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J Biol Chem, Vol. 273, Issue 52, 35238-35244, December 25, 1998
From the GRK2 is a ubiquitous member of the G
protein-coupled receptor kinase (GRK) family and has been shown to play
a key role in determining the desensitization and resensitization
patterns of a variety of G protein-coupled receptors. In this report,
we show that GRK2 is actively degraded by the proteasome proteolytic
pathway, unveiling a new mechanism for the rapid regulation of its
expression levels. Interestingly, activation of
Agonist-promoted desensitization of G protein-coupled receptors
(GPCR)1 involves rapid
phosphorylation of the activated receptor by a family of specific G
protein-coupled receptor kinases (GRK), followed by binding to the
phosphorylated receptor of regulatory proteins termed arrestins,
leading to uncoupling from G proteins (1, 2). GRK2 is the most
ubiquitous member of this kinase family and has been shown to play a
key regulatory physiological role in the heart and other tissues (3,
4). GRK2 may also play a role in facilitating the transient
agonist-induced GPCR internalization allowing receptor
dephosphorylation and recycling (5-7). Thus, GRK2 contributes to
determine the rate and extent of both GPCR desensitization and
resensitization. This fact, together with the recently suggested
participation of GRKs and arrestins in the GPCR-mediated
mitogen-activated protein kinase cascade (8) and the ability of GRK2 to
phosphorylate tubulin (9), further stresses the relevance of GRK2 in
GPCR signaling.
Changes in GRK2 expression may therefore alter the efficacy of GPCR
signal transduction systems. In this regard, GRK2 expression levels
have been reported to increase in human heart failure (10), hypertension (11) or upon lymphocyte activation (3), or to decrease
rapidly in several rat tissues during the perinatal
period.2 Despite recent
advances in the understanding of the mechanisms regulating the activity
and subcellular distribution of GRK2 (1, 2, 6, 12, 13), very little is
known about the mechanisms that regulate the kinase cellular
complement. In this study, we sought to characterize the degradation
process of GRK2 protein and its potential modulation. Our results
clearly show that GRK2 is rapidly degraded by the proteasome pathway
and that kinase degradation is modulated by activation of
Cell Treatments and Metabolic Labeling--
HEK-293 cells
(American Type Culture Collection, Manassas, VA) were transfected with
pCDNA3-GRK2, pCDNA3-GRK2-K220R, and/or pws636-SF
Immunoprecipitation--
Cells were washed and lysed in RIPA
buffer (50 mM Tris, pH 7.5, 300 mM NaCl, 1%
Triton X-100, 0.1% SDS, and 0.5% deoxycholate, with protease
inhibitors) for 120 min at 4 °C with continuous rocking and then
centrifuged (15,000 × g, 15 min). Protein extracts were immunoprecipitated with the specific GRK2 polyclonal antibody AbFP1 (12), followed by incubation with protein A-Sepharose for 1 h. Immunoprecipitates were resolved in 10% SDS-PAGE. After fluorography (Amplify, Amersham Pharmacia Biotech), the gel was dried
and exposed at Western Blotting--
Cells lysates were obtained exactly as
described (12) and resolved in 7% SDS-PAGE. Proteins were transferred
to nitrocellulose membranes and probed with GRK2 Ab9 polyclonal
antibody raised against recombinant GRK2 as described (5, 12), and the
blot was developed using a chemiluminiscent method (ECL, Amersham
Pharmacia Biotech). In other experiments, blots were analyzed using the GRK2 polyclonal antibodies AbFP2 or affinity-purified Ab1 (17), raised
against different regions of bovine GRK2 (see "Results"). Densitometric analysis of the blots was performed as above.
In Vitro GRK2 Degradation Assays--
Bovine GRK2 was
overexpressed in Sf9 cells using the baculovirus expression
system and purified as described previously (12). Purification of a 20 S proteasome (MCP) from rat liver was as described (18, 19). The
in vitro proteolytic assay was performed by incubating 2 µg of purified MCP with 1.25 µg of GRK2 exactly as reported (18,
19). Proteins were resolved in 10% SDS-PAGE and analyzed by Coomassie
Blue staining. Alternatively, GRK2 samples (10 µg) undigested or
following digestion with proteasome for 60 min were injected on a gel
filtration HPLC column (TSK 5000, Beckman, Palo Alto, CA) (18).
Fractions in the ~80-kDa range were tested for either the presence of
GRK2 and proteolytic fragments in Western blot or for GRK2 activity as
detailed below.
Determination of GRK2 Activity--
GRK2 samples obtained by gel
filtration on an HPLC column (see above) were diluted 1:10, and GRK2
activity was assessed by the rhodopsin phosphorylation assay as
described (12). Relative GRK2 activity was estimated by densitometric
analysis of the autoradiography.
Ubiquitination of GRK2--
HEK-293 cells were transiently
transfected by the calcium phosphate precipitation method with
different combinations of expression vectors for GRK2,
Desensitization and Adenylyl Cyclase Assays--
HEK-293 cells
stably expressing GRK2 and Internalization Assays--
Following preincubation with 30 µM lactacystin or vehicle (control) in DMEM for 3 h,
cells were challenged with 0.1 or 1 µM isoproterenol for
2 min at 37 °C, washed by centrifugation, and labeled at 4 °C
with M1 FLAG antibody. Receptor sequestration was then quantitated by
flow cytometry essentially as described (5). The fraction of
sequestered receptors was calculated by comparing the signal obtained
in the absence or presence of agonist.
GRK2 Is Degraded by the Proteasome in Vivo and in Vitro--
The
GRK2 degradation rate was measured by pulse and chase analysis using
HEK-293 cells that stably express GRK2. In such cells, neither the
subcellular localization pattern of the kinase nor the ratio between
subcellular compartments was altered when compared with endogenous GRK2
(14). A rapid decay in the 35S-labeled GRK2
immunoprecipitated by a specific kinase antibody is shown in Fig.
1A, indicating that GRK2 is a
short-lived protein with a half-life estimated at ~60 min. A similar
value was obtained for the endogenous GRK2 in identical experiments
performed in Jurkat cells (data not shown), ruling out the possibility
that overexpression might alter the normal rate of kinase proteolysis. To ascertain which mechanism was responsible for the rapid GRK2 degradation, pulse and chase experiments were performed with specific inhibitors of different proteolytic pathways (Fig. 1B).
Treatment with the cysteine protease inhibitor ALLN clearly prevents
GRK2 degradation after 4 h of chase when compared with control
conditions, at concentrations (50 µM) in which it
inhibits the proteasome pathway but not at doses (1 µM)
in which only nonproteasomal proteases are blocked (22), thus excluding
the involvement of the latter. The presence of leupeptin, an inhibitor
of protein degradation in lysosomes, did not alter GRK2
degradation. These results suggested that the proteasome pathway
was involved in the rapid GRK2 proteolysis. Consistently, the addition
of the highly specific proteasome inhibitor lactacystin fully blocks
kinase degradation in intact cells (Fig. 1B).
To further address the role of the proteasome in regulating GRK2
stability, in vitro degradation experiments were conducted with recombinant GRK2 and purified 20 S proteasome from rat liver (18).
The purified MCP preparations were judged homogeneous by SDS-PAGE
analysis (Fig. 2A). Under the
conditions tested, recombinant GRK2 (indicated as the 80-kDa protein in
Fig. 2A) is indeed rapidly degraded, whereas GRK2 incubated
for 2 h without MCP remains unaltered. To exclude the possibility
that a minor contaminant protease present in the incubation could
contribute to GRK2 proteolysis, we tested different MCP preparations
for their ability to degrade GRK2, obtaining essentially the same
results (data not shown). Moreover, different doses of the specific
proteasome inhibitor lactacystin clearly prevent GRK2 cleavage as
compared with control (Fig. 2B). Altogether, our data
indicate that the kinase is a target for the proteasome, in
vivo as well as in vitro. Interestingly, our in
vitro GRK2 degradation experiments reveal the presence of a ~74-kDa band that appears as GRK2 decays (Fig. 2, A and
C). This protein appears to represent an intermediate
proteolytic product (also degraded by MCP following longer incubation
times), whose formation is blunted in the presence of lactacystin (Fig.
2C). In a first attempt to determine whether the MCP
initially digests the NH2- or the COOH-terminal region of
GRK2 to yield the 74-kDa protein, we performed immunoblot analysis of
GRK2 preparations incubated with or without MCP, using different GRK2
antibodies. Both intact GRK2 and the 74-kDa band are detected
efficiently by the polyclonal antibody Ab9 raised against recombinant
GRK2 and AbFP2, a polyclonal antibody against the COOH terminus
(residues 436-689) (Fig. 2C). Moreover, antibody Ab1, which
recognizes a more restricted epitope (GRK2 residues 648-655) that
would be eliminated in an initial COOH-terminal cleavage, yields the
same results (Fig. 2C, right panel), strongly
suggesting that GRK2 is initially digested at its NH2
terminus.
The generation of the 74-kDa band as a result of GRK2 degradation
in vitro raised the question of whether this cleavage
product could be also observed in vivo. Fig.
3A indicates that the 74-kDa band is indeed immunodetected when measuring steady-state protein levels in cellular lysates from HEK-293 cells stably overexpressing GRK2 alone or with
Most proteins targeted to the proteasome pathway undergo
polyubiquitination (23, 24). To investigate the possible role of
ubiquitination in basal and induced degradation of GRK2 in intact
cells, we transiently transfected HEK-293 cells with different combinations of a tagged ubiquitin construct, GRK2, and
Given that Blocking GRK2 Degradation Alters
Previous reports have provided evidence that GRK2 plays a facilitatory
role in receptor internalization (5, 6, 28). Consistently, the blocking
of kinase degradation with lactacystin markedly enhanced (up to
2.6-fold) receptor sequestration after a short term (2 min) challenge
with isoproterenol (Fig. 5, right panel). These results are
in agreement with our previous report (5) and further indicate that the
impairment of the normal kinase turnover has important functional
consequences on receptor desensitization and internalization processes.
Changes in the cellular complement of GRK2 can modulate the
desensitization/resensitization processes of a variety of GPCR (7, 29)
and may prove critical in determining the specificity of regulation of
certain receptors (4, 7, 30). In this report, we show that GRK2
displays a rapid turnover through the ubiquitin-dependent
proteasome pathway. This proteolytic pathway is responsible for the
rapid degradation of an increasing number of cell cycle regulators and
key signaling proteins (see Ref. 31 for a recent review). Changes in
the stability of proteins as a consequence of dysregulated proteasome
complex activity can lead to an impairment in the cellular functions
mediated by these proteins (31). In this line, our data indicate that
GRK2 degradation is modulated by GPCR activation and that blocking of
kinase degradation strongly affects its role as regulator of GPCR function.
Proteins targeted to the proteasome pathway usually undergo
polyubiquitination (23, 24), although this is not an absolute requirement (16, 32, 33). We show that GRK2 ubiquitination and
proteolysis by the proteasome pathway is increased upon agonist activation of GPCR, although the kinase also exhibits weak
ubiquitination under unstimulated conditions, suggesting that common
mechanisms can regulate signal-dependent and basal GRK2
degradation in contrast to I In contrast to most proteasome substrates that undergo a complete and
processive degradation, proteolytic processing of GRK2 by the
proteasome yields a 74-kDa fragment that transiently accumulates in vivo as well as in vitro. The generation of
this product could only be a "marker" of the initial steps of GRK2
degradation, like the 42-kDa fragment of cyclin B (34), and/or have an
unknown physiological role. Consistent with the latter possibility, the 74-kDa fragment indeed displays kinase activity toward a G
protein-coupled receptor substrate. Interestingly, its ability to
phosphorylate rhodopsin is clearly lower than that of the full-length
kinase. In this regard, it has been recently reported that GRK2 is also able to phosphorylate other proteins different from G protein-coupled receptors, such as tubulin (9). It is tempting to speculate that the
proteolytic removal of a NH2-terminal domain of GRK2 by the
proteasome both alters the kinase efficacy toward GPCR and modifies
kinase activity toward other cellular substrates.
We provide evidence that Several recent reports suggest that both the extent and rate of
receptor internalization as well as the pattern of
desensitization/resensitization processes may vary depending on the
cellular content of GRK2 and In summary, our results indicate that receptor activation leads not
only to changes in GRK2 activity and subcellular localization but also
promotes enhanced GRK2 proteolysis through a
ubiquitin-dependent proteasome pathway.
Activity-dependent regulation of GRK2 stability may provide
an important mechanism for modulating its expression levels,
contributing to the regulation of desensitization/resensitization patterns of GPCR. Changes in GRK2 levels promoted by the activation of
a given receptor may also modify the efficacy of the cellular response
to subsequent challenges by other agonists acting through different
GPCR. This new pathway of receptor-dependent modulation of
the stability of a regulatory protein may therefore have important physiological consequences.
We thank Dirk Bohmann for tagged ubiquitin
constructs, A. de Blasi for the Ab1 antibody, J. L. Benovic for
the gift of GRK2 and GRK2-K220R cDNAs and the Ab9 antibody, and S. Cotecchia for the constitutively active *
This work was supported by Grant PM95-0033 from the
Ministerio de Educación y Cultura, Grant AE 00213/95 from the
Comunidad de Madrid, and Grant BMH4-98-3566 from the European Union
(to F. M.) and grants from Comision Interministerial Ciencia y
Tecnología and Fundación Ramón Areces (to J. G. C.). The Centro de Biología Molecular holds an
institutional grant from the Fundación Ramón Areces.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.
¶
To whom correspondence should be addressed. Tel.:
34-91-397-48-65; Fax: 34-91-397-47-99; E-mail:
fmayor{at}cbm.uam.es.
The abbreviations used are:
GPCR, G
protein-coupled receptor; ALLN, N-acetyl-Leu-Leu-norleucinal; 2
P. Penela and F. Mayor, Jr., manuscript in preparation.
3
A. Ruiz-Gómez, unpublished data.
4
P. Penela, A. Muñoz, and F. Mayor, Jr.,
manuscript in preparation.
Degradation of the G Protein-coupled Receptor Kinase 2 by the
Proteasome Pathway*
,,¶
Departamento de Biología Molecular,
Centro de Biología Molecular "Severo Ochoa," Consejo
Superior de Investigaciones Científicas-Universidad
Autónoma de Madrid, Universidad Autónoma, E-28049 Madrid,
Spain and the § Departamento de Bioquímica e
Instituto de Investigaciones Biomédicas, Facultad de Medicina,
Universidad Autónoma, E-28029 Madrid, Spain
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
2-adrenergic receptors (2AR) markedly increases GRK2 ubiquitination and degradation through the
proteasome pathway. In addition, blocking GRK2 degradation notably
alters 2AR signaling and internalization, consistent with a relevant physiological role for GRK2 proteasomal degradation. Activity-dependent modulation of GRK2 cellular levels
emerges as an important mechanism for modulating the cellular response to agonists acting through G protein-coupled receptors.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
2-adrenergic receptors (2AR). Regulation
of GRK2 stability may provide an important mechanism for modulating the
cellular levels of this kinase and contribute to the regulation of
desensitization/resensitization patterns of GPCR, as suggested by the
fact that blockade of GRK2 degradation alters 2AR
signaling as well as receptor internalization.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
2AR by the calcium phosphate precipitation method and selected for stable expression as described (5). In some experiments, cells were transiently transfected as reported (14) with different combinations of GRK2 and 2AR, as well as constitutively
active 2AR (15) or a COOH-terminal truncated mutant
(amino acids 1-354) of the mouse
2AR.3 For
metabolic labeling, cells were kept for 2 h in methionine and
cysteine-free Dulbecco's modified Eagle's medium (DMEM) and then
incubated for 15-30 min in DMEM supplemented with 250 µCi/ml of
[35S]methionine and [35S]cysteine labeling
mixture (NEN Life Science Products).The plates were washed with
phosphate-buffered saline and chased for the indicated times in DMEM
plus 10% fetal bovine serum. The proteolysis inhibitors lactacystin
(Calbiochem, La Jolla, CA), ALLN (Sigma), and leupeptin (Boehringer
Mannheim) were added 90 min before metabolic labeling and maintained
during the chase periods (16). Pulse-labeled cells expressing
2AR were challenged with isoproterenol (Sigma) or
vehicle during chase periods in DMEM supplemented with 20 mM HEPES (pH 7.5) and 1 mM ascorbic acid (Sigma).
70 °C. Band density was quantitated by laser densitometric analysis.
2AR, and HA-ubiquitin (20). Cells were treated with
isoproterenol or vehicle for 30 min, and ubiquitin conjugates were
purified by immunoprecipitation with a specific monoclonal antibody
against the HA epitope (Boehringer Mannheim). The presence of
ubiquitinated GRK2 was subsequently analyzed by Western blotting with
Ab9 antibody as described above. Similar GRK2 and ubiquitin expression
levels were confirmed in cell lysates by dot blot and Western blot
analysis using their specific antibodies. 2AR levels were determined by radioligand binding as described (5).
2AR or transiently
transfected with GRK2 and wild-type 2AR or COOH-terminal
truncated 2AR were subcultured (6 × 105 cells) for 12 h before preincubation with 30 µM lactacystin or vehicle (90 min, 37 °C) and
maintained in its presence through the following steps. Subsequently,
cells were incubated with 10 µM isoproterenol or vehicle
(30 min, first challenge). Afterward, cells were extensively washed
with phosphate-buffered saline and incubated with DMEM for 30 min
(resensitization period) before adding 10 µM
isoproterenol (10 min, second challenge). Lysates were obtained and
assessed in triplicate for cAMP levels as described (21). Data were
corrected according to the total protein amount in each sample and
compared with values obtained in control conditions (not treated with
lactacystin and not undergoing the first challenge with agonist).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
GRK2 degradation is a rapid process mediated
by a proteasome-dependent pathway. A, GRK2
turnover was determined by pulse and chase experiments in stably
transfected HEK-293 cells. Cells (10 × 106) were
processed as described under "Experimental Procedures."
35S-Labeled GRK2 immunoprecipitated by the anti-GRK2
antibody AbFP1 was resolved in SDS-PAGE followed by fluorography
(top) and densitometry. Data (mean ± S.E.) from three
or four independent experiments are shown in the plot. B,
cells (2 × 106) were pulsed and chased with or
without (control) ALLN, leupeptin, or lactacystin (see "Experimental
Procedures"). GRK2 was immunoprecipitated and analyzed as in
panel A. Results are representative of two independent
experiments performed in duplicate.
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Fig. 2.
Limited proteolysis of GRK2 by purified 20 S
proteasome. A, recombinant GRK2 (80-kDa band) was
incubated for the indicated times with or without (C120)
purified 20 S proteasome (MCP) and resolved in SDS-PAGE followed by
Coomassie Blue staining. An intermediate degradation product of 74 kDa
is detected. MCP components are observed at the bottom of
the gel. B, GRK2 was digested with MCP (control) or
MCP plus different concentrations of lactacystin, resolved in 7%
SDS-PAGE and analyzed as in panel A. C, GRK2
digested with (+MCP) or without MCP (C60) was
resolved in 7% SDS-PAGE and immunoblotted with anti-GRK2 polyclonal
antibodies Ab9 (dilution, 1:1000), AbFP2 (dilution, 1:600), and
affinity-purified Ab1 (dilution, 1:20). Results are representative of
two or three independent experiments.
2AR (Fig. 3A, lanes
4 and 5), whereas it is undetectable in untransfected
cells (Fig. 3A, lane 1). Notably, the 74-kDa
fragment seems to be more abundant in cells coexpressing GRK2 and a
constitutively activated 2AR mutant (15) (lane
2 versus lane 3 in Fig. 3A). Moreover, we
are able to immunoprecipitate the 74-kDa protein with a GRK2-specific
antibody (Fig. 3B) but not with a preimmune serum (data not
shown), when cells are metabolically labeled for periods longer than
those used in pulse and chase experiments. These data indicate that the
partial digestion of GRK2 by the proteasome takes place in
situ and suggest that receptor activation might play some role in
the process to render this product. We next determined the functional
activity of the 74-kDa fragment by testing its ability to phosphorylate
rhodopsin, a well known GRK2 substrate (12). For this purpose, kinase
samples, undigested or digested with MCP, were purified by HPLC
chromatography to remove MCP prior to the phosphorylation assay. The
eluted fractions were resolved by SDS-PAGE and analyzed in Western blot
(Fig. 3C). Most GRK2 was cleaved by MCP to yield the 74-kDa
fragment. As can be seen in Fig. 3D, MCP-digested fractions
(open circles) have less capacity to phosphorylate rhodopsin
than undigested fractions (filled circles). Samples treated
with MCP still display undigested GRK2 (~27%), which may account for
the remaining activity. The decrease in activity (a 44% reduction) is
lower, however, than that expected in such a case (a 73% reduction)
indicating that the 74-kDa fragment can indeed phosphorylate rhodopsin,
although with less efficiency than GRK2.
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Fig. 3.
The 74-kDa GRK2 fragment is detected in
intact cells and displays reduced kinase activity in vitro.
A, lysates from cells expressing GRK2 were analyzed in
immunoblots with a kinase antibody (Ab9). Lanes 1-3,
HEK-293 cells transiently transfected with GRK2, 2AR, or
constitutively active 2AR mutant constructs. Lanes
4 and 5, HEK-293 cell clones stably expressing GRK2
alone or with 2AR. B, cells as in Fig.
1A were metabolically labeled for 1 h (lane
1) or 2 h (lane 2) and immunoprecipitated with
anti-GRK2 AbFP1 antibody. C, GRK2 digested with MCP
(open symbols) or undigested (control, filled
symbols) was subjected to HPLC chromatography and fractions
resolved in SDS-PAGE and developed with anti-GRK2 antibody Ab9
(upper panel), quantified, and plotted. The sum of GRK2
control fractions reactivity was considered as 100% of
immunoreactivity. Squares, GRK2; triangles,
74-kDa protein. D, activity of the fractions
assessed in panel C was determined using a rhodopsin
phosphorylation assay. Data are represented as the percentage of total
GRK2 activity in all fractions. Open circles, undigested
GRK2; filled circles, MCP-digested GRK2. Results in all
panels are representative of two independent experiments.
2AR Activation Increases GRK2 Ubiquitination and
Degradation--
To explore whether receptor activation can modulate
kinase turnover, we treated HEK-293 cells stably coexpressing GRK2 and 2AR with the -agonist isoproterenol (10 µM) or vehicle during different chase periods after
35S metabolic labeling (Fig.
4A). Under control conditions
(Fig. 4A, ISO), the levels of GRK2 decay
essentially as described in cells expressing the kinase alone (see also
Fig. 1A). Agonist stimulation (Fig. 4A,
+ISO) clearly enhances the GRK2 degradation rate as compared
with control (27% GRK2 remaining after 1 h of ISO treatment
versus 54% in untreated cells; p < 0.05, data indicate the means ± S.E. of three to five independent
experiments). GRK2 half-life thus decreases from 60 min (basal
conditions) to ~30 min (stimulated turnover). The presence of
lactacystin or ALLN completely abrogates GRK2 degradation in
isoproterenol-treated cells (Fig. 4B), as they do in
unstimulated cells. This ruled out the possibility that the increase in
kinase processing triggered by receptor stimulation might arise from
the activation of an additional proteolytic pathway other than
proteasome-dependent degradation.
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Fig. 4.
GRK2 turnover is modulated by receptor
stimulation. A, HEK-293 cells stably coexpressing GRK2
and 2AR were pulsed (15 min), chased in the presence of
ISO (open circles) or vehicle (filled circles)
and processed as in Fig. 1A. B, pulse and chase
experiments were performed as in panel A in the presence or
absence of lactacystin or ALLN, and cells were chased (3 h) with
isoproterenol or vehicle. C, HEK-293 cells were transiently
transfected with the indicated combinations of GRK2,
2AR, and HA-tagged ubiquitin (HA-Ubi)
constructs and challenged with ISO or vehicle for 30 min. Ubiquitin
conjugates were immunoprecipitated with anti-HA antibody (0.3 µg/106 cells) and immunoblotted with anti-GRK2 antibody
Ab9 (upper panel). Polyubiquitinated GRK2 conjugates are
marked with a line (right side). The two upper
bands in all lanes correspond to proteins unspecifically cross-reacting
with the antibodies used. Ubiquitin (middle panel) and GRK2
(lower panel) levels were analyzed by dot blot and Western
blot, respectively. D, HEK-293 cells stably expressing
2AR and mutant GRK2-K220R were pulse-labeled and chased
as described for Fig. 1A (upper panel). The
effect of isoproterenol on GRK2-K220R turnover (lower panel)
was determined as in panel A. Lanes C, control;
lanes I, isoproterenol. Experiments are representative of
two to five independent experiments.
2AR. In cells in which ubiquitin was overexpressed with
the kinase and the receptor, a band smear corresponding to
multi-ubiquitinated GRK2 (marked with a line in the
right side of the upper panel in Fig.
4C) was clearly detected upon receptor stimulation with isoproterenol, whereas GRK2 ubiquitination was much lower in untreated cells (Fig. 4C, upper panel). Taken together, our
data suggest that the extent of GRK2 ubiquitination is
stimulation-dependent and probably accounts for the
enhanced agonist-induced kinase degradation by the proteasome pathway.
2AR occupancy by agonist promotes very rapid
translocation of GRK2 to the plasma membrane and kinase activation (1,
2, 6, 13), we tested whether the activity of the kinase might also
affect its turnover. To this end, HEK-293 cells stably expressing
2AR and a dominant-negative GRK2 mutant (GRK2-K220R) (25) were metabolically labeled and chased in the presense (Fig. 4D, lanes I) or absence (Fig. 4D,
lanes C) of isoproterenol. Agonist stimulation promotes no
change in mutant kinase levels (Fig. 4D). More surprisingly,
the amount of GRK2-K220R does not appear to decay even after 8 h
of chase in the absence of agonist (Fig. 4D, upper
panel), indicating that the mutant protein is far more stable than
the wild type.
2AR
Signaling--
The rapid and agonist-modulated degradation of GRK2 by
the proteasome would provide a mechanism to regulate the cellular
complement of GRK2 and, therefore, GPCR desensitization patterns. In
this regard, we used HEK-293 cells stably expressing both
2AR and GRK2 to test the effects of blocking GRK2
degradation with the specific proteasome inhibitor lactacystin on the
subsequent cell response to the -adrenergic agonist isoproterenol
and on the 2AR desensitization process. In control
cells, agonist challenge promotes a 1.8 ± 0.1-fold increase in
cAMP levels over basal values. Pretreatment with isoproterenol for 30 min induces the typical decrease in responsiveness to a subsequent
agonist challenge (23 ± 7% decrease) (Fig.
5, left panel) and also a
decrease in GRK2 levels (45 ± 9% of control), consistent with
our previous data. Interestingly, inhibition of GRK2 degradation with
lactacystin results in a clear increase in GRK2 protein levels both in
control and isoproterenol-pretreated cells (Fig. 5, inset,
lanes 2 and 4) and reduces
2-adrenergic receptor signaling by 33 ± 2 and 44 ± 1%, respectively (Fig. 5, left panel). Because
cellular 2AR levels remain unchanged with lactacystin
treatments (see legend to Fig. 5), these data suggest that alteration
of the kinase/receptor ratio clearly affects signal transduction. Other
factors may also contribute to the reduction in 2AR
signaling in the presence of lactacystin, because regulatory subunits
of cAMP-dependent protein kinase (26, 27) and other
components of the transduction cascade could be degraded by the
proteasome pathway. However, adenylyl cyclase activation by forskolin
is not modified in the presence of lactacystin, and the same results
were obtained in the presence of cAMP phosphodiesterase inhibitors
(data not shown). Moreover, lactacystin treatment does not affect cAMP
production by a transiently transfected truncated 2AR,
which lacks phosphorylation sites for GRK2 but retains those for
cAMP-dependent protein kinase and its ability to modulate
adenylyl cyclase (Fig. 5, left panel), indicating that
changes in GRK2 levels have a direct and crucial effect in the observed
alteration in 2AR responsiveness.
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Fig. 5.
Effect of blocking GRK2 degradation on
2-adrenergic receptor signaling and
internalization. Left panel, stably transfected HEK-293
cells used in Fig. 4 or cells transiently transfected with GRK2 and a
COOH-terminal truncated mouse 2AR were preincubated with
lactacystin (Lac) or vehicle (control), pretreated with
isoproterenol or vehicle (control) for 30 min, resensitizated, and
finally challenged with isoproterenol for 10 min to both assess
receptor-dependent adenylyl cyclase activity and GRK2
levels by immunoblot (inset). Data are expressed as fold
stimulation of cAMP levels over basal values in control cells (18 ± 4 pmol/mg protein). Fold stimulation in control conditions was taken
as 100%. Treatments did not significantly alter total receptor levels
in stable or transfected cells (28.6 ± 0.6 and 1.2 ± 0.3 pmol/mg protein, respectively). Data are the means ± S.E. from
two or three experiments performed in duplicate. Right
panel, HEK-293 cells stably transfected with GRK2 and an
epitope-tagged 2AR receptor were incubated with
lactacystin or vehicle (control) for 3 h and receptor
internalization in response to a 2-min challenge with the indicated
concentrations of agonist analyzed by flow cytometry (see
"Experimental Procedures"). Receptor internalization under control
conditions was taken as 100%. Results are the means ± S.E. of
three independent experiments performed in duplicate.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
degradation (16). On the other
hand, the ability of purified 20 S proteasome to digest GRK2 indicates
that a direct interaction between the kinase and proteasome subunit(s)
can occur. It is feasible that an undetermined structural motif in GRK2
contributes to target the protein to this degradation machinery, in
addition to ubiquitination.
2AR activation promotes an
increase in GRK2 ubiquitination and degradation. Several hypotheses can be considered to explain this observation. First, GRK2-mediated phosphorylation promotes arrestin binding to the agonist-activated receptor, leading to receptor uncoupling and internalization (1, 5,
28). Data suggest the formation of a multimolecular complex (receptor/GRK2/-arrestin) at specialized locations of the plasma membrane (5, 35-37) that will be internalized after recruitment of
different proteins of the endocytic machinery. It is possible that
interaction of GRK2 with molecules of this multimolecular complex plays
a role in its signal-dependent degradation by locating the
kinase in close proximity with the molecules responsible for its
ubiquitination and/or allowing conformational changes that unmask
kinase domains directly targeted for ubiquitination. In this regard, it
is worth noting that the yeast G protein-coupled receptor Ste2p is
ubiquitinated at the plasma membrane even if endocytosis is blocked
(38, 39). Alternatively, the higher level of GRK2 ubiquitination due to
agonist receptor activation might result from second messenger-mediated
mechanisms or as a consequence of stimulated GRK2 activity leading to
phosphorylation of unknown factors different from GPCR or to transient
increases in kinase autophosphorylation, improving GRK2 modification by ubiquitination. Data obtained with the GRK2-K220R mutant are consistent with a strong relationship between the stability of GRK2 and its cellular function; the slow turnover of GRK2-K220R as compared with
wild-type GRK2 is largely unaffected by receptor activation, the mutant
is deficient in kinase activity, and it strongly impairs -arrestin
binding and the subsequent internalization (1, 28, 40).
-arrestin (7, 30, 35). Our data are
consistent with these reports and, further, suggest that a tight
regulation of GRK2 cellular levels by agonists is needed for normal
receptor function and modulation. Indeed, the rise in GRK2 complement
by blockade of its normal degradation pathway clearly enhanced the
initial rate of receptor sequestration, according to the facilitatory role of GRK2 previously reported in this process (5, 28). More
interestingly, receptor response to agonists is markedly affected in
these conditions, indicating that in our cellular system a moderate
increase of GRK2 over its steady-state levels is enough to evoke a
strong change in GPCR signaling, putting forward the relevance of an
adequate ratio between the receptor and its regulatory proteins. In
this context, it is tempting to suggest that the increased
down-regulation of kinase levels observed after sustained GPCR
stimulation would help to preserve some degree of response to
additional activation by agonists. In this regard, it is worth noting
that GRK2 levels rapidly decrease in several tissues during the rat
perinatal period,2 a physiological situation characterized
by a dramatic and prolonged surge in plasma catecholamines. On the
other hand, the impaired protein degradation by the proteasome pathway
described in pathological conditions such as hypothyroidism (41) could
explain the increased GRK2 levels detected by our laboratory in several
rat hypothyroid tissues4 and
contribute to the reported attenuation of adrenergic responses in this situation.
ACKNOWLEDGEMENTS
2AR cDNA. We
also thank A. Morales and M. Sanz for secretarial assistance, C. Mark
for editorial help, and Prof. F. Mayor for continuous encouragement.
FOOTNOTES
2AR, 2-adrenergic receptor; DMEM, Dulbecco's modified
Eagle's medium; GRK, G protein-coupled receptor kinase; ISO, isoproterenol; MCP, multicatalytic proteinase complex; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid
chromatography; HA, hemagglutinin.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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