From the Departments of Biochemistry,
§ Psychiatry, and
Neurosciences, Case Western Reserve
University Medical School, Cleveland, Ohio 44106 and the
¶ Kimmel Cancer Center, Thomas Jefferson University Medical
School, Philadelphia, Pennsylvania 19107
Received for publication, August 2, 2000, and in revised form, November 6, 2000
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ABSTRACT |
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5-Hydroxytryptamine 2A
(5-HT2A) receptors, a major site of action of
clozapine and other atypical antipsychotic medications, are,
paradoxically, internalized in vitro and in
vivo by antagonists and agonists. The mechanisms responsible for
this paradoxical regulation of 5-HT2A receptors are
unknown. In this study, the arrestin and dynamin dependences of
agonist- and antagonist-mediated internalization were investigated in
live cells using green fluorescent protein (GFP)-tagged
5-HT2A receptors (SR2-GFP). Preliminary experiments indicated that GFP tagging of 5-HT2A receptors had no
effect on either the binding affinities of several ligands or agonist
efficacy. Likewise, both the native receptor and SR2-GFP were
internalized via endosomes in vitro. Experiments with a
dynamin dominant-negative mutant (dynamin K44A) demonstrated that both
agonist- and antagonist-induced internalization were
dynamin-dependent. By contrast, both the agonist- and
antagonist-induced internalization of SR2-GFP were insensitive to three
different arrestin (Arr) dominant-negative mutants (Arr-2 V53D,
Arr-2-(319-418), and Arr-3-(284-409)). Interestingly, 5-HT2A receptor activation by agonists, but not
antagonists, induced greater Arr-3 than Arr-2 translocation to the
plasma membrane. Importantly, the agonist-induced internalization of
5-HT2A receptors was accompanied by differential sorting of
Arr-2, Arr-3, and 5-HT2A receptors into distinct plasma
membrane and intracellular compartments. The agonist-induced
redistribution of Arr-2 and Arr-3 into intracellular vesicles and
plasma membrane compartments distinct from those involved in
5-HT2A receptor internalization implies novel roles for
Arr-2 and Arr-3 independent of 5-HT2A receptor
internalization and desensitization.
5-Hydroxytryptamine 2A
(5-HT2A)1
receptors are essential for the actions of 5-HT in a wide variety of
physiological processes, including vascular and nonvascular smooth
muscle contraction and platelet aggregation (1). 5-HT2A
receptors also mediate the actions of hallucinogens such as lysergic
acid diethylamide, 4-iodo-2,5-dimethoxyamphetamine, and
N,N-dimethyltryptamine by activating
5-HT2A receptors located on cortical pyramidal neurons (2,
3). Additionally, atypical antipsychotic drugs such as clozapine,
risperidone, and olanzapine may mediate some of their unique actions
via antagonism of 5-HT2A receptors (4).
For many years, it has been clear that a large number of
G-protein-coupled receptors (GPCRs), including opiate receptors (5-7) and 5-HT2A receptors are subject to unique modes of regulation
compared with other GPCRs. Thus, as previously reported, both agonists and antagonists induce down-regulation and internalization of 5-HT2A receptors in vitro and in vivo
(14-16). Since antagonist binding does not lead to receptor
activation, it is difficult to imagine how antagonists could induce
either internalization or arrestin binding, assuming that the process
delineated for Accordingly, in this set of studies, we examined the arrestin and
dynamin sensitivities for the agonist- and antagonist-mediated internalization of 5-HT2A receptors in vitro. As
we report, both the agonist- and antagonist-induced internalization of
5-HT2A receptors are dynamin-dependent and
arrestin-independent. Furthermore, we report that even though the
agonist-induced internalization is arrestin-independent, activation of
5-HT2A receptors leads to a redistribution of Constructs and Antibodies--
A green fluorescent protein
(GFP)-tagged rat 5-HT2A fusion protein (SR2-GFP) was
constructed by amplifying the entire coding region of the
5-HT2A receptor including 15 base pairs of the
5'-untranslated region in-frame using Pfu polymerase and
subcloning it into the vector pEGFP-N2 (CLONTECH).
Clones containing inserts in the appropriate orientation were verified
by automated sequencing (Cleveland Genomics, Inc.) of the entire
insert. A rat arrestin-2 dominant-negative mutant (Arr-2 V53D) was
provided by Dr. Marc Caron (Duke University); the carboxyl-terminal
arrestin-2 (Arr-2(319-418); CT-Arr-2) and arrestin-3
(Arr-3(284-409); CT-Arr-3) dominant-negative mutants were prepared as
previously detailed, as were Arr-2 and Arr-3 full-length eukaryotic
expression vectors (12, 17, 18). Mono- and polyclonal anti-GFP
antibodies were from CLONTECH; polyclonal 5-HT2A receptor amino terminus-specific and
arrestin-3-specific antibodies have been previously described (14, 17).
A rabbit polyclonal CT-Arr-2-specific antibody was generated using a
glutathione S-transferase fusion protein containing residues
357-418 of bovine arrestin-2 as antigen. The rat 5-HT2A
receptor was FLAG epitope-tagged on the amino-terminal extracellular
domain by subcloning into pCMV-TAG2B (Stratagene) with the
introduction of a consensus Factor Xa cleavage site (IEGR)
between the FLAG epitope and the amino terminus of 5-HT2A
to yield FLAG-2A. In brief, polymerase chain reaction amplification was
performed using the oligonucleotide primers
5'-aaaggatccatcgagggccgcggaggtatggaaattctttgtgaag-3' and 5'-tttggatcctcacacacagctaaccttttc-3', introducing a BamHI
cleavage site for in-frame insertion into pCMV-TAG2B. The
entire insert was subjected to automated sequencing to confirm
that the FLAG tag was in-frame and that no polymerase chain
reaction-induced mutations occurred during the amplification.
Transfection and Expression of 5-HT2A Receptors and
Arrestins in HEK-293 Cells--
HEK-293 cells were plated onto
6-well, 35-mm plates at ~0.5 × 106 cells/ml in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
fetal calf serum and antibiotics. At 24 h after plating, cells were transfected with 2 µg of DNA using Fugene 6 (Roche Molecular Biochemicals) exactly as described by the manufacturer. For
cotransfection experiments, the total amount of DNA transfected was
kept constant by the addition of empty vector (pcDNA3). At 24 h after transfection, cells were split into 24-well plates containing
polylysine-coated coverslips and grown for an additional 24 h in
DMEM supplemented with 10% dialyzed serum. The next day, the medium
was removed; and after washing with serum-free medium, the cells were
incubated overnight with serum-free DMEM to remove 5-HT present in the serum.
Immunocytochemistry--
Following various treatments, cells
were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS)
for 30 min, lightly permeabilized on ice (0.2% Triton X-100 in PBS)
for 20 min, and incubated with blocking buffer (1% bovine serum
albumin and 1% gelatin in PBS) for 1 h. Cells were then incubated
overnight with the following antibodies diluted in blocking buffer: a
5-HT2A receptor amino terminus-specific antibody (Ab51;
1:3000 dilution) (14), mono- and polyclonal anti-GFP antibodies (1:5000
dilution), anti-CT-Arr-2 antibody (1:3000 dilution), and a monoclonal
anti-arrestin-2 antibody (1:3000 dilution) (19). After warming to room
temperature and washing with PBS, cells were incubated with a 1:200
dilution of either BODIPY-FL-labeled goat anti-mouse antibody or
Texas Red-labeled goat anti-rabbit antibody (diluted in blocking
buffer) for 1 h. Cells were then washed with PBS and mounted for
fluorescent confocal microscopic evaluation as previously detailed
(14). For confocal microscopy, all images were taken at an overall
magnification via the microscope of ×1000; and in selected images,
electronic magnification greater than ×1000 was obtained.
Live Cell Confocal Microscopy--
For these studies, HEK-293
cells were plated onto sterile 35-mm cell wells that contained
coverslips affixed to the bottom in DMEM with 10% fetal calf serum.
Cells were then transfected as described above, incubated with DMEM
containing 10% dialyzed fetal calf serum for 24 h, and switched
to serum-free DMEM for an additional 24-h incubation. On the day of the
experiment, cells were switched to indicator-free HEPES-buffered
serum-free DMEM and placed on the heated stage of a Zeiss confocal
microscope. In addition to a heated stage, a plastic tent enclosed the
stage and objective so that a stream of heated air maintained the
microscope objective and air surrounding the stage at 37 °C for the
duration of the experiment. Images were automatically collected at 30-s intervals, and subsequent image sequences were used to compose movies
using Quick-time Pro.
Internalization Assays--
Internalization was quantified as
previously described using a computer-based image analysis system (14,
15). Typically, 30-100 cells were quantified per experiment in a
blinded fashion, and all experiments were replicated at least three
times. Data were analyzed via Student's t test for
comparison of independent mean values, with p < 0.05 considered significant.
Surface Biotinylation Assays--
Surface biotinylation was done
as described previously (15) with modifications. In brief, HEK-293
cells were cotransfected with FLAG-2A and either Arr-2 or CT-Arr-2 as
described above in 100-mm dishes. The next day, cells were split onto
6-well polylysine-coated plates with DMEM + 10% dialyzed fetal calf
serum; and 24 h later, the medium replaced with serum-free DMEM.
The next day, cells were incubated with 5-HT (10 µM) or
vehicle (PBS) for 15 min, placed on ice, rinsed with 2 ml of ice-cold
biotinylation buffer (10 mM boric acid, 154 mM
NaCl, 7.2 mM KCl, and 1.8 mM CaCl2, pH 8.4), and then surface-biotinylated in a total volume of 1.0 ml with
0.8 mM biotin disulfide N-hydroxysuccinimide
ester for 15 min on ice. The reaction was quenched with 2 ml of
quenching buffer (0.192 M glycine, 25 mM Tris,
1.8 mM CaCl2, and 154 mM NaCl, pH
8.3), and the cells were then lysed in 1 ml of lysis buffer (20 mM HEPES, 0.1% SDS, 1% Nonidet P-40, 0.5% deoxycholate, and 1× complete protease inhibitor mixture (
For Western blot analysis, blots were blocked with Tris-buffered saline
with Tween 20 (150 mM NaCl, 50 mM Tris-Cl, and
0.1% Tween 20, pH 7.40; TBST) containing 5% nonfat dry milk (blocking buffer) for 1 h at room temperature. Blots were then
incubated with a 1:2000 dilution of a monoclonal anti-FLAG antibody
(Sigma) in blocking buffer for 2 h. Following extensive washing
with Tris-buffered saline, blots were incubated with secondary antibody
(peroxidase-conjugated goat anti-mouse, 1:2000; Accurate Chemical and
Science Corp.) in blocking buffer for 1 h, washed
extensively with TBST, and washed twice with Tris-buffered saline.
Blots were then incubated for 1 min with chemiluminescence substrate
and visualized and quantified using a chemiluminescence imaging system
(Kodak Digital Science).
Characterization of GFP-tagged 5-HT2A
Receptors--
In initial studies, we characterized the pharmacology
and functional activity of GFP-tagged 5-HT2A receptors
(SR2-GFP) and compared SR2-GFP with wild-type receptors expressed in
HEK-293 cells. Table I shows that SR2-GFP
and wild-type 5-HT2A receptors have similar expression
levels and agonist and antagonist pharmacologies. These results
demonstrate that GFP tagging does not lead to detectable changes in
5-HT2A receptor pharmacology or functional activity. Our
prior studies of agonist-induced internalization of 5-HT2A receptors suggested an endosome-mediated process (14). To verify that
GFP tagging did not alter the trafficking of 5-HT2A
receptors, we performed dual-label, real-time experiments with SR2-GFP
and Texas Red-labeled transferrin. Preliminary studies (data not shown) indicated that SR2-GFP was internalized coincident with transferrin, in
agreement with our prior studies on the native receptor (14).
Agonist- and Antagonist-mediated Internalization Are
Dynamin-dependent--
We next cotransfected SR2-GFP with a
dynamin dominant-negative mutant (dynamin K44A) to investigate the
dynamin dependences of agonist- and antagonist-mediated
internalization. For these studies, image sequences were converted to
Quick-time Movie Format (see Supplemental
Material). These image sequences clearly demonstrate that 5-HT induced
a rapid internalization of SR2-GFP in live cells (Image Sequence 1 in
Supplemental Material) and that the process of internalization was
abolished by cotransfection with dynamin K44A (Image Sequence 2 in
Supplemental Material). These image sequences also show that 5-HT
induced a change in shape (Image Sequence 1 in Supplemental Material),
which was unaffected by cotransfection with dynamin K44A (Image
Sequence 2 in Supplemental Material). Further image sequences of live
cells (Image Sequence 3 in Supplemental Material) demonstrate that
clozapine induced internalization of 5-HT2A receptors
without inducing a change in shape. By contrast, cells exposed to
vehicle (Image Sequence 4 in Supplemental Material) showed neither
shape change nor receptor internalization. These results indicate that
shape change and agonist-mediated receptor internalization are
independent processes. Analysis of a large number of cells from several
experiments is shown in Fig. 1. As shown,
5-HT and clozapine induced similar degrees of internalization and were
both equally sensitive to dynamin K44A. These results indicate that
both agonist- and antagonist-induced internalization of
5-HT2A receptors are dynamin-dependent.
Agonist- and Antagonist-induced Internalization Are
Arrestin-independent--
We next examined the effects of agonist and
antagonist exposure on arrestin subcellular distribution using Arr-2
and SR2-GFP. As shown in Fig. 2, agonist
exposure induced minimal Arr-2 translocation. Similar results were
obtained with cells expressing GFP-Arr-2 and native 5-HT2A
receptors (data not shown). After longer time periods of agonist
exposure, a redistribution of Arr-2 became evident. As shown in Fig. 2,
by 5 min of agonist exposure, a punctate redistribution of Arr-2 in the
cytoplasm became evident, which was pronounced by 15 min of agonist
exposure. At this time period as well, punctate accumulations of Arr-2
at the plasma membrane were also evident, although it was clear that
these punctate accumulations were distinct from those containing
SR2-GFP. No redistribution of Arr-2 was seen in cells that were exposed
to 5-HT but not cotransfected with 5-HT2A receptors (data
not shown).
We next examined the arrestin dependences of agonist- and
antagonist-induced internalization using three dominant-negative arrestins: 1) the carboxyl-terminal clathrin-binding domain of arrestin-2 (Arr-2-(319-418); CT-Arr-2) (20, 21); 2) the Arr-2 V53D
mutant, which has been used by many others (22); and 3) an arrestin-3
dominant-negative mutant (Arr-3-(284-409); CT-Arr-3) (17). As shown in
Fig. 3, the agonist quipazine (100 µM) and the antagonist clozapine (10 µM)
both induced internalization of SR2-GFP, which was unaffected by
coexpressing CT-Arr-2.
We also quantified results from a large number of cells that were
transfected with SR2-GFP + pcDNA3 (empty vector; negative control),
SR2-GFP + dynamin K44A (positive control), or SR2-GFP + CT-Arr-2. As
shown in Fig. 4, cotransfection with
CT-Arr-2 had no significant effect on either agonist- or
antagonist-induced internalization of SR2-GFP. Similar negative results
were found with the dominant-negative mutant Arr-2 V53D (data not
shown). As a positive control, the dynamin K44A mutant inhibited both agonist- and antagonist-induced internalization. Fig.
5 shows that CT-Arr-3, an Arr-3
dominant-negative mutant, also had no effect on agonist-induced
internalization of 5-HT2A receptors.
A closer examination of the confocal images revealed a distinctive
redistribution of Arr-2, Arr-3, and 5-HT2A receptors. Thus, after 5 min of agonist exposure, a redistribution of CT-Arr-2 to the
plasma membrane and distinct presumably endocytotic vesicles was
apparent. As shown at the 5-min time point in Fig.
6, minimal colocalization of CT-Arr-2 and
SR2-GFP was seen in endocytotic vesicles, even though there
was apparent translocation of CT-Arr-2 to the plasma
membrane. Interestingly, the CT-Arr-2 immunofluorescent puncta were
clearly distinct from those in which SR2-GFP was localized (Fig. 6).
Separate dual-label studies using a polyclonal CT-Arr-2-specific antibody and a monoclonal anti-AP-2 antibody indicated that the vesicles in which CT-Arr-2 is localized are AP-2 containing organelles (data not shown), in verification of prior studies (18).
Next, we examined the effect of quipazine on Arr-3 translocation. As
shown in Fig. 7, quipazine (100 µM) induced a modest and rapid translocation of Arr-3 to
the plasma membrane of cells transfected with GFP-Arr-3 and native
5-HT2A receptors. At later time periods, a redistribution
of GFP-Arr-3 into intracellular vesicles distinct from those containing
5-HT2A receptors was evident (Fig. 7). No colocalization of
GFP-Arr-3 and 5-HT2A receptors to intracellular vesicles
was evident at any of the time points studied. Additionally, clozapine
exposure led to neither GFP-Arr-3 translocation nor colocalization of
GFP-Arr-3 with 5-HT2A receptors (Fig. 7). These results
indicate that the agonist-induced internalization of 5-HT2A
receptors leads to a redistribution of GFP-Arr-3 to subcellular domains
that are distinct from those occupied by 5-HT2A receptors.
These results also indicate that clozapine induces internalization of
5-HT2A receptors without inducing Arr-3 translocation. Control experiments were performed with HEK-293 cells cotransfected with GFP-Arr-3 and
Additionally, we sought to determine the effect of CT-Arr-2 on
5-HT2A receptor internalization using a biochemical assay
of internalization. For these studies, we cotransfected HEK-293 cells with FLAG-2A and either Arr-2 or CT-Arr-2. Internalization of FLAG-2A
was quantified by measuring the loss of surface-biotinylated FLAG-2A by
Western blot analysis. As shown in Fig.
8, FLAG-2A was internalized to a similar
extent in cells cotransfected with either Arr-2 (61 ± 7%) or
CT-Arr-2 (59 ± 6%). Additionally, the extent of internalization
induced by 5-HT (61%) was quite similar to that measured by our image
analysis technique (45-61%).
To determine whether the differential sorting of 5-HT2A
receptors, Arr-2, and CT-Arr-2 was due to some artifact related to GFP
tagging of 5-HT2A receptors, we did additional experiments using a FLAG-tagged 5-HT2A receptor (FLAG-2A). For these
experiments, we cotransfected FLAG-2A with either Arr-2 or CT-Arr-2 and
visualized FLAG-2A with a monoclonal anti-FLAG antibody and
Arr-2/CT-Arr-2 with a polyclonal CT-Arr-2-specific antibody. As shown
in Fig. 9 (A-C), after 5 min
of 5-HT exposure, differential sorting of FLAG-2A and Arr-2 was seen. A
similar pattern of differential sorting was seen for FLAG-2A and
CT-Arr-2 (Fig. 9, D-F) following 5 min of 5-HT exposure.
These studies indicate that FLAG-2A and SR2-GFP are sorted in an
identical manner.
The major findings of this study are that 1) agonist- and
antagonist-induced internalization of 5-HT2A receptors in
HEK-293 cells are dynamin-dependent and
arrestin-independent and 2) the agonist-induced internalization of
5-HT2A receptors leads to a differential sorting of
5-HT2A receptors and In addition to these novel observations, the results are important
because they clarify a long-standing conundrum related to the study of
5-HT2A receptors. Since 1980 (24), it has been noted by a
large number of investigators that various 5-HT2A
antagonists can induce 5-HT2A receptor down-regulation
in vivo and in vitro (see Refs. 1 and 25 for
reviews). Our prior studies (26), as well as those of others (16), have
clearly demonstrated that the antagonist-induced down-regulation of
5-HT2A receptors occurs without significant changes in
5-HT2A receptor gene transcription. These results imply
that the antagonist-induced down-regulation of 5-HT2A
receptors occurs via post-transcriptional mechanisms. Our recent
in vivo and in vitro studies, in which we
demonstrated that antagonists induce 5-HT2A receptor
internalization in vivo and in vitro, imply that
antagonist-mediated internalization is a prominent pathway for
5-HT2A receptor down-regulation (15).
Studies delineating the cellular mechanisms responsible for GPCR
internalization, however, have implied that antagonists cannot induce
internalization and/or down-regulation because the internalization process is dependent upon receptor activation, followed by subsequent receptor phosphorylation and arrestin binding. 5-HT2A
antagonists do not activate 5-HT2A receptors
using any measure of receptor activity (phosphatidylinositol
hydrolysis or arachidonic acid release) and do not induce shape change
(present findings). In fact, clozapine, the antagonist used in these
studies, has been demonstrated to be a potent antagonist with
negative intrinsic activity at 5-HT2A
receptors (27). Thus, it is difficult to reconcile a large number of
prior findings that 5-HT2A antagonists can induce receptor
internalization and down-regulation with most of the current models of
GPCR regulation.
Our present results, which indicate that both the clozapine- and
quipazine-induced internalization are sensitive to a dynamin dominant-negative mutant (dynamin K44A) and insensitive to three different arrestin dominant-negative mutants (Arr-2 V53D,
Arr-2-(319-418), and Arr-3-(284-409)), show that 5-HT2A
agonists and antagonists induce internalization via an
arrestin-independent pathway. Since the pathway is
dynamin-sensitive and since 5-HT2A receptors internalize via clathrin-coated vesicles (28) and transferrin-containing endosomes
(Ref. 14 and present results), it is clearly an endosome-mediated process. It is unlikely that internalization occurs via caveolae since
the present studies demonstrated that agonist-induced internalization occurs coincident with transferrin receptor internalization, which is a
well accepted marker of endosome-mediated internalization. Additionally, our prior studies demonstrated that internalized receptors do not colocalize with caveolin in vitro (14,
28).
Our results also show that, although arrestins are not involved in
5-HT2A receptor internalization, activation of
5-HT2A receptors by agonists induces a
translocation/redistribution of Arr-2 and Arr-3. Intriguingly, although
Arr-2 and truncated Arr-2 (Arr-2-(319-418)) were redistributed to
intracellular vesicles, minimal colocalization with 5-HT2A
receptors was detected. Prior studies have implicated the C terminus of
Arr-2 as containing both clathrin-binding (20) and AP-2 adaptor
protein-binding (13) motifs. Our results imply that the C terminus of
Arr-2 may be recruited to distinct plasma membrane and endocytic
subcellular domains independently of GPCR internalization. This
conclusion is supported by preliminary findings from triple-label
studies in which we found that Arr-2 and CT-Arr-2 are redistributed, in
part, to AP-2 containing vesicles that are distinct from those
containing 5-HT2A receptors (data not shown).
Quite recent studies (29) suggest that at least two distinct classes of
GPCRs exist, depending upon their affinities for Arr-2 and Arr-3. The
5-HT2A receptor appears to fall into Group B since
5-HT2A receptors induce a very modest redistribution of Arr-2 and Arr-3. Prior studies by us (19) have demonstrated that Arr-2
and Arr-3 can bind to the purified third intracellular loop of the
5-HT2A receptor in vitro and that
5-HT2A receptors are colocalized with Arr-2 and Arr-3 in
some, but not all, cortical neurons. The present studies imply that
unknown neuron-specific factors regulate the interactions of Arr-2 and
Arr-3 with 5-HT2A receptors in cortical neurons because
minimal colocalization of Arr-2/Arr-3 and 5-HT2A receptors
was evident in HEK-293 cells.
If Arr-2 and Arr-3 are induced to redistribute by 5-HT2A
receptor stimulation, but fail to regulate internalization, what is the
role for redistribution of Arr-2 to punctate domains on the plasma
membrane and distinct intracellular vesicles? The present studies do
not answer these important questions, although work in progress
suggests that the C terminus of Arr-2 may regulate mitogen-activated
protein kinase (p42/44) activation via a pathway distinct from that
used to internalize 5-HT2A
receptors.2
Several control experiments were performed to verify that the results
we obtained were not due to artifacts related to GFP tagging of
5-HT2A receptors or to the presence of endogenous 5-HT receptors in HEK-293 cells. Thus, we found that native
5-HT2A receptors and FLAG-tagged and GFP-tagged
5-HT2A receptors were differentially sorted from Arr-2 and
Arr-3 during endocytosis. Additionally, we verified that CT-Arr-2 did
not inhibit 5-HT2A receptor internalization using a
biochemical assay of internalization (surface biotinylation). In fact,
the extent of internalization induced by 5-HT was the same whether
internalization was measured by confocal microscopy or by surface
biotinylation (~60% in both cases). Finally, in untransfected cells,
no redistribution of Arr-2 or Arr-3 was seen after 5-HT exposure.
Taken together, these results demonstrate that 5-HT2A
receptors may induce plasma membrane and intracellular sorting of Arr-2 and Arr-3 distinct from that of internalized 5-HT2A
receptors. Importantly, internalization per se is not a
sufficient signal for Arr-2 or Arr-3 redistribution since clozapine,
which induces internalization but does not activate 5-HT2A
receptors, does not alter the subcellular distribution of Arr-2 or
Arr-3. Additionally, our results demonstrate that the
dynamin-dependent, arrestin-independent modes of GPCR
internalization occur coincident with redistribution of Arr-2 and Arr-3
to the plasma membrane. Taken together, these results are sufficient to
conclude that novel internalization-independent modes of arrestin
redistribution occur following 5-HT2A receptor activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-adrenergic receptors (8, 9), are found in coated vesicles and
various intracellular vesicles associated with endocytic pathways. Studies with the
-adrenergic receptor in particular have delineated a general pathway for agonist-mediated internalization by which agonist-induced activation of receptors leads to receptor
phosphorylation and then the binding of arrestin to phosphorylated
receptors (10, 11). Arrestin binding appears to facilitate
translocation of phosphorylated GPCRs to clathrin-coated pits and the
eventual internalization of GPCRs via the endosome pathway.
Interactions of the C terminus of arrestin with clathrin (12) and the
AP-2 adaptor protein (13) appear to be essential for arrestin-mediated internalization of many GPCRs.
-adrenergic receptors is universal for GPCRs.
Arr-1
(Arr-2),
-Arr-2 (Arr-3), and truncated Arr-2 (Arr-2(319-418)) into
plasma membrane domains and intracellular vesicles distinct from those
containing 5-HT2A receptors. Our results imply a novel and
distinct sorting pathway for Arr-2 and Arr-3 that occurs independently
of 5-HT2A receptor internalization.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EDTA), pH 7.40) for 15 min on ice. After clarification of lysates by centrifugation (14,000 × g for 20 min at 4 °C), lysates were
incubated in a total volume of 1.0 ml with 50 µl of
streptavidin-agarose for 2 h at 4 °C with constant mixing.
Biotinylated proteins were then purified by centrifugation, followed by
three washes with lysis buffer at 4 °C. After the final wash,
biotinylated FLAG-2A was liberated from the agarose by the addition of
100 µl of SDS sample buffer and heating at 65 °C for 1 min.
Samples containing equivalent quantities of protein were then run on
10% SDS-polyacrylamide gels and transferred to nitrocellulose as
previously described (15).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Pharmacological characterization of SR2-GFP expressed in HEK-293
cells
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Fig. 1.
Agonist- and antagonist-induced
internalization of 5-HT2A receptors are
dynamin-dependent. Cells transfected with SR2-GFP + pcDNA3 (empty vector control) or SR2-GFP + dynamin K44A were
exposed to vehicle, 5-HT (10 µM for 15 min), or clozapine
(10 µM for 30 min). Cells were then fixed and prepared
for immunofluorescent confocal microscopy, and internalization was
quantified as described under "Experimental Procedures." Data
represent means ± S.E. of internalized receptor from a large
number of cells (100-200 cells) from a representative experiment that
was replicated three times. *, p < 0.01 versus control (Student's t test).
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Fig. 2.
Agonist-induced 5-HT2A receptor
internalization results in a differential redistribution of
arrestin-2. Cells were transfected with SR2-GFP
(green) and Arr-2 (red), exposed to 5-HT (10 µM) for various time periods, and then prepared for
immunofluorescent confocal microscopy as described under
"Experimental Procedures." Data represent representative images
taken from an experiment that was replicated three times with identical
results. Arrows indicate differential localization of Arr-2
and SR2-GFP. With the exception of the series of panels on the right,
all images were taken from the middle of representative cells. The last
series of panels shows a higher power view taken at the level of the
plasma membrane from a representative cell after 15 min of 5HT
exposure.
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Fig. 3.
Dominant-negative arrestin does not alter
agonist- or antagonist-induced internalization of 5-HT2A
receptors. For these experiments, cells were transfected with
SR2-GFP + Arr-2-(319-418), stimulated with quipazine
(QUIP; 100 µM) or clozapine (CLOZ;
10 µM) for various time periods, and then prepared for
confocal microscopy as described under "Experimental Procedures." A
polyclonal CT-Arr-2-specific antibody was used with a Texas Red-labeled
secondary antibody to visualize Arr-2-(319-418) (red),
whereas SR2-GFP (green) was visualized using GFP
fluorescence. Images are shown from the middle of representative cells
from a typical experiment that was replicated with identical results
four times.
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Fig. 4.
Dynamin-dependent,
arrestin-independent internalization of 5-HT2A
receptors. Shown are the means ± S.E. from a
representative experiment in which 100-200 cells were imaged and
internalization of SR2-GFP was analyzed as described under
"Experimental Procedures." Cells were transfected with
SR2-GFPA ± pcDNA3 (empty vector control), dynamin K44A
(positive control), or Arr-2-(319-418).
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Fig. 5.
Agonist-induced internalization of
5-HT2A receptors is insensitive to an arrestin-3
dominant-negative mutant. HEK-293 cells were transfected
with pRcCMV-SR2 + GFP-Arr-3 or pRcCMV-SR2 + Arr-3-(284-409). Shown are
the means ± S.E. from experiments in which 50-100 cells were
imaged and internalization of the 5-HT2A receptors was
quantified as described under "Experimental Procedures."
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Fig. 6.
Arrestin-2 carboxyl terminus is
differentially sorted from internalized 5-HT2A
receptors. Shown is a close-up view of a high power
(magnification × 1000) image of a representative HEK-293 cell
cotransfected with SR2-GFP (green) and Arr-2(319-418)
(red), stimulated with 5-HT (10 µM) for 5 min,
and prepared for confocal microscopy as described under "Experimental
Procedures." Arrowheads demonstrate Arr-2(319-418)
immunoreactive puncta; arrows represent SR2-GFP
immunoreactive vesicles. The experiment was replicated three times with
identical results.
2-adrenergic receptors. In agreement
with prior studies (23), isoproterenol (10 µM) exposure
induced a rapid translocation of GFP-Arr-3 to the plasma membrane (data not shown). Additionally, in cells transfected only with GFP-Arr-3, 5-HT exposure did not change GFP-Arr-3 distribution (data not shown).
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Fig. 7.
Agonist-induced 5-HT2A receptor
internalization leads to a differential sorting of arrestin-3 and
5-HT2A receptors. For these experiments, HEK-293 cells
were cotransfected with pRcCMV-SR2 to visualize
5-HT2A receptors (red) and with GFP-Arr-3
(GFP- Arr2) (green) to visualize
Arr-3, stimulated with quipazine (QUIP; 100 µM) or clozapine (CLOZ; 10 µM)
for various time periods, and then prepared for confocal microscopy as
described under "Experimental Procedures." The inset of
the 2-min time period shows magnified images of the plasma membrane
from the same cells. Arrows indicate 5-HT2A
receptor immunoreactivity, whereas arrowheads represent
GFP-Arr-3 fluorescence. The experiment was replicated three times with
identical results.
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Fig. 8.
CT-Arr-2 does not inhibit 5-HT2A
receptor internalization as assessed by surface biotinylation.
HEK-293 cells were cotransfected with FLAG-2A and either Arr-2 or
CT-Arr-2 and prepared for surface biotinylation as described under
"Experimental Procedures." 15 min after vehicle ( ) or 5-HT (10 µM; +) exposure. The upper panel
shows mean percent internalization (loss of surface receptors) for
n = three separate determinations, whereas the
lower panel shows a representative Western blot. The
experiment was replicated twice.
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Fig. 9.
Differential sorting of FLAG-2A, Arr-2, and
CT-Arr-2 following 5-HT exposure in HEK-293 cells. For these
studies, HEK-293 cells were cotransfected with FLAG-2A and either Arr-2
(A-C) or CT-Arr-2 (D-F), stimulated for 5 min
with 10 µM 5-HT, and then prepared for immunofluorescent
confocal microscopy as described under "Experimental
Procedures." FLAG-2A was visualized using a mouse monoclonal
anti-FLAG antibody and a BODIPY-FL-tagged anti-mouse secondary
antibody, whereas Arr-2 and CT-Arr-2 were visualized using rabbit
polyclonal anti-CT-Arr-2 antibodies and Texas Red-tagged anti-rabbit
secondary antibodies. The inset shows a magnified image of
the plasma membrane of a typical cell. Arrows indicate
FLAG-2A immunofluorescence, whereas the arrowhead indicates
the one vesicle in which CT-Arr-2 and FLAG-2A were colocalized. The
experiment was replicated with identical results three times.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-arrestin-1 (Arr-2) and
-arrestin-2 (Arr-3) into distinct plasma membrane and intracellular compartments. These results demonstrate that novel sorting pathways exist for arrestins and 5-HT2A receptors. Additionally,
since 5-HT2A receptor activation leads to a redistribution
of Arr-2 and Arr-3, our findings imply that internalization-independent functions exist for Arr-2 and Arr-3.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants RO1MH61887, RO1MH57635, and KO2MH01366 and by a National Alliance for Research on Schizophrenia and Depression independent investigator award (to B. L. R.).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.
The on-line version of this article (available at
http://www.jbc.org) contains Image Sequences 1-4 in
Quick-time Movie Format.
** To whom correspondence should be addressed: Dept. of Biochemistry, Rm. W438, Case Western Reserve University Medical School, 10900 Euclid Ave., Cleveland, OH 44106-4935. Tel.: 216-368-2730; Fax: 216-368-3419; E-mail: roth@biocserver.cwru.edu.
Published, JBC Papers in Press, November 7, 2000, DOI 10.1074/jbc.M006968200
2 J. Woods and B. L. Roth, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: 5-HT2A, 5-hydroxytryptamine 2A; 5-HT, 5-hydroxytryptamine; GPCR, G-protein-coupled receptor; Arr, arrestin; GFP, green fluorescent protein; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; BODIPY-FL, 4,4- difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid, succinimidyl ester.
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