From the Department of Psychiatry and Department of Cellular and Molecular Pharmacology, Program in Cell Biology, University of California, San Francisco, California 94143-0984 and § Department of Anesthesiology, University of California, Los Angeles, California 90024
Received for publication, October 10, 2000, and in revised form, February 1, 2001
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ABSTRACT |
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We have observed an unexpected type of
nonreciprocal "cross-regulation" of the agonist-induced endocytosis
of G protein-coupled receptors by clathrin-coated pits.
Isoproterenol-dependent internalization of
G protein-coupled receptors
(GPCRs)1 are regulated by a
set of highly conserved molecular mechanisms (1-3). Because most cells express multiple types of GPCR that serve distinct functions, the
specificity of these regulatory mechanisms is of great physiological interest. Extensive studies have led to the classification of distinct
"heterologous" and "homologous" mechanisms of GPCR regulation. An example of a heterologous regulatory mechanism is rapid
desensitization of the Many GPCRs, such as the prototypic Arrestins play a highly conserved role in promoting endocytosis of
various GPCRs by clathrin-coated pits (11). Recent studies indicate
that particular GPCRs differ substantially in their effects on the
intracellular trafficking of arrestins. Many G protein-coupled receptors (including the cDNA Constructs and Mutagenesis--
The coding sequence of
the human V2R was amplified by PCR and inserted into a tagging vector
containing the influenza hemagglutinin signal sequence and the FLAG
epitope (21). The N-terminal epitope-tagged receptor sequence was
subcloned into the mammalian expression vector pcDNA3.1
(Invitrogen). The human Cell Lines and Transfection--
HEK293 cells (ATCC) were
cultured in Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum, 100 units/ml penicillin, and 0.1 mg/ml streptomycin
(University of California San Francisco Cell Culture Facility) in a
humidified incubator with 7% CO2 at 37 °C. Cells were
transiently transfected using calcium phosphate co-precipitation (23).
For generation of stable transformants, cells after transfection were
selected for resistance to 0.5 mg/ml G418 (Geneticin, Life
Technologies, Inc.). Single clones were isolated after 2-3 weeks of
culture and screened for expression by immunofluorescence microscopy
and radioligand binding assays.
Immunofluorescence Microscopy--
Cells were grown on glass
coverslips in 6-well tissue culture plates. For co-localization of
FLAG-tagged V2R and HA-tagged Co-localization of Membrane Preparation--
Individual cell clones stably
expressing the Radioligand Binding Assays--
To assess expression levels for
the V2R, 10 µg of membrane protein were incubated in 125 µl of V2
binding buffer (50 mM Hepes/NaOH, pH 7.4, 5 mM
MgCl2, 0.2% bovine serum albumin) with 10 nM
[3H-Arg8]vasopressin ([3H]AVP)
(PerkinElmer Life Sciences, 68.5 Ci/mmol) for 30 min at 30 °C.
Nonspecific binding was determined in the presence of 4 µM nonlabeled AVP. The binding reaction was stopped by
rapid filtration over GF/C glass-fiber filters (Whatman) using a
Brandel cell harvester. Filters were washed 3 times with ice-cold V2
filtration buffer (20 mM Hepes/NaOH, pH 7.4, 2 mM MgCl2, 0.02% bovine serum albumin). For the
Scoring of Receptor Localization by Fluorescence
Microscopy--
Coverslips were processed for indirect
immunofluorescence staining, as above, and coded such that the identity
and treatment conditions of the specimen were not specified.
Examination of coded specimens by epifluorescence microscopy was
performed by a second individual not familiar with the details of the
experiment. The localization of fluorochromes representing individual
receptors were classified in multiple cells examined at random,
positive for expression of both receptors. Cells with detectable
expression of only one of the two receptors were excluded from the
scoring. Immunostaining was classified according to the following
criteria: non-internalized localization (bright staining around the
cell periphery with <10 immunoreactive puncta visualized within the cytoplasm); intermediate appearance (10-20 internal puncta);
internalized (>20 immunoreactive puncta resolved within the
cytoplasm). At least 25 cells per coverslip were scored by these
criteria, and all specimens from an individual experiment were scored
in a single session before the code was broken and results were
tabulated. For studies employing GFP-tagged arrestins, receptor
localization for GFP positive and negative cells were scored in the
same sitting, and the specificity of observed effects was confirmed by
transfection of GFP not fused to Estimation of Receptor Internalization by Fluorescence Flow
Cytometry--
A previously established (26) flow cytometric assay was
used to quantitate immunoreactive receptors present on the surface of
intact cells after dissociation from the cell culture dish. All data
points represent quantitation of 20,000 cells using a FACScan cytometer
(Becton Dickenson) performed in triplicate (representing three
independently treated dishes) for each experiment. The extent of
receptor internalization was calculated according to the
agonist-induced reduction in mean surface immunoreactivity (26).
Figures indicate mean ± S.D. for results compiled from three
separate experiments.
We chose to examine membrane trafficking of the Both the 2-adrenergic receptors in stably transfected
HEK293 cells was specifically blocked (>65% inhibition) by
vasopressin-induced activation of V2 vasopressin receptors co-expressed
at similar levels. In contrast, activation of
2
receptors caused no detectable effect on V2 receptor internalization in
the same cells. Several pieces of evidence suggest that this
nonreciprocal inhibition of endocytosis is mediated by
receptor-specific intracellular trafficking of
-arrestins. First,
previous studies showed that the activation of V2 but not
2 receptors caused pronounced recruitment of
-arrestins to endocytic membranes (Oakley, R. H., Laporte, S. A., Holt, J. A., Barak, L. S., and Caron, M. G. (1999) J. Biol. Chem. 274, 32248-32257). Second,
overexpression of arrestin 2 or 3 (
-arrestin 1 or 2) abolished the
V2 receptor-mediated inhibition of
2 receptor
internalization. Third, mutations of the V2 receptor that block
endomembrane recruitment of
-arrestins eliminated the V2
receptor-dependent blockade of
2 receptor
internalization. These results identify a novel type of heterologous
regulation of G protein-coupled receptors, define a new functional role
of receptor-specific intracellular trafficking of
-arrestins, and suggest an experimental method to rapidly modulate the functional activity of
-arrestins in intact cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
2-adrenergic receptor
(
2AR) mediated by the cyclic AMP-dependent protein kinase. Activated cAMP-dependent protein kinase can
phosphorylate both the
2AR as well as a number of other
GPCRs irrespective of whether or not these receptors have bound
agonist. A well characterized mechanism of homologous regulation is
desensitization of the
2AR mediated by G protein-coupled
receptor kinases. In general G protein-coupled receptor kinases
preferentially phosphorylate ligand-activated receptors without
affecting other GPCRs present in the same cells that are not activated
by their respective agonist (4-6).
2AR, are regulated by
agonist-induced endocytosis via clathrin-coated pits (7-10). This process is promoted by G protein-coupled receptor kinase-mediated phosphorylation of receptors followed by membrane recruitment of
nonvisual arrestins (or
-arrestins), which link receptors to the
clathrin/AP-2 endocytic coat (9, 11, 12). Because high affinity
interaction of GPCRs with arrestins is promoted by both G
protein-coupled receptor kinase-mediated phosphorylation and an
agonist-induced conformation of the receptor protein (13, 14),
endocytosis by clathrin-coated pits is thought to represent a highly
homologous mechanism of GPCR regulation (8, 11). Consistent with this,
distinct GPCRs, even when co-expressed at high levels, are endocytosed
by coated pits in a highly selective manner after activation by their
respective agonist (15).
2AR) recruit arrestins to the
plasma membrane but do not remain associated with arrestins after
endocytosis (16, 17). In contrast, the V2 vasopressin receptor (V2R)
has been shown to mediate recruitment of arrestins both to the plasma membrane and to V2R-containing endocytic vesicles (17). This receptor-specific difference in the endomembrane recruitment of arrestins is mediated by a persistent phosphorylation of internalized V2Rs, which inhibits recycling of receptors to the plasma membrane and
causes a prolonged state of receptor desensitization (17, 18).
Endosome-associated arrestins have been proposed to play an important
role in determining the specificity of downstream signal transduction
by endocytosed GPCRs (19, 20). However, to our knowledge no previous
studies have examined the possibility that arrestin trafficking may
modulate the specificity of GPCR endocytosis itself. In the present
study we have observed an unexpected type of heterologous and
nonreciprocal inhibition of
2AR endocytosis mediated by
agonist-induced activation of the V2R. This heterologous inhibition is
dependent on endomembrane recruitment of
-arrestins, suggesting a
novel role of receptor-specific trafficking of arrestins in modulating
endocytosis of certain GPCRs.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
2AR and the murine
-opioid receptor were tagged in the N-terminal extracellular domains with the
HA epitope (YPYDVPDYA) and subcloned into pcDNA3.1, as previously described (22). Two truncated forms of the V2R were obtained through
oligonucleotide-directed mutagenesis using the polymerase chain
reaction with 3' antisense primers incorporating a stop codon followed
by a suitable restriction site (345T and 362T: stop codons substituted
for residues at positions 345 and 362 of the human V2R amino acid
sequence, respectively) (18). PCR products were subcloned into
pcDNA3.1. GFP-tagged arrestin 3 (
-arrestin 2) was a generous
gift of Dr. Marc Caron. A C-terminally EE epitope-tagged version of
arrestin 2 (
-arrestin-1, a generous gift of Dr. Jeffrey Benovic) was
constructed by ligating a synthetic adaptor oligonucleotide encoding
the epitope tag sequence and cloning into pcDNA3. All constructs
were verified by DNA sequencing (University of California San Francisco
Biomolecular Resource Center).
2AR or HA-tagged
-opioid receptor, cells were incubated with the monoclonal
anti-epitope antibodies anti-FLAG M1 (Sigma) and anti-HA HA.11
(Covance), respectively, both at a 1:500 dilution for 30 min at
37 °C. Cells were then treated under the respective conditions with
agonists, fixed using 4% formaldehyde in phosphate-buffered saline,
and permeabilized with blocking buffer (3% nonfat dry milk, 0.1%
Triton X-100, 50 mM Tris/HCl, pH 7.5, 1 mM
CaCl2). Bound M1 antibody was detected using incubations
with a rabbit anti-mouse IgG2b subtype-specific linker
antibody (1:800 dilution, 45 min at room temperature) followed by a
Texas Red-conjugated donkey anti-rabbit antibody (1:1000 dilution, 20 min at room temperature). Bound HA.11 antibody was detected using
fluorescein isothiocyanate-conjugated goat anti-mouse IgG1
subtype-specific antibody (1:800 dilution, 20 min at room temperature).
Immuno-stained coverslips were mounted on microscopy slides and
examined by epifluorescence or confocal microscopy (see below).
-Arrestin with Vasopressin V2 Receptor and
2-Adrenergic Receptor--
Cells stably co-expressing
FLAG epitope-tagged V2R and HA epitope-tagged
2AR were
transiently transfected with an expression plasmid encoding GFP-tagged
-arrestins as described previously (24). Cells were incubated with
the monoclonal anti-FLAG M1 antibody at a 1:500 dilution for 30 min at
37 °C. Cells were treated and fixed as mentioned above. FLAG
epitope-tagged V2R was detected using a
6-((7-amino-4-methylcoumarin-3-acetyl)amino) hexanoic acid,
succinimidyl ester (AMCA-S)-conjugated goat anti-mouse secondary antibody (Molecular Probes, OR, diluted 1:250, blue fluorescence). The
2AR was detected by staining with a rabbit polyclonal
antibody raised against the C-terminal 15-amino acid residues of the
human
2AR (22) diluted 1:500 and a Texas-Red conjugated
anti-rabbit secondary antibody (Jackson Immunoresearch Laboratories,
diluted 1:500, red fluorescence).
-Arrestin 2-GFP was detected by
GFP fluorescence (green fluorescence). Immuno-stained coverslips were examined by epifluorescence microscopy using a Nikon 60× NA1.4 objective and Chroma filter sets optimized for these fluorophores. Confocal microscopy was performed using a Bio-Rad MRC1000 and a Zeiss
100X NA1.3 objective. In all experiments, minimal bleed-through between
channels was confirmed using single-labeled control specimens.
2AR and/or the V2R were grown on 10-cm
dishes to confluency. Cells were lifted in phosphate-buffered saline
containing 2 mM EDTA and centrifuged at 500 × g for 10 min. Pellets were resuspended in 1 ml of lysis buffer (5 mM Hepes/NaOH, pH 7.4, 5 mM EDTA, 5 mM EGTA, 0.5 mM Pefabloc SC) and
homogenized using a glass Dounce homogenizer. The suspension was
centrifuged for 15 min at 20,000 × g, and the pellets
were resuspended and homogenized as above. The final pellet was
resuspended in 200 µl of membrane buffer (20 mM
Hepes/NaOH, pH 7.4, 1 mM EDTA, 5 mM
MgCl2, 0.5 mM Pefabloc SC). Protein
concentration was estimated by the method of Bradford (25). Membranes
were stored frozen at
80 °C.
2AR binding assay, 10 µg of membrane protein were
incubated in 500 µl of
2 binding buffer (75 mM Tris/HCl, pH 7.4, 12.5 mM MgCl2,
1 mM EDTA, 0.2% bovine serum albumin) with 10 nM [3H]dihydroalprenolol (Amersham Pharmacia
Biotech, 88 Ci/mmol) for 90 min at 25 °C. Nonspecific binding was
determined in the presence of 10 µM nonlabeled
alprenolol. The binding reaction was stopped by rapid filtration over
GF/C glass-fiber filters (Whatman). Filters were washed 3 times with
ice-cold
2 filtration buffer (75 mM Tris/HCl, pH 7.4, 12.5 mM MgCl2, 1 mM EDTA). Filter-bound radioactivity was counted by liquid
scintillation. All tests were performed in triplicate.
-arrestin. Numbers in figures
represent the mean ± S.D. for results from a representative
experiment, and all reported experiments were performed independently
at least three times with similar results.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
2AR
and V2R in HEK293 cells because both receptors stimulate adenylyl
cyclase activity via the Gs-signaling pathway and undergo
agonist-induced endocytosis by an arrestin-dependent
mechanism mediated by clathrin-coated pits, yet these receptors are
well established to differ substantially in their effects on the
intracellular trafficking of
-arrestins in this cell type (17). A
functional HA-tagged
2AR and FLAG-tagged V2R were
co-expressed in HEK293 cells by stable transfection. Because a single
selection marker (neomycin) was used to isolate stably transfected
cells after transfection of separate plasmids encoding the
2AR and V2R, many cell populations obtained by this method expressed either the
2AR or V2R but not both.
Dual label fluorescence microscopy was used to identify stably
transfected cell populations that expressed both the
2AR
and V2R in the majority (~70%) of cells. A cell clone expressing
both receptors at moderate levels (3.8 ± 0.3 pmol/mg for the V2R
and 4.7 ± 0.2 pmol/mg for the
2AR, respectively)
was selected from this group for further analysis because these levels
of expression are comparable to those used previously for studies of
GPCR endocytosis and arrestin trafficking in HEK293 cells (16, 17,
22).
2AR and V2R were visualized by fluorescence
microscopy in the plasma membrane of cells incubated in the absence of
agonist (Fig. 1, panels a and
b). In the presence of saturating concentrations (10 µM) of the
2AR agonist isoproterenol
(ISO),
2AR redistributed from the plasma membrane to
endocytic vesicles within 15 min, whereas co-expressed V2Rs remained in
the plasma membrane and did not exhibit any detectable internalization
(Fig. 1, panels c and d). Conversely, in the
presence of saturating concentrations (10 µM) of the V2R
agonist AVP, V2Rs were selectively endocytosed without any detectable
internalization of co-expressed
2ARs (Fig. 1,
panels e and f). Agonist-induced endocytosis of the
2AR in HEK293 cells is mediated by clathrin-coated
pits, as indicated by morphological studies and the effects of
biochemical and genetic inhibitors of clathrin-coated pit function
(7-9, 22). Recent studies indicate that agonist-induced endocytosis of
the V2R is also mediated by clathrin-coated pits (17, 27). Consistent
with this, we confirmed that AVP-induced internalization of the
FLAG-tagged V2R was blocked by mild hypertonicity (0.45 M
sucrose) and was also specifically inhibited by overexpression of K44E
(dominant-negative) mutant dynamin (not shown).
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Fig. 1.
Cellular trafficking of the V2R and
2AR upon agonist treatment. HEK293 cells stably
expressing both FLAG epitope-tagged V2R and HA epitope-tagged
2AR at equal levels were treated for 20 min at 37 °C
with 10 µM either no agonist (a and
b), ISO alone (c and d), AVP alone
(e and f), or both ISO and AVP (g and
h). The distribution of both receptors after treatment was
visualized by immunofluorescence microscopy. The upper
panels show the localization of the V2R, and the lower
panels show the localization of the
2AR in the same
field. Black arrows in panels g and h
indicate a cell co-expressing both receptors, and white
arrows indicate a cell expressing only the
2AR
without any detectable V2R.
In contrast to the ability of the 2AR or V2R to
endocytose selectively when activated separately by ISO or AVP,
respectively, surprising results were observed in cells exposed to a
saturating concentration (10 µM) of both ISO and AVP.
Under these conditions, pronounced internalization of the V2R was still
observed, but co-expressed
2ARs remained in the plasma
membrane and appeared to be completely resistant to agonist-induced
internalization (Fig. 1, panels g and h, the
black arrow indicates an example of a cell co-expressing
both receptors). Indeed, the only cells in which
2AR
internalization was observed under these conditions were the minority
of cells in the transfected population (~10-20% of total, depending
on the cell line) that expressed only
2AR without any
detectable V2R (white arrow in Fig. 1, panels g
and h, indicates an example of such a cell). Quantitation of
multiple cells (selected at random in coded specimens, see
"Experimental Procedures") confirmed these observations (Fig.
2A). Similar results were also
observed in transiently transfected cells that vary more widely in
2AR and V2R expression levels (see below). Transient transfection of Chinese hamster ovary cells yielded comparable results,
demonstrating that the inhibition can be observed in more than one cell
type (not shown).
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The heterologous inhibition of 2AR internalization
caused by activation of co-expressed V2Rs was further confirmed using an established flow cytometric assay (26) applied to stably transfected
cells expressing both receptors at closely similar levels (~4
pmol/mg), as assessed by radioligand binding assays (see
"Experimental Procedures"). Consistent with observations made by
fluorescence microscopy, AVP and ISO specifically promoted internalization of the V2R and
2AR, respectively, in a
strictly homologous manner when added separately to the culture medium (Fig. 2B, first, second, fourth, and fifth
bars). In contrast, in cells exposed to both AVP and ISO,
ISO-induced activation of the
2AR was markedly and
specifically inhibited (Fig. 2B, third and sixth bars). The
magnitude of AVP-induced inhibition of
2AR internalization measured by this assay was ~65% and was
statistically highly significant (Fig. 2B legend), whereas
ISO-induced activation of the
2AR caused no detectable
effect on AVP-induced internalization of the V2R. Furthermore, the
extent of V2R-mediated inhibition of
2AR internalization
measured in these experiments was underestimated because of the
presence of a subpopulation of stably transfected cells expressing
2AR without detectable V2R (~15% of total), in which
no AVP-induced inhibition of
2AR internalization is observed (Fig. 1, panels g and h). A similar
extent of inhibition of
2AR internalization was observed
60 min after incubation of co-transfected cells with ISO and AVP (not
shown). When stimulated separately, the extent of internalization of
both receptors reached steady state within 15 min after the addition of
agonist (AVP or ISO, respectively). Taken together, these observations
suggest that AVP-induced activation of the V2R in these cells causes a nearly complete blockade of the internalization of co-expressed
2ARs.
The nonreciprocal nature of the cross-inhibition of 2AR
internalization induced by V2R activation was particularly remarkable because both receptors were expressed at closely similar levels (see
above), and the flow cytometric analysis indicated that the
2AR (when activated individually in the absence of V2R
activation) can internalize in these cells to a significantly greater
extent at steady state than the V2R (Fig. 2B). To begin to
examine the mechanism of the selective V2R-mediated endocytic
inhibition, we examined its generality to several other receptors that
endocytose via clathrin-coated pits. AVP-induced activation of the V2R
caused no detectable inhibition of constitutive endocytosis of
endogenously expressed transferrin receptors, as visualized by
fluorescence microscopy using Texas Red-conjugated transferrin (not
shown). However, V2R activation did cause a nearly complete blockade of etorphine-stimulated internalization of co-expressed HA-tagged
-opioid receptor, which was similar to that observed for the
2AR and was also nonreciprocal (Fig.
3). Together, these observations indicate
that the V2R-mediated inhibition is specific to the mechanism mediating
agonist-induced endocytosis of certain GPCRs and does not reflect a
more general inhibition or saturation of the clathrin-mediated endocytic pathway.
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Opioid receptors, like the 2AR, recruit
-arrestins to
the plasma membrane of transfected HEK293 cells but fail to mediate detectable endomembrane recruitment of arrestins (28-31). In contrast, the V2R recruits
-arrestins both to the plasma membrane and to V2R-containing endocytic vesicles (17). These observations suggest that
the nonreciprocal inhibition of
2AR internalization by
V2R activation might be mediated by receptor-specific trafficking of
arrestins to endomembranes. To begin to test this hypothesis, we
examined the effect of overexpressing
-arrestins on the V2R-mediated blockade of
2AR internalization. Cells co-expressing
FLAG-tagged V2R and HA-tagged
2AR were transiently
transfected with a plasmid encoding a GFP-tagged version of arrestin 3 (
-arrestin 2) (24). Then cells were incubated as above in the
presence of both AVP and ISO, and triple-color fluorescence microscopy
was used to visualize the subcellular distribution of V2R,
2AR, and GFP-tagged arrestin 3 in the same cells. In
contrast to the complete lack of detectable
2AR
internalization in cells expressing
-arrestins at endogenous levels
(Figs. 1 and 2), internalization of the
2AR was readily
observed under the same conditions in cells overexpressing GFP-tagged
arrestin 3 (Fig. 4A). Many of
the punctate structures containing
2ARs observed in
cells overexpressing
-arrestins could be resolved from the plasma
membrane by confocal optical sectioning and were inaccessible to
antibody in nonpermeabilized cells (not shown), demonstrating that
these structures represent bona fide
2AR-containing endocytic vesicles (rather than clusters of receptors present in the plasma membrane). These results were confirmed in multiple cells examined in coded specimens (Fig. 4B). Similar results were observed when transiently
transfecting both receptors into cells stably overexpressing an EE
epitope-tagged arrestin 2 (
-arrestin 1, not shown). Significantly,
overexpression of
-arrestins has little effect on ISO-induced
internalization of the
2AR in HEK293 cells not
expressing the V2R (32). Together these results suggest that
overexpression of
-arrestins is sufficient to specifically
"rescue" the V2R-mediated inhibition of
2AR
internalization. Furthermore we observed that
2ARs and
V2Rs visualized in arrestin-transfected cells were observed in an
overlapping population of endocytic vesicles that colocalized
extensively with GFP-tagged arrestin 3 (Fig. 4A,
corresponding arrows in each panel indicate
examples of such colocalized vesicles in a representative high-power
field including several arrestin-transfected cells). These results
demonstrate that once the V2R-mediated "sequestration" of arrestin
activity is overcome, the co-expressed
2AR and V2R can
enter a similar endocytic pathway.
|
To examine whether endomembrane trafficking of -arrestins is
actually necessary for the V2R-mediated inhibition of
2AR internalization, we used a mutational strategy to
disrupt V2R-mediated recruitment of
-arrestins to endocytic
vesicles. The wild type V2R is highly resistant to dephosphorylation in
endocytic vesicles, leading to endomembrane recruitment of arrestins
and inhibited recycling of receptors (17, 18). Truncation mutations
(362T and 345T) of the cytoplasmic tail create functional receptors
that undergo agonist-induced endocytosis with similarly rapid kinetics
as the wild type receptor (18) but exhibit distinct defects in
agonist-dependent phosphorylation/dephosphorylation. The
345T mutant receptor does not exhibit detectable phosphorylation after
exposure to agonist; the 362T mutant receptor undergoes agonist-induced
phosphorylation but is rapidly dephosphorylated after endocytosis (17,
18). Significantly, neither mutant receptor mediates detectable
endomembrane recruitment of
-arrestins (17). We confirmed this
result (not shown) and then compared the ability of FLAG-tagged
versions of full-length, 362T and 345T mutant V2Rs to mediate
AVP-induced inhibition of the
2AR (co-expressed in
HEK293 cells without overexpression of
-arrestins). Consistent with
observations in stably transfected cells (Figs. 1 and 2), activation of
the full-length V2R strongly inhibited agonist-induced internalization
of co-expressed
2ARs in transiently transfected HEK293
cells (Fig. 5A, panels
a and b). In contrast, neither the 345T (Fig.
5A, panels c and d) nor the 362T (Fig.
5A, panels e and f) mutant receptor
mediated detectable inhibition of
2AR internalization
when examined under the same conditions. The ability of these mutations
to abrogate the AVP-induced inhibition of
2AR
internalization was not a result of differences in expression levels of
the mutant receptors, as immunofluorescence staining intensity present
in individual transfected cells (estimated using an electronic camera
connected to the fluorescence microscope, not shown) confirmed that
345T, 362T, and full-length V2Rs were expressed over a similar range of
expression levels in individual transfected cells. The differential
effects of mutant V2Rs on internalization of the co-expressed
2AR were confirmed by examining multiple cells at random
in coded specimens (Fig. 5B). We also observed extensive
colocalization of the
2AR with both the 362T and 345T
mutant receptors in endocytic membranes visualized in the same cells by
dual label fluorescence microscopy (arrows in Fig.
5A indicate examples of such colocalized endocytic
vesicles). These results further confirm that, once the
2AR endocytic inhibition is rescued either by
overexpression of
-arrestins (Fig. 4) or by mutations of the V2R
that block endomembrane recruitment of
-arrestins (Fig. 5), the
2AR can enter a similar endocytic pathway as the
V2R.
|
We conclude that AVP-induced activation of the V2R mediates a
pronounced heterologous inhibition of agonist-induced endocytosis of
the 2AR as well as certain other GPCRs that endocytose
via clathrin-coated pits. This inhibition is clearly not reciprocal, because activation of the
2AR with saturating
concentrations of agonist does not detectably inhibit agonist-induced
endocytosis of the V2R, even in cells in which both receptors are
expressed at closely similar levels and in which steady state
internalization of the
2AR (induced by ISO in the
absence of AVP) is significantly greater than that of the V2R. The
nonreciprocal nature of this inhibition is correlated with a previously
described difference in the effects of the
2AR and V2R
on intracellular trafficking of
-arrestins (17). We established that
overexpression of
-arrestins is sufficient to rescue the
heterologous inhibition of
2AR internalization mediated
by the wild type V2R. Moreover, mutations of the V2R that prevent
endomembrane recruitment of
-arrestins abrogate the AVP-induced
inhibition of
2AR internalization in cells expressing
-arrestins at endogenous levels. Thus we propose that
receptor-specific intracellular trafficking of
-arrestins
depletes the functional pool of cytoplasmic
-arrestins below
that required to promote ligand-induced endocytosis of certain GPCRs.
Whereas receptor-specific differences in the intracellular trafficking
of various GPCRs have been examined in considerable detail, relatively
little is known about the functional consequences of receptor-specific
differences in the intracellular trafficking of -arrestins. Recent
studies suggest that
-arrestins associated with endocytic membranes
may play an important role in organizing or modulating downstream
signal transduction via endocytosed GPCRs (19, 20, 33). However, to our
knowledge the present results provide the first direct evidence for a
specific functional consequence of
-arrestin trafficking in
regulating GPCR endocytosis itself. Furthermore, we believe these
results provide the first data implicating
-arrestins in a
heterologous form of GPCR regulation, which may complement previous
studies suggesting heterologous regulation of certain G protein-coupled
receptor kinases (34, 35).
In this study we have focused primarily on V2R-mediated effects on
regulated internalization of a subset of GPCRs, which have been
established most definitively to endocytose in HEK293 cells by
clathrin-coated pits. Therefore further studies will be necessary to
examine the generality of our observations to other cell types and
other GPCRs, particularly receptors that are endocytosed by "alternative" mechanisms (8, 36) or further differ in their effects
on intracellular trafficking of -arrestins (16, 37). It will
also be important to determine whether the mechanism characterized in
the present study is capable of mediating cross-inhibition of other
important arrestin-dependent functions such as functional uncoupling of receptors from heterotrimeric G proteins or
receptor-mediated signaling via mitogenic kinase cascades. The levels
of receptor expression examined in the present studies, although
moderate compared with other studies of transfected cells, may still be considerably higher than observed in native tissues. Thus the relevance
of the present observations to endogenously expressed GPCRs remains to
be determined. Nevertheless, as the process of endomembrane recruitment
of
-arrestins can be observed in natively expressing neurons (37),
we anticipate that the heterologous regulatory mechanism described in
the present study may be of considerable physiological importance. For
example it is tempting to speculate that this mechanism may be relevant
to the recently reported ability of the V2R to bypass desensitization
of myocardial adrenergic receptors in an experimental model of
congestive heart failure (38). The potential physiological relevance of
this regulatory mechanism notwithstanding, the present results also establish a novel experimental method by which an important functional activity of cytoplasmic
-arrestins can be modulated acutely in living cells. We anticipate that this approach may be useful as an
adjunct to previously described methods (e.g. dominant
loss-of-function mutations (39, 40), antisense knockdown (41, 42), and gene knockout approaches (43, 44)) that typically allow cellular
-arrestins to be manipulated only over a much longer time scale.
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ACKNOWLEDGEMENTS |
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We thank Dr. J. L. Benovic for providing cDNA encoding arrestin 2 and Dr. M. G. Caron for providing cDNA encoding GFP-arrestin 3. We thank Dr. B. Kobilka for use of his cell harvester and for valuable discussion.
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FOOTNOTES |
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* These studies were supported by research grants from the National Institutes of Health.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 during part of these studies by a postdoctoral
fellowship from the American Heart Association. To whom correspondence should be addressed: Advanced Medicine, Inc., Dept. of Biochemistry, 901 Gateway Blvd., South San Francisco, CA 94080. Tel.: 650-808-6088; Fax: 313-557-2727; E-mail: uklein@advmedicine.com.
Published, JBC Papers in Press, February 8, 2001, DOI 10.1074/jbc.M009214200
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ABBREVIATIONS |
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The abbreviations used are:
GPCR, G
protein-coupled receptor;
HEK, human embryonic kidney;
2AR,
2-adrenergic receptor;
V2R, V2
vasopressin receptor;
PCR, polymerase chain reaction;
HA, hemagglutinin;
GFP, green fluorescent protein;
AVP, [Arg8]vasopressin;
ISO, isoproterenol.
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