the state of receptor/G protein
coupling. This may not be the case when 2-AR
internalization is examined by using a hydrophilic agonist to quantify
cell surface receptor density. It is possible that agonist treatment
could reduce agonist binding of desensitized plasma membrane
2a-AR even in the absence of receptor internalization.
The physiological or functional significance of the large intracellular
pool of the 2c-AR subtype is not known at this time. Both we and others have reported this unique intracellular distribution in a variety of cell lines, including COS-7, HEK 293, MDCK II, Rat1
fibroblasts, and NRK (16, 17). Moreover, this subcellular distribution
is not a species-specific idiosyncrasy to the mouse 2c-AR because the wild-type human receptor shows similar
intracellular localization when transfected in COS-7 or HEK-293 cells
(data not shown). Wozniak and Limbird (17) have shown that in MDCK II
cells, this distribution is independent of receptor expression levels;
consistent with our findings that a relatively small proportion of the
2c-AR is targeted to the plasma membrane (Fig. 4, B-D), the authors showed that this receptor is targeted directly to the
basolateral membrane of MDCK II cells. It is also noteworthy that when
2c- and 2a-ARs are coexpressed in the
same cell, these differences in receptor sorting are maintained (Fig.
4, C and D). The 2c-AR may require a specific chaperone
protein for efficient targeting to the plasma membrane, or it may
require a specific protein that anchors the receptor to the
cytoskeleton. In the latter case, one would expect the receptor to
traffic normally to the plasma membrane but not be retained. However,
this mechanism is not consistent with the results of experiments using
the thrombin/flag epitope-tagged receptor (Fig. 8) that demonstrate no
detectable cycling of receptor between intracellular compartment and
the plasma membrane.
We noted that plasma membrane 2c-AR was more easily
visualized in NRK cells (Figs. 7, 8, and 10) than in HEK 293 cells. It may be that NRK cells are somewhat better in translocating the 2c-AR to the plasma membrane than are HEK 293 cells;
however, this may also be due to the fact that the NRK cells are larger and very flat, permitting better visualization of a larger amount of
plasma membrane in a single plane of focus.
We were unable to detect agonist-induced internalization of the
2c-AR using conventional immunocytochemical techniques
(Fig. 1). This is in part due to the large preexisting pool of
intracellular 2c-AR. However, using a cleavable
thrombin/flag epitope on the amino terminus of the 2c-AR
allowed us to identify that some receptor is internalized with
prolonged agonist treatment (Fig. 8, E and F). This was confirmed by
prelabeling the amino-terminal M1 flag epitope of plasma membrane
2c-AR with antibody before agonist treatment (Fig. 10).
The differences in intracellular trafficking observed for the
2-AR subtypes is somewhat unexpected considering the
high degree of amino acid identity and the functional similarity with
respect to ligand-binding properties and G protein coupling. The
functional significance of differences in receptor trafficking is
unknown but may be more important in vivo in highly
differentiated cells. It has been proposed that receptor subtypes may
be targeted to specific plasma membrane microdomains in which receptors
are found in close proximity with specific G proteins and effector
enzymes (25). Thus, unique trafficking behavior of receptors may enable receptors to couple to specific effector systems.
Summary.
We reported that in several cell lines, the three
2-ARs display unique patterns of subcellular
distribution and sorting. At steady state, the 2a- and
2b-AR subtypes are localized in the plasma membrane.
Agonist treatment induces internalization of the 2b-AR
into endosomes, presumably using the same cellular machinery as the
2-AR, whereas no accumulation of 2a-AR in
endosomes could be detected after agonist stimulation. In contrast, at
steady state the 2c-AR is targeted to the plasma
membrane; however, a significant proportion of this receptor is
localized in an intracellular pool that has not yet been delivered to
the plasma membrane. Through colocalization studies, we determined that
the intracellular pool of 2c-ARs is predominantly
localized in the endoplasmic reticulum and cis/medial Golgi.
Agonist treatment of the 2c-AR results in some receptor
internalization from the plasma membrane. The functional significance
of the observed differences in subcellular sorting of the three
relatively highly homologous 2-ARs remains to be
established.
| |
Footnotes |
|---|
Received October 28, 1996; Accepted February 10, 1997
Send reprint requests to: Dr. Brian K. Kobilka, B159 Beckman Center, Stanford University, Stanford, CA 94305. E-mail: kobilka{at}cmgm.stanford.edu
| |
Abbreviations |
|---|
AR, adrenergic receptor; HEK, human embryonic kidney; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; M6PR, mannose-6-phosphate receptor; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; CHO, Chinese hamster ovary.
| |
References |
|---|
|
Summary
Introduction
Procedures
Results
Discussion
References
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M. J. Orsini, J.-L. Parent, S. J. Mundell, and J. L. Benovic Trafficking of the HIV Coreceptor CXCR4. ROLE OF ARRESTINS AND IDENTIFICATION OF RESIDUES IN THE C-TERMINAL TAIL THAT MEDIATE RECEPTOR INTERNALIZATION J. Biol. Chem., October 22, 1999; 274(43): 31076 - 31086. [Abstract] [Full Text] [PDF]
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N. L. Schramm and L. E. Limbird Stimulation of Mitogen-activated Protein Kinase by G Protein-coupled alpha 2-Adrenergic Receptors Does Not Require Agonist-elicited Endocytosis J. Biol. Chem., August 27, 1999; 274(35): 24935 - 24940. [Abstract] [Full Text] [PDF]
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S. W. Edwards and L. E. Limbird Role for the Third Intracellular Loop in Cell Surface Stabilization of the alpha 2A-Adrenergic Receptor J. Biol. Chem., June 4, 1999; 274(23): 16331 - 16336. [Abstract] [Full Text] [PDF]
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W. Zhang, V. Klimek, J. T. Farley, M.-Y. Zhu, and G. A. Ordway alpha 2C Adrenoceptors Inhibit Adenylyl Cyclase in Mouse Striatum: Potential Activation by Dopamine J. Pharmacol. Exp. Ther., June 1, 1999; 289(3): 1286 - 1292. [Abstract] [Full Text]
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L. Prezeau, J. G. Richman, S. W. Edwards, and L. E. Limbird The zeta Isoform of 14-3-3 Proteins Interacts with the Third Intracellular Loop of Different alpha 2-Adrenergic Receptor Subtypes J. Biol. Chem., May 7, 1999; 274(19): 13462 - 13469. [Abstract] [Full Text] [PDF]
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J. L. DeGraff, A. W. Gagnon, J. L. Benovic, and M. J. Orsini Role of Arrestins in Endocytosis and Signaling of alpha 2-Adrenergic Receptor Subtypes J. Biol. Chem., April 16, 1999; 274(16): 11253 - 11259. [Abstract] [Full Text] [PDF]
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J.-L. Parent, P. Labrecque, M. J. Orsini, and J. L. Benovic Internalization of the TXA2 Receptor alpha and beta Isoforms. ROLE OF THE DIFFERENTIALLY SPLICED COOH TERMINUS IN AGONIST-PROMOTED RECEPTOR INTERNALIZATION J. Biol. Chem., March 26, 1999; 274(13): 8941 - 8948. [Abstract] [Full Text] [PDF]
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 R. G. Vickery and M. von Zastrow Distinct Dynamin-dependent and -independent Mechanisms Target Structurally Homologous Dopamine Receptors to Different Endocytic Membranes J. Cell Biol., January 11, 1999; 144(1): 31 - 43. [Abstract] [Full Text] [PDF]
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M. J. Orsini and J. L. Benovic Characterization of Dominant Negative Arrestins That Inhibit beta 2-Adrenergic Receptor Internalization by Distinct Mechanisms J. Biol. Chem., December 18, 1998; 273(51): 34616 - 34622. [Abstract] [Full Text] [PDF]
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 M. Vrecl, L. Anderson, A. Hanyaloglu, A. M. McGregor, A. D. Groarke, G. Milligan, P. L. Taylor, and K. A. Eidne Agonist-Induced Endocytosis and Recycling of the Gonadotropin-Releasing Hormone Receptor: Effect of {beta}-Arrestin on Internalization Kinetics Mol. Endocrinol., December 1, 1998; 12(12): 1818 - 1829. [Abstract] [Full Text]
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T. Awaji, A. Hirasawa, M. Kataoka, H. Shinoura, Y. Nakayama, T. Sugawara, S.-i. Izumi, and G. Tsujimoto Real-Time Optical Monitoring of Ligand-Mediated Internalization of {alpha}1b-Adrenoceptor with Green Fluorescent Protein Mol. Endocrinol., August 1, 1998; 12(8): 1099 - 1111. [Abstract] [Full Text]
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O. Vogler, G. S. Bogatkewitsch, C. Wriske, P. Krummenerl, K. H. Jakobs, and C. J. van Koppen Receptor Subtype-specific Regulation of Muscarinic Acetylcholine Receptor Sequestration by Dynamin. DISTINCT SEQUESTRATION OF m2 RECEPTORS J. Biol. Chem., May 15, 1998; 273(20): 12155 - 12160. [Abstract] [Full Text] [PDF]
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 J. G. Richman and J. W. Regan alpha 2-Adrenergic receptors increase cell migration and decrease F-actin labeling in rat aortic smooth muscle cells Am J Physiol Cell Physiol, March 1, 1998; 274(3): C654 - C662. [Abstract] [Full Text] [PDF]
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C. Saunders and L. E. Limbird Disruption of Microtubules Reveals Two Independent Apical Targeting Mechanisms for G-protein-coupled Receptors in Polarized Renal Epithelial Cells J. Biol. Chem., July 25, 1997; 272(30): 19035 - 19045. [Abstract] [Full Text] [PDF]
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C. M. Hurt, F. Y. Feng, and B. Kobilka Cell-type Specific Targeting of the alpha 2c-Adrenoceptor. EVIDENCE FOR THE ORGANIZATION OF RECEPTOR MICRODOMAINS DURING NEURONAL DIFFERENTIATION OF PC12 CELLS J. Biol. Chem., November 3, 2000; 275(45): 35424 - 35431. [Abstract] [Full Text] [PDF]
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A.-L. Matharu, S. J. Mundell, J. L. Benovic, and E. Kelly Rapid Agonist-induced Desensitization and Internalization of the A2B Adenosine Receptor Is Mediated by a Serine Residue Close to the COOH Terminus J. Biol. Chem., August 3, 2001; 276(32): 30199 - 30207. [Abstract] [Full Text] [PDF]
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J. G. Richman, A. E. Brady, Q. Wang, J. L. Hensel, R. J. Colbran, and L. E. Limbird Agonist-regulated Interaction between alpha 2-Adrenergic Receptors and Spinophilin J. Biol. Chem., April 27, 2001; 276(18): 15003 - 15008. [Abstract] [Full Text] [PDF]
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K. L. Pierce, A. Tohgo, S. Ahn, M. E. Field, L. M. Luttrell, and R. J. Lefkowitz Epidermal Growth Factor (EGF) Receptor-dependent ERK Activation by G Protein-coupled Receptors. A CO-CULTURE SYSTEM FOR IDENTIFYING INTERMEDIATES UPSTREAM AND DOWNSTREAM OF HEPARIN-BINDING EGF SHEDDING J. Biol. Chem., June 15, 2001; 276(25): 23155 - 23160. [Abstract] [Full Text] [PDF]
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