(Received for publication, January 13, 1997, and in revised form, February 10, 1997)
From the Departamento de Biología Molecular and Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma) Universidad Autónoma de Madrid, 28049 Madrid, Spain
Rapid regulation of G protein-coupled receptors
appears to involve agonist-promoted receptor phosphorylation by G
protein-coupled receptor kinases (GRKs). This is followed by binding of
uncoupling proteins termed arrestins and transient receptor
internalization. In this report we show that the -adrenergic
receptor kinase (
ARK-1 or GRK2) follows a similar pattern of
internalization upon agonist activation of
2-adrenergic receptors (
2AR) and
that
ARK expression levels modulate receptor sequestration. Stable
cotransfected cells expressing an epitope-tagged
2AR and
ARK-1 show an increased rate and extent of
2AR
internalization compared with cells expressing receptor alone.
Moreover, subcellular gradient fractionation studies suggest that
ARK colocalizes with the internalized receptors. In fact, double
immunofluorescence analysis using confocal microscopy shows extensive
colocalization of
2AR and
ARK in intracellular vesicles upon receptor stimulation. Our results confirm a functional relationship between receptor phosphorylation and sequestration and
indicate that
ARK does not only translocates from the cytoplasm to
the plasma membrane in response to receptor occupancy, but shares
endocytic mechanisms with the
2AR. These data suggest a
direct role for
ARK in the sequestration process and/or the involvement of receptor internalization in the intracellular
trafficking of the kinase.
A general feature of G protein-coupled receptors
(GPCR)1 is the existence of complex
regulatory mechanisms that modulate receptor responsiveness and which
underlie important physiological phenomena such as signal integration,
plasticity, and desensitization. The molecular mechanisms of
desensitization have been investigated using the
2-adrenergic receptor (
2AR) as the main
model system. Work from several laboratories has shown that rapid,
short term
2AR desensitization is due to functional
uncoupling from G proteins as a consequence of receptor
phosphorylation. Agonist occupancy triggers phosphorylation of the
receptor by the
-adrenergic receptor kinase (
ARK-1), a
serine/threonine kinase that specifically phosphorylates the
COOH-terminal cytoplasmic domain of the receptor.
ARK1 is a member
of a family of G protein-coupled receptor kinases (GRKs), which
phosphorylate different GPCRs, and it is now also termed GRK2. The
phosphorylated
2AR interacts with additional regulatory proteins, the
-arrestins, which block signal transduction. The uncoupled receptors are subsequently removed from the plasma membrane in a process termed internalization or sequestration (1-4).
Despite the fact that agonist-promoted sequestration is a common
feature of many GPCRs, the molecular mechanisms involved have remained
elusive and controversial. However, recent data have shed new light
into this field. It has been suggested that sequestration plays a key
role in resensitizing uncoupled GPCRs by allowing the dephosphorylation
and recycling of functional receptors back to the plasma membrane
(5-8). On the other hand, the internalization compartments of
2AR and other GPCRs have been identified as early
endosomes (9-12). Finally, receptor phosphorylation and subsequent
-arrestin binding have been shown to facilitate the process of
sequestration, leading to the suggestion that
-arrestin may play a
direct role as an adaptor molecule for receptor trafficking (14-16).
It should be noted that agonist occupancy of GPCRs does not only
promote changes in the subcellular distribution of the receptor. Upon
receptor activation, ARK transiently translocates to the plasma
membrane (17-20), in a process that seems to be facilitated by
interactions of COOH-terminal regions of the kinase with G protein
subunits (21-23). On the other hand, we have shown recently that a significant amount of
ARK is associated to internal,
microsomal membranes (24-26). However, very little is known about the
mechanisms governing such complex subcellular distribution. In
particular, the way
ARK is recycled after phosphorylating GPCR in
the plasma membrane and its possible relationship with the subsequent
receptor sequestration have not been investigated.
In this context, we have examined the effects of ARK overexpression
on the internalization parameters of epitope-tagged
2ARs and investigated the changes in the subcellular localization of
ARK
that take place during the sequestration process. Our results indicate
a close relationship between the intracellular dynamics of
2ARs and that of the kinase as a consequence of receptor
activation.
All recombinant DNA procedures were
carried out following standard protocols. A cDNA encoding bovine
ARK-1 (donated by Dr. J. L. Benovic, Jefferson University,
Philadelphia) was cloned into the mammalian expression vector pREP4
(Invitrogen). A cDNA encoding the human
2AR modified
to incorporate the Signal FLAG (SF) epitope at the amino terminus (gift
of Dr. B. L. Kobilka, Stanford University, Ref. 27) was cloned into
pREP4. After cleavage, the FLAG epitope can be specifically detected
using the anti-FLAG M1 monoclonal antibody (IBI).
Human embryonic kidney cells
(HEK-EBNA 293) were obtained from Invitrogen and maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum and 0.2 mg/ml geneticin (Sigma). Cells were transfected
with pREP4-ARK1 and/or pREP4-SF
2AR by the calcium
phosphate precipitation method. For selection of stable transfected
cells, 0.25 mg/ml hygromycin (Calbiochem) was added to the culture
medium following transfection. Colonies originating from single cells
were subcloned into 96-well tissue culture plates and screened for
ARK and
2AR protein expression by immunofluorescence,
immunoblot analysis, and [3H]dyhydroalprenolol binding
(see below). The level of
2AR expression in the selected
clonal cell lines was between 0.7 and 2 pmol/mg of whole cell protein.
GRK2 levels were in the range of ~25 pmol/mg of protein (~20-fold
higher than those of control cells). Experiments were performed in
different clonal cell lines. In addition, similar experiments were
carried out in HEK-293 cells transiently expressing both
ARK-1 and
2AR.
Receptor sequestration was quantitated by flow cytometry essentially as described previously (7). Following treatment in the presence of 0-10 µM isoproterenol (Sigma) for the indicated times at 37 °C in 96-well culture plates (50,000 cells/well), the cells were quickly chilled, washed by centrifugation, and labeled at 4 °C for 60 min with M1 FLAG antibody (1:2000 dilution) in phosphate-buffered saline supplemented with 2% fetal bovine serum. After washing by centrifugation in the same vehicle, the cells were subsequently labeled with a 1:100 dilution of fluorescein-labeled goat anti-mouse antibody (Amersham Corp.). Cells were washed and fixed in 3.6% formaldehyde and the fluorescence present at the cell surface analyzed within 1 h on a Coulter scientific flow cytometer. Base-line cell fluorescence intensity was determined with washed unlabeled cells and cells labeled only with the goat anti-mouse fluorescein-conjugated antibodies. The fraction of sequestered receptors was then calculated by comparing the signal obtained in the absence or presence of agonist.
Subcellular FractionationAliquots of HEK-293 cells
expressing both ARK-1 and
2AR were incubated at
37 °C for 20 min in the presence or absence of 10 µM
isoproterenol in the culture medium. The reaction was stopped by
addition of ice-cold phosphate-buffered saline. The same number of
control and treated cells (obtained from one 10-cm dish per assay) were
harvested and resuspended at 2-3 × 106 cells/ml in
homogenization buffer (0.25 M sucrose, 10 mM
Hepes, pH 7.2, 1 mM EDTA, 1 mM benzamidine, 100 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor) and homogenized in ice in a Dounce
homogenizer (40 strokes). Particulate fractions were obtained by
centrifuging postnuclear supernatants (10 min, 750 × g
at 4 °C) at 250,000 × g for 30 min at 4 °C.
Gradient fractionation of membrane fractions was performed as described (28), with some modifications. Briefly, pellets were resuspended in the
same volume of homogenization buffer and mixed with Percoll (from a
stock solution containing 90% Percoll (Sigma) in 0.25 M
sucrose) and bovine serum albumin to a final concentration of 27%
(v/v) Percoll and 4 mg/ml bovine serum albumin in a final volume of
11.5 ml. The mixture was layered over a 1-ml cushion of 2.5 M sucrose and centrifuged at 29,000 × g
for 90 min at 4 °C in a 50Ti rotor (Beckman). Fractions of 0.4 ml
were collected from the bottom of the tube and tested for
2AR binding activity by
[3H]dyhydroalprenolol binding as described (28) and for
ARK protein by Western blot. Samples to be analyzed by Western blot
were diluted with the same volume of SDS buffer, incubated for 30 min
at 4 °C, and centrifuged at 250,000 × g for 45 min
at 4 °C to sediment the Percoll. Fractions were resolved by 7.5%
SDS-polyacrylamide gel electrophoresis, electroblotted onto
nitrocellulose filters, and probed with AB9, a polyclonal antibody
raised against purified recombinant bovine
ARK (generous gift of Dr.
J. L. Benovic, Jefferson University) as described (25, 26), and
developed using a chemiluminiscent method (ECL, Amersham).
Densitometric analysis of the blots was performed using a Molecular
Dynamics laser densitometer.
Cells overexpressing ARK-1
and epitope-tagged
2AR were grown on glass coverslips
and incubated in the presence or absence of the desired concentrations
of isoproterenol or other modulators for various times at 37 °C.
After treatment, cells were fixed in 4% formaldehyde in
phosphate-buffered saline and permeabilized with 0.2% Nonidet P-40,
5% dry non-fat milk, and 50 mM Tris-HCl, pH 7.4. Polyclonal
ARK antibody AbFP1 (raised against a fusion protein
containing amino acids 50-145 of bovine
ARK1) (1:500, see Ref. 25)
and M1 monoclonal anti-FLAG antibody to detect the tagged
2AR were then applied to the specimen in the same blocking medium. After 60 min, the samples were extensively washed, and
bound antibodies were detected using species-specific antibodies labeled with different fluorochromes (fluorescein-labeled goat anti-rabbit and Texas Red-labeled goat anti-mouse (Amersham) at a
dilution of 1:100 for 30-45 min. In some experiments, colocalization of internalized
2AR with transferrin receptors was
established by serial double labeling immunofluorescence using M1
anti-FLAG and anti-transferrin receptor monoclonal antibodies. Confocal microscopy was performed using a Zeiss LSM 320 confocal laser scan
microscope and conventional immunofluorescence microscopy by using a
Zeiss Axiovert 35 microscope with 63 × NA 1.3 and 100 × NA
1.3 oil-immersion lenses. Absence of signal crossover was established
using single-labeled samples.
To investigate the relationship between
agonist-dependent receptor phosphorylation by ARK and
the internalization process, we used flow cytometry to measure the
extent of epitope-tagged
2AR remaining at the cell
surface (7, 11) after treating with the
-agonist isoproterenol
HEK-293 cells stably expressing similar levels of
2AR
alone or in combination with
ARK1. Fig. 1 shows that
the extent of receptor internalization attained at low concentrations
of agonist (left and middle panels) is markedly increased in cells cotransfected with
ARK. At a high concentration of isoproterenol (10 µM, right panel) there is
only a slight effect of
ARK cotransfection on the extent of
internalization, although the rate of the process appears to be
slightly enhanced with respect to control cells. An increased
kinase/receptor ratio in the cotransfected cells would increase the
proportion of phosphorylated receptors in response to a given
concentration of agonist, thus leading to the observed increase in the
extent of receptor internalization noted at low concentrations of
agonists, as reported previously for m2 muscarinic acetylcholine
receptors (14). The fact that previous reports (13, 29, 30) have failed
to show an increased internalization of wild-type
2AR as
a consequence of
ARK overexpression may be ascribed to the fact that
only high agonist doses and long times of treatment (30 min) were
investigated. Nevertheless,
ARK overexpression has been shown to be
able to rescue the sequestration of internalization-deficient
2AR mutants (13, 15) and a dominant negative mutant of
ARK decreased both wild-type and mutant
2AR agonist-induced receptor internalization (13, 14). In line with these
data, our results support a functional relationship between
2AR phosphorylation by
ARK and receptor
sequestration.
We next investigated if changes in the subcellular distribution of the
kinase can be observed during agonist-induced 2AR endocytosis. Using subcellular gradient fractionation, a clear change
in the pattern of
2AR binding in particulate fractions can be detected in HEK-293 cells transfected with both
2AR and
ARK in the presence of 10 µM
isoproterenol (compare ISO versus CONTROL in Fig.
2A). Interestingly, a relative increase in
ARK protein is noted in the same fractions enriched in internalized
2AR in the agonist-treated cells (Fig. 2, B
and C). Fraction 24 (showing most of the internalized
receptors) contains 1.88 ± 0.23-fold more
ARK protein than the
average of fractions 22-25 in the isoproterenol-treated cells,
compared with 0.98 ± 0.07 in fraction 24 of control cells
(average ± S.E. of three experiments, p < 0.05).
Consistent with an agonist-dependent redistribution of
ARK, a decrease in the proportion of kinase associated to plasma
membrane fractions is noted (Fig. 2C, small arrow). These results suggested that
ARK may colocalize with receptors during the
internalization process. Both
2AR and GRK2 levels in
fraction 24 have been estimated to increase in treated cells in the
range of 10-20 pmol (data not shown). Unfortunately, a more detailed quantitative analysis of the stoichiometry of
2AR and
GRK2 in internalized vesicles is not possible using this experimental approach.
To further study the changes in subcellular distribution taking place
upon ligand binding, we performed double immunofluorescence confocal
microscopy studies. Cells were transfected with epitope-tagged 2AR and
ARK, so the localization of both proteins can
be analyzed in the same samples by using monoclonal antibodies that
recognize the receptor tag and specific polyclonal antibodies raised
against
ARK (25) coupled to different chromophores. Fig.
3A shows that in control conditions the
receptor is located in the plasma membrane whereas
ARK (Fig.
3B) displays a diffuse cytoplasmic localization as well as a
plasma membrane staining. The plasma membrane localization of
ARK
can be detected even in the absence of agonists in cells overexpressing
2AR, probably as a consequence of the basal activity of
receptors2; the same effect was observed
for
-arrestin localization in similar experimental conditions (31).
After agonist stimulation (0.1 or 10 µM isoproterenol for
C and D and E and F in Fig.
3, respectively), the
2AR distribution is markedly and
gradually changed (Fig. 3, C and E). A similar
punctate pattern, intracellular structures or vesicles, can be observed
for
ARK, with extensive colocalization with the receptor (Fig. 3,
D and F). These data were further confirmed by
image merging (not shown). Similar results were obtained in transiently
transfected cells using either confocal or conventional double labeling
immunoflourescence microscopy (not shown). It should be noted that
sequestered receptors colocalize with transferrin receptors in
endocytic vesicles (not shown), in agreement with previous reports
investigating
2AR internalization (10, 11). It is worth
noting that immunoflourescence studies appear to show a more clear and
extensive colocalization of internalized receptor and kinase than
anticipated by the gradient fractionation data. This could be ascribed
to a better preservation in the former experimental approach of GRK2
association to endosomal vesicles, which could be partially lost during
cell lysis and fractionation procedures given the peripheral nature of
kinase association to membranes (18, 25), as well as to the favored
visualization of structures displaying concentrated antigens (either
2AR or
ARK) by the indirect immunofluorescence
technique. The marked change in
ARK subcellular distribution does
not appear to be a consequence of signaling pathways downstream
receptor activation, since treatment of cells transfected only with the
kinase with forskolin or aluminum fluoride does not promote any
apparent changes in the
ARK localization pattern (data not shown).
The presence of
2AR and
ARK in intracellular vesicles
is not detected when agonist treatment is performed at low temperature,
in line with previous observations (Ref. 10 and data not shown). It is
also worth noting that the colocalization with the
2AR
during the sequestration process is not extended to other proteins
involved in signal transduction, since receptor-activated
G
s does not colocalize with
2AR in
endosomes (32).
Taken together, our results confirm that ARK expression levels can
modulate the extent of receptor sequestration at certain agonist
concentrations and, more importantly, show that
ARK does not only
translocates to the plasma membrane upon receptor activation, but
colocalizes with
2AR during receptor internalization.
Previous experiments have indicated that
2AR and other
GPCRs are internalized via the clathrin-coated vesicle-mediated
endocytic pathway. Sequestered receptors have been shown to colocalize
with endosomal markers, such as transferrin, rab 5, or clathrin
(10-12, 31), and dynamin is essential for
2AR (but not
angiotensin AT1A receptor) internalization (16). Very
recent reports have focused on the role of the uncoupling protein
-arrestin in this process. Overexpression of
-arrestin rescues
the sequestration of internalization-defective
2AR
mutants, and dominant negative arrestins inhibit wild-type
2AR sequestration (15, 16), thus suggesting that these
uncoupling proteins would act as adaptor molecules by helping to target
GPCRs to the endocytic machinery. In fact, during the writing process
of this manuscript, a report showing an interaction of
-arrestin
with clathrin "in vitro" and
agonist-dependent colocalization of
2AR,
-arrestin, and clathrin has been published (31). In this context,
the fact that the expression of wild-type or dominant negative
ARK
facilitates or decreases internalization, respectively (this report and
Refs. 13, 14, 30, and 33; see above), may be explained by a facilitation of the binding to the phosphorylated receptor of endogenous
-arrestin, which would then directly mediate the
internalization process. However, the colocalization of
ARK with
2AR in endocytic vesicles that we report here may
suggest that the kinase, in addition to
-arrestin, plays a direct
role in receptor sequestration, either by contributing to a correct
conformation of the various domains of the receptor involved in
sequestration (34, 35) as a consequence of the kinase interaction with
cytoplasmic receptor domains other than the phosphorylation sites
(reviewed in Ref. 2) or by direct interaction of
ARK with as yet
unidentified proteins of the endocytic machinery.
Alternatively, or in addition, the presence of ARK in the
internalization vesicles may indicate that the endocytic system plays a
role in the recycling of the kinase that translocated to the plasma
membrane upon receptor stimulation. Such rapid kinase sequestration
would be in agreement with the transient nature of its agonist-induced
association with the plasma membrane (17-20) and may contribute to the
modulation of
ARK subcellular distribution (24-26). It is also
possible that
ARK serves other unknown cellular functions in the
internalization vesicles. The recently reported functional relationship
between
ARK and heterotrimeric G proteins in intracellular
organelles (26) and the role of these G proteins in regulating
intracellular trafficking is an intriguing possibility in this regard
(36 and references therein).
The coexistence of 2AR and the regulatory proteins
ARK and
-arrestin in the same cellular structures during
agonist-induced receptor internalization raises important questions to
be addressed in future research. Whether
ARK (and
-arrestin) are
bound (simultaneously or not) to the
2AR or to other
components of the endocytic vesicles (G protein
subunits,
lipids, etc.) should be investigated. On the other hand, and in line
with the recent report by Benovic and colleagues (31), the
identification of additional cellular proteins able to interact with
ARK and
-arrestin (or receptor-kinase-arrestin complexes), and
the characterization of its functional relevance, may help to better
understand the internalization pathways of GPCRs and their
physiological role in the modulation of cellular responses to
messengers.
We thank Drs. B. K. Kobilka and J. L. Benovic for experimental tools and suggestions, M. Sanz for skillful secretarial assistance, Drs. A. Aragay and C. Murga for critical reading of the manuscript, and Prof. F. Mayor for continuous encouragement.