From the Howard Hughes Medical Institute, Departments of Medicine
and Biochemistry, Duke University Medical Center, Durham, North
Carolina 27710 and the Howard Hughes Medical Institute,
Vollum Institute, Oregon Health Sciences University, Portland, Oregon
97201
Received for publication, October 5, 2000, and in revised form, January 18, 2001
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ABSTRACT |
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The Hormonal signaling through G protein-coupled receptors
(GPCRs)1 is attenuated during
prolonged exposure to agonist, a process known as desensitization (1).
One of the initial events in this multistep process is the
phosphorylation of agonist-occupied receptor molecules. Two families of
kinases are responsible for the phosphorylation of GPCRs, the second
messenger-activated kinases (PKA and PKC) and the GPCR kinases (GRKs
1-7) (2-8). Although the second messenger-activated kinases are
capable of phosphorylating receptors in the absence or presence of
agonist, the GRKs specifically phosphorylate only agonist-occupied
receptors (5, 9). Moreover, phosphorylation by GRKs leads to the
recruitment of the arrestin proteins to the phosphorylated receptors,
preventing further coupling to G proteins (1). In the case of the
prototypic The second messenger-activated kinases are constitutively localized to
subcellular structures via their interactions with anchoring proteins
(17). In the case of PKA, its localization is achieved through a
family of A kinase-anchoring proteins (AKAPs) that were originally
identified by their ability to interact with PKA regulatory subunits II
(18). The AKAPs are responsible for the localization of PKA to
particular subcellular compartments, including the plasma membrane,
mitochondria, post-synaptic densities, and centrosomes (19-27). Some
of the AKAPs have been shown to exist in complexes containing a number
of other signaling molecules, including the
N-methyl-D-aspartate receptor, ion channels,
GPCRs, and protein phosphatases (27-33). These discoveries have led to the hypothesis that AKAPs may act as nodes at which cross-talk between
different signaling events may be coordinated (34). One family member,
AKAP79 (human AKAP79, rodent AKAP150, and bovine AKAP75), binds not
only to PKA but also to PKC and the protein phosphatase 2B (20,
34-37). AKAP79 has also been shown to be responsible for the
association of PKA with the plasma membrane and with integral membrane
proteins including the Materials
GRK2 was purified from baculovirus-infected Sf9 cells as
described previously (38). Bovine GRK2ct (residues 467-689) was expressed as a glutathione S-transferase (GST) fusion
protein in bacteria and purified as described previously (13).
Purification of rod outer segment membranes (39), G Plasmid Constructs
Construction of the pcDNA3-Bovine GRK2 Mutant
S685A--
pcDNA1-bovine GRK2 (43) was restriction digested
with HindIII and XbaI, and the insert was ligated
into the pcDNA3 vector (Invitrogen). A single point mutation
changing serine 685 to alanine was introduced by the polymerase chain
reaction using the primers 5'-TCCCCAACCGCCTCGAGTGGC-3' and
5'-CTAGTCTAGATCAGAGGCCGTTGGCGGCGCCGCGC-3'. The polymerase chain
reaction product was restriction digested with XhoI and
XbaI and used to replace the equivalent fragment in the
pcDNA3 construct. The sequence and orientation of the clone were
confirmed by automated DNA sequence analysis.
Construction of a COOH-terminal Truncated GRK2 Methods
Cell culture, Immunoprecipitations, and
Immunoblotting--
HEK293 cells were grown at 37 °C in minimal
essential medium containing 10% fetal bovine serum and 1×
penicillin/streptomycin (Life Technologies, Inc.) under 5%
CO2. Cells at 60% confluence were transfected with up to 5 µg of plasmid DNA and 15 µl of Fugene 6 (Roche Molecular
Biochemicals). Two days after transfection, cells were lysed in
radioimmune precipitation buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 5 mM EDTA, 1% v/v Nonidet
P-40, 0.5% w/v sodium deoxycholate, 10 mM NaF, 10 mM Na2-pyrophosphate, 0.1% w/v SDS, 5 µg/ml
aprotinin, 150 µg/ml benzamidine, 5 µg/ml leupeptin, 4 µg/ml
pepstatin, and 20 µg/ml phenylmethylsulfonyl fluoride). If
cross-linking of proteins was necessary before immunoprecipitation, cells were incubated at room temperature for 20 min in
phosphate-buffered saline containing 10 mM HEPES, pH 7.4, and 1 mg/ml dithiobis(succinimidyl propionate) before lysis in
radioimmune precipitation buffer (45). After removal of insoluble cell
debris by centrifugation, protein concentrations were equalized in all
samples, and FLAG epitope-tagged proteins were immunoprecipitated for
15 h with 40 µl of a 1:1 slurry of M2 anti-FLAG antibody
covalently coupled to Sepharose beads. The beads were washed four times
with radioimmune precipitation buffer, and bound proteins were eluted
in 50 µl of 2× SDS-PAGE sample buffer (100 mM Tris, pH
7.2, 4% w/v SDS, 200 mM dithiothreitol, 20% v/v glycerol,
20 µg/ml bromphenol blue with 5% v/v Receptor Sequestration Assay--
COS7 cells expressing
FLAG- Receptor Phosphorylation Assay--
Receptor phosphorylation was
assessed after the labeling of the intracellular ATP pool of HEK293
cells stably transfected with FLAG- In Vitro PKA-mediated Phosphorylation of Full-length and
Fragments of GRK2--
One microgram of GRK2 or GRK2 GRK2-mediated Phosphorylation of Protein
Substrates--
In vitro phosphorylation of rhodopsin,
tubulin, and the peptide substrate RRRREEEEESAAA by GRK2 was performed
as described previously (47). The reactions used 25 ng of GRK2 to
phosphorylate 2 µg of rhodopsin or 100 ng of GRK2 to phosphorylate
either 0.2 µg of tubulin or 1 mM peptide substrate.
In Vitro Binding of G It has been shown previously that the protein kinase A-anchoring
protein AKAP79 can directly interact with and regulate the phosphorylation of the 2 adrenergic receptor (
2AR) undergoes
desensitization by a process involving its phosphorylation by both
protein kinase A (PKA) and G protein-coupled receptor kinases (GRKs).
The protein kinase A-anchoring protein AKAP79 influences
2AR
phosphorylation by complexing PKA with the receptor at the membrane.
Here we show that AKAP79 also regulates the ability of GRK2 to
phosphorylate agonist-occupied receptors. In human embryonic kidney 293 cells, overexpression of AKAP79 enhances agonist-induced
phosphorylation of both the
2AR and a mutant of the receptor that
cannot be phosphorylated by PKA (
2AR/PKA
). Mutants of
AKAP79 that do not bind PKA or target to the
2AR markedly inhibit
phosphorylation of
2AR/PKA
. We show that PKA directly
phosphorylates GRK2 on serine 685. This modification increases G
subunit binding to GRK2 and thus enhances the ability of the kinase to
translocate to the membrane and phosphorylate the receptor. Abrogation
of the phosphorylation of serine 685 on GRK2 by mutagenesis (S685A) or
by expression of a dominant negative AKAP79 mutant reduces
GRK2-mediated translocation to
2AR and phosphorylation of
agonist-occupied
2AR, thus reducing subsequent receptor
internalization. Agonist-stimulated PKA-mediated phosphorylation of
GRK2 may represent a mechanism for enhancing receptor phosphorylation
and desensitization.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 adrenergic receptor (
2AR), agonist stimulation leads
to the recruitment of cytosolic GRK2 to the plasma membrane where it
binds to and phosphorylates the receptor. The mechanisms by which
different GRKs are recruited to the receptor varies, but for both GRK2
and GRK3, recruitment is achieved through the binding of phospholipids and G
subunits to the COOH-terminal pleckstrin homology domain of
the kinase (10-16). As the recruitment requires the presence of
free G
subunits, the binding of the kinase to the receptor only
occurs at times when the receptor is signaling through coupling to G
proteins. In this manner, the kinase is only delivered at times when
there are agonist-occupied receptor substrates for it to phosphorylate.
2AR (27). It has been demonstrated that the
overexpression of AKAP79 with the
2AR enhances receptor
phosphorylation and, furthermore, that mutants of AKAP79, which fail to
bind to the
2AR or to PKA, are effective at reducing
phosphorylation. In this manner, AKAP79 may be acting as a
scaffold which coordinates the events involved in receptor
desensitization. To investigate this novel function of AKAP79 further,
we set out to test whether another event involved in
2AR
desensitization, i.e. receptor phosphorylation by GRK2, is
modulated by PKA scaffolding.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits,
(40) and tubulin (41) was described previously. AKAP79, AKAP79pro, and AKAP79108-427 mammalian expression constructs were
described previously (27, 37). Mammalian expression constructs of FLAG
epitope-tagged
2 adrenergic receptor (
2AR) and
2AR/PKA
mutant receptor (PKA phosphorylation sites serines 261, 262, 345, and
346 all mutated to alanines) were described previously (42). The PKA
catalytic subunit was purchased from Promega, and the anti-G
antibody was from Perkin Elmer Life Sciences. The anti-GRK2
monoclonal and AKAP79 polyclonal antibodies were described previously
(20, 42). M2 anti-FLAG antibody conjugated to Sepharose beads and M2
antibody were from Sigma. Unless otherwise stated, the chemicals were
from Sigma.
19 Baculovirus
Plasmid--
The RsrII/BamHI restriction
fragment from a GRK2
19 construct (construct 2 in Ref. 13 encoding
full-length GRK2 with a stop codon inserted at codon 671) was used to
replace the equivalent fragment in pVL1392-GRK2(S670A) (44). The
orientation and sequence of the clone were confirmed by automated DNA
sequence analysis. Purification of the expressed protein from
Sf9 cells was identical as that described for full-length GRK2
(38).
-mercaptoethanol for
cross-linked samples) for 10 min at 95 °C. Samples were resolved on
10% or 4-20% polyacrylamide gels (Novex) and transferred to nitrocellulose filters for immunoblotting. Filters were blocked with
5% w/v fat-free milk powder in Tris-buffered saline with Tween 20 (20 mM Tris, pH 7.4, 500 mM NaCl, 0.1% v/v Tween
20) and incubated overnight at 4 °C with appropriate primary
antiserum. After thorough washing in Tris-buffered saline with
Tween 20, filters were incubated for 1 h with horseradish
peroxidase-conjugated anti-rabbit or mouse secondary antibody (Amersham
Pharmacia Biotech), washed again with Tris-buffered saline with Tween
20, immersed in ECL reagent (Amersham Pharmacia Biotech), and exposed
to x-ray film.
2AR/PKA
alone or coexpressing AKAP79 with GRK2 or GRK2S685A
were stimulated with 10 µM isoproterenol for 30 min.
Agonist-induced receptor internalization was measured as the loss of
cell surface FLAG epitopes available for M2 antibody binding by
detection of a fluorescently labeled secondary antibody as described
previously (46).
2AR or FLAG-
2AR/PKA
with
[32P]orthophosphate (PerkinElmer Life Sciences) as
described previously (43). Cells were labeled for 1 h at 37 °C
in phosphate-free Dulbecco's modified Eagle's medium containing 10 mM HEPES, pH 7.4, 100 µCi/ml
[32P]orthophosphate, and 1 µg/ml microcystin L-R
(Calbiochem). Cells were stimulated with 10 µM
isoproterenol for 5 min, washed twice with ice-cold phosphate-buffered
saline buffer, solubilized in 750 µl of radioimmune precipitation
buffer, and equivalent protein amounts were subjected to
immunoprecipitation. Samples were resolved on 10% polyacrylamide gels
and dried under vacuum. Radioactive bands were visualized and
quantified using a PhosphorImager (Molecular Dynamics) and by exposure
to x-ray film. The levels of receptor expression were measured by flow
cytometry by detecting cell surface-bound anti-epitope tag M2
antibodies and fluorescein-conjugated secondary antibodies (46). All
phosphorylation levels were normalized to receptor expression and are
shown as stimulated-basal values.
19 purified
from baculovirus-infected Sf9 cells or GRK2ct purified by
cleavage with thrombin from bacterially expressed GST fusion protein
(13) was incubated with 1 unit of PKA catalytic subunit (Promega) in
phosphorylation reaction buffer containing 20 mM Tris, pH
7.5, 10 mM MgCl2, 2 mM EDTA, 1 mM dithiothreitol, 60 µM
[
32P]ATP (~1000 cpm/pmol), 1 µg/ml phospholipids
(crude preparation containing 20% w/v
L-
-phosphatidylcholine, Sigma), and 0.8 µM G
subunits. After 30 min of incubation at 30 °C, reactions
were stopped by the addition of an equal volume of 2× SDS-PAGE sample buffer. Samples were boiled for 10 min and resolved on 4-20% gradient polyacrylamide gels. Radioactive bands in dried gels were visualized and quantified using a PhosphorImager and by exposure to x-ray film.
to GST-GRK2ct--
One microgram of
GST-GRK2ct (residues 467-689 of bovine GRK2) bound to
glutathione-conjugated Sepharose 4B beads (Calbiochem) was incubated
with or without PKA as described above. After 30 min of incubation at
30 °C, beads were washed extensively with phosphate-buffered saline
containing 0.01% v/v lubrol to remove all the PKA and ATP. Four
micrograms of G
from bovine brain was then added and incubated at
4 °C for 2 h. After extensive washes with phosphate-buffered
saline containing 0.01% lubrol, 2× sample buffer was added, and
proteins were eluted by boiling for 10 min. Samples were resolved on
4-20% polyacrylamide gels, transferred to nitrocellulose filters, and
immunoblotted with an antibody recognizing G
subunits.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2AR (27). In HEK293 cells overexpressing FLAG
epitope-tagged
2AR, the coexpression of AKAP79 increases
2AR
phosphorylation by 40%, whereas the coexpression of the AKAP79 mutant
AKAP79108-427, which does not bind to the receptor or
localize PKA to the membrane, impairs receptor phosphorylation by 50%
(Fig. 1A). To determine whether this enhancement of
2AR phosphorylation is a result of AKAP79 directly facilitating phosphorylation of the receptor by PKA,
agonist-induced phosphorylation of a FLAG epitope-tagged
2AR mutant
lacking all PKA phosphorylation sites (
2AR/PKA
) was measured.
Surprisingly, the coexpression of wild type AKAP79 enhances
agonist-induced phosphorylation of
2AR/PKA
by 40% (Fig. 1B). In contrast, cells transfected with either of two
AKAP79 mutants that are unable to target PKA to the plasma membrane, AKAPpro and AKAP108-427, exhibit a 55-70% decrease in
2AR/PKA
phosphorylation. AKAP79pro is targeted to the plasma
membrane and can bind to
2AR but is unable to bind to the PKA
regulatory subunit, whereas AKAP79108-427 binds to PKA but
lacks the receptor and membrane-targeting domain (27, 37). Although
GRK2 and AKAP79 do not interact directly (data not shown), when GRK2
and AKAP79 are overexpressed together a 2.3-fold increase in
2AR/PKA
phosphorylation is observed compared with a 1.4-fold
increase by overexpression of GRK2 alone (Fig. 1C).
Overexpression of either of the two AKAP79 mutants with GRK2 inhibits
receptor phosphorylation by 40-60% compared with control cells
expressing only endogenous levels of GRK2 (Fig. 1C).
View larger version (30K):
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Fig. 1.
Effects of PKA anchoring on
2AR and
2AR/PKA
phosphorylation. A,
agonist-stimulated
2AR phosphorylation is regulated by AKAP79.
HEK293 cells overexpressing FLAG-
2AR were transiently transfected
with AKAP79 or the mutant AKAP79108-427 and incubated in
[32P]orthophosphate. Cells were stimulated with 10 µM isoproterenol (iso) for 5 min, and
FLAG-
2AR was immunoprecipitated and resolved by SDS-PAGE.
Upper panel shows a representative autoradiogram
(n = 4) with basal (
) and isoproterenol-stimulated
(+)
2AR phosphorylation in transfected cells as
indicated. AKAP79 and AKAP79108-427 expression was
detected in cell lysates by immunoblotting (IB, lower
panel). B, agonist-stimulated
2AR/PKA
phosphorylation is regulated by AKAP79. HEK293 cells expressing
FLAG-
2AR/PKA
were transiently transfected with AKAP79, AKAP79pro,
or AKAP79108-427, and isoproterenol-stimulated
phosphorylation of
2AR/PKA
was measured as described earlier. The
graph shows the mean levels of phosphorylation of
2AR/PKA
relative to control transfected cells. AKAP79, AKAP79pro,
and AKAP79108-427 expression were detected in cell lysates
by immunoblotting (lower panel). C, AKAP79
regulates
2AR/PKA
phosphorylation by GRK2. HEK293 cells expressing
2AR/PKA
were transiently transfected with GRK2, AKAP79, AKAP79pro,
and AKAP79108-427 as indicated. Isoproterenol-stimulated
phosphorylation of
2AR/PKA
was measured as described earlier.
Expression of AKAP79, its mutants, and GRK2 was confirmed by
immunoblotting (lower panels). D,
2AR/PKA
cannot be phosphorylated by PKA. FLAG-
2AR or FLAG-
2AR/PKA
was
transfected into HEK293 cells. Cells were subsequently stimulated with
10 µM isoproterenol or 10 µM vasoactive
intestinal peptide (VIP) for 5 min, and 32P
incorporation was measured. The graphs show the mean ± S.E. of three experiments.
These results suggest that AKAP79 does not affect 2AR
phosphorylation by simply enhancing direct receptor phosphorylation by
PKA. Rather, they suggest that AKAP79 enhances GRK2-mediated phosphorylation of the
2AR. To ensure that PKA is not capable of
phosphorylating the
2AR/PKA
mutant, the phosphorylation of the
wild type receptor and
2AR/PKA
was compared following activation of PKA by stimulation of a coexpressed Gs-coupled receptor (Fig. 1D). In the absence of its ligand, the
2AR can be
phosphorylated by PKA but not by the GRKs as they specifically
phosphorylate only agonist-occupied receptors. Stimulation of
endogenous vasoactive intestinal peptide receptors leads to the
phosphorylation of wild type
2AR to 20% of the level observed with
isoproterenol stimulation (Fig. 1D). This level is
consistent with previous reports for PKA phosphorylation of the
2AR
in HEK293 cells (48). The
2AR/PKA
mutant, however, shows no
vasoactive intestinal peptide-induced phosphorylation (Fig.
1D), confirming that the removal of the PKA phosphorylation
sites renders the receptor incapable of being further phosphorylated by
PKA. Taken together, the regulation of
2AR/PKA
phosphorylation by
wild type and mutants of AKAP79 indicates that AKAP79 may indirectly
regulate GRK2 activity by a mechanism requiring the anchoring of PKA to
the plasma membrane.
To further test the ability of PKA to affect GRK2 activity against
receptor substrates, we measured the effect of PKA on GRK2-mediated phosphorylation of rhodopsin in vitro. Incubation with PKA
and 167 nM G subunits or with GRK2 alone results in
little or no phosphorylation of rhodopsin (Fig.
2). However, with the addition of
increasing concentrations of G
subunits, GRK2 phosphorylation of
rhodopsin is greatly increased and is further enhanced with the
addition of PKA. As PKA does not significantly phosphorylate rhodopsin
nor is its activity regulated by G
subunits, the observed increase in phosphorylation is attributed to enhanced GRK2 activity toward rhodopsin.
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It has been reported that PKC can directly phosphorylate GRK2 at a site
within the carboxyl terminus and that this facilitates GRK2
translocation to the plasma membrane (49, 50). To investigate whether a
similar mechanism exists for the phosphorylation of GRK2 by PKA, we
tested the ability of PKA to phosphorylate purified full-length GRK2
and fragments of GRK2 (Fig.
3A). In the presence of
phospholipids that activate GRK2, bovine GRK2 purified from Sf9
cells shows weak autophosphorylation (a stoichiometry of 0.03 mol of
phosphate/mol of protein, Fig. 3B). Incubation of GRK2 with
PKA leads to a significant increase in phosphorylation (0.25 mol of
phosphate/mol of protein), which is further enhanced by the addition of
0.8 µM G subunits to 0.7 mol of phosphate/mol of
protein. A fragment of the carboxyl terminus of GRK2 (residues 467-689, GRK2ct), which encompasses the pleckstrin homology domain and
G
binding domain, also acts as a good PKA substrate (0.4 mol of
phosphate/mol of protein), and this phosphorylation is markedly
enhanced by the addition of G
subunits (0.7 mol of phosphate/mol
of protein, Fig. 3B). Furthermore, a GRK2 mutant that lacks
19 residues at the carboxyl terminus is unable to be phosphorylated by
PKA (Fig. 3B).
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Examination of the extreme COOH-terminal sequence of GRK2 reveals three
potential sites of PKA phosphorylation, serines 670, 676, and 685. Although the phosphorylation of serine 670 by Erk1/Erk2 was reported
previously to inactivate GRK2 (44), no studies have investigated
whether phosphorylation of serines 676 and 685 can affect GRK2
activity. The site of PKA phosphorylation on GRK2 was mapped by
incubating the GRK2 carboxyl terminus fragment with PKA and
[-32P]ATP followed by tryptic digestion and subsequent
high pressure liquid chromatography fractionation and sequencing of
radiolabeled peptides. This yields one major phosphopeptide
corresponding to residues 678-689 of GRK2 containing a single site of
phosphorylation at serine 685. Taken together, these data show that PKA
is capable of phosphorylating GRK2 at a specific site within the
carboxyl terminus and that this phosphorylation is markedly enhanced by the presence of G
.
PKA phosphorylation of GRK2 could lead to enhanced phosphorylation of
receptors by two mechanisms; either the catalytic activity of GRK2 is
enhanced by its phosphorylation or GRK2 targeting to the plasma
membrane is increased, possibly in a G
subunit-dependent manner. To test whether PKA
phosphorylation of GRK2 leads to a direct enhancement of GRK2 catalytic
activity, the ability of GRK2 to phosphorylate nonreceptor substrates
(a peptide substrate and tubulin) was compared with and without
phosphorylation of GRK2 by PKA (Fig. 4).
Incubation with PKA alone weakly phosphorylates tubulin and fails to
phosphorylate the peptide substrate (data not shown), whereas
incubation with GRK2 leads to significant phosphorylation of both
substrates (0.25 mol of phosphate/mol of protein for the peptide
substrate (Fig. 4A) and 0.3 mol of phosphate/mol of protein
for tubulin (Fig. 4B)). Incubation of the substrates with
both PKA and GRK2 under conditions that permit GRK2 phosphorylation by
PKA shows no increase in the level of substrate phosphorylation,
indicating that PKA phosphorylation of GRK2 does not directly affect
its catalytic activity.
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To test whether PKA phosphorylation of GRK2 enhances the interaction
between GRK2 and G subunits, a GST fusion of the carboxyl terminus of GRK2 immobilized on glutathione-Sepharose beads was phosphorylated by PKA and then incubated with purified G
subunits. After washing, the amount of G
bound to the fusion
protein was monitored by Western blotting for G
subunits (Fig.
5). Before phosphorylation by PKA, the
GST-GRK2ct is capable of binding G
subunits. After
phosphorylation, the binding of G
is increased by 64 ± 25%, whereas GST alone binds G
very poorly. This finding shows
that phosphorylation by PKA indeed enhances the ability of GRK2 to bind
G
subunits, and this could represent the mechanism by which GRK2
phosphorylation of receptors is regulated by factors affecting PKA
activity in cells.
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Upon agonist stimulation, GRK2 translocates to the plasma membrane by
virtue of its interaction with G subunits (13-16, 51, 52). If
the stability of this interaction is partly dependent on PKA-mediated
phosphorylation of GRK2, the removal of PKA from the membrane should
also inhibit GRK2 translocation. To test this theory, the ability of
GRK2 to interact with the
2AR in cells coexpressing AKAP79 was
examined (Fig. 6). Cells coexpressing the
FLAG epitope-tagged
2AR, GRK2, and either AKAP79 or
AKAP79108-427 were stimulated for 5 min with 10 µM isoproterenol. Cells were then treated with a cell
permeant cross-linker, and the
2AR was immunoprecipitated from the
cell lysates. The amount of GRK2 present in the immune complex was
detected by Western blotting. Overexpression of
AKAP79108-427 impairs GRK2 translocation to
2AR by
40 ± 15% (Fig. 6A), demonstrating that a mutant
AKAP79 that reduces PKA association with the plasma membrane does
indeed decrease GRK2 association with the
2AR.
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The role that PKA phosphorylation of GRK2 plays in the colocalization
of GRK2 with the 2AR was further demonstrated by mutating serine 685 in GRK2 to alanine (GRK2S685A) and comparing it to the wild type kinase
in its ability to associate with the receptor. The GRK2S685A mutant
lacking the PKA phosphorylation site of the native GRK2 should not show
PKA-mediated enhancement of translocation. Indeed, a 40% impairment in
the agonist-stimulated association of GRK2S685A with
2AR compared
with wild type GRK2 is observed (Fig. 6B).
We also predicted that coexpression AKAP79 with GRK2S685A, unlike with
wild type GRK2, should not enhance 2AR/PKA
phosphorylation. When tested in vitro against nonreceptor substrates, the
mutant and wild type GRK2 showed no detectable differences in their
catalytic activities (data not shown). Coexpression of wild type GRK2
with AKAP79 enhances receptor phosphorylation and subsequently leads to
enhanced levels of receptor internalization when compared with control
transfected cells (Fig. 7A).
Coexpression of GRK2S685A with AKAP79, however, fails to significantly
enhance
2AR/PKA
phosphorylation, confirming that prevention of
PKA-mediated phosphorylation of GRK2 at serine 685 impairs the ability
of the kinase to both associate with and phosphorylate the
2AR in
response to agonist. As a consequence of enhancing agonist-induced
2AR/PKA
phosphorylation by the coexpression of GRK2 and AKAP79,
receptor internalization is increased (Fig. 7B).
Coexpression of GRK2S685A and AKAP79, however, does not increase
receptor internalization, mirroring the failure of the mutant kinase to
enhance receptor phosphorylation.
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DISCUSSION |
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The 2AR exists in a complex with AKAP79/AKAP150 and associated
signaling proteins including PKA in rat brain tissue (27). Enhancement
or perturbation of the complex in HEK293 cells leads to alterations in
phosphorylation of the
2AR. This originally was assumed to occur
primarily through the effects of PKA on the receptor. However, we now
show that receptor phosphorylation by GRK2 is also affected by AKAP79.
In HEK293 cells, the relative contributions of PKA and GRK2 to
agonist-induced
2AR phosphorylation are ~20 and 80%, respectively
(48). When AKAP79 is overexpressed in cells, a 40% enhancement in the
level of receptor phosphorylation is observed, whereas the disruption
of this complex with a mutant of AKAP79 reduces receptor
phosphorylation by 50% (Fig. 1A). If the ability of AKAP79
to affect
2AR phosphorylation relates entirely to the
phosphorylation of the receptor by PKA, then at most a 20% reduction
in phosphorylation would be expected. This disparate finding led us to
explore the possibility that AKAP79 somehow regulates the ability of
GRKs to phosphorylate their receptor substrates. We confirmed this
observation by demonstrating that phosphorylation of a mutant
2AR
that is not a PKA substrate is also regulated by AKAP79 (Fig. 1,
B-D). Furthermore, in vitro phosphorylation of
rhodopsin by GRK2 is enhanced in the presence of PKA and G
subunits, even though rhodopsin itself is not a PKA substrate (Fig.
2).
The mechanism underlying these findings appears to be the ability of
PKA to phosphorylate GRK2 at a specific residue, serine 685 (Fig. 3).
Phosphorylation at this site does not alter the kinase activity of GRK2
per se (Fig. 4). Rather, it leads to the enhanced binding of
GRK2 to G subunits, which should increase the association of the
kinase with receptor substrates (Fig. 5). We also demonstrate that the
ability of PKA to phosphorylate GRK2 in vitro is enhanced in
the presence of G
subunits, suggesting that in cells PKA may
preferentially phosphorylate those GRK2 molecules already complexed
with G
subunits at the membrane. Therefore, phosphorylation by
PKA may be involved both in promoting translocation of GRK2 to the
membrane and in maintaining GRK2 in a G
-bound form once there.
We confirmed the role of PKA phosphorylation in GRK2 membrane
translocation by showing that the mutation of the PKA phosphorylation site in GRK2 (serine 685 to alanine) diminishes the ability of GRK2 to
be recruited to 2AR and phosphorylate the
2AR (Figs. 6 and 7).
The phosphorylation of GRK2 by PKA was also shown to have a functional
consequence as overexpression of GRK2, but not GRK2S685A, leads to
enhanced receptor internalization in response to agonist (Fig.
7B). Tethering of PKA to the
2AR via AKAP79, therefore,
may represent a regulatory loop whereby agonist-stimulated coupling of
the
2AR to Gs, resulting in the production of cAMP, is attenuated by
the cAMP-activated kinase PKA phosphorylating both the receptor and the
major desensitizing kinase, GRK2. This process leads to
increased translocation of GRK2 to the receptor by enhanced binding to
G
subunits, resulting in further receptor phosphorylation,
desensitization, and internalization (Fig.
8).
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A similar mechanism may also exist for the recruitment of GRK2 to the
receptor following phosphorylation of GRK2 by PKC. Earlier studies have
shown that activation of the cellular pool of PKC with phorbol
12-myristate 13-acetate leads to the translocation of cytosolic GRK2 to
the plasma membrane (49, 50). A site of robust PKC phosphorylation on
GRK2 has been mapped partially to within the COOH-terminal domain, and
it has been demonstrated that such phosphorylation leads to enhanced
G-dependent GRK2 activity toward a purified
receptor substrate.
AKAP79 acts as a scaffolding protein for both PKA and PKC, associating
both kinases with the plasma membrane and the 2AR. Therefore, it is
feasible that AKAP79 also modulates the ability of PKC to affect
membrane recruitment of GRK2. If this is the case, AKAP79 might
represent a general scaffold for regulating membrane recruitment of
GRK2 by second messenger-activated kinase phosphorylation. It will be
interesting to determine whether the site of PKC phosphorylation on
GRK2 is the same as or different from that of PKA and whether such
phosphorylation also enhances G
binding. It is also worthy to
note that GRK5 can be phosphorylated by PKC at sites near the carboxyl
terminus, but unlike GRK2, this phosphorylation inhibits GRK5 activity
(53). Similarly, phosphorylation of GRK2 by Erk1 or Erk2 on serine 670 leads to an almost complete inhibition of the kinase (44). This may
represent a mechanism by which GRK2 is returned to an inactive state
after it has phosphorylated agonist-occupied receptors. PKC
phosphorylation of GRK5, therefore, seems more analogous to the
Erk1/Erk2 phosphorylation of GRK2 rather than to GRK2 phosphorylation
by either PKA or PKC. These marked differences in regulatory
phosphorylation imply that the roles of these two GRKs in GPCR
regulation are very different.
It has been reported recently that gravin (AKAP250) can also form
complexes with the 2AR receptor and that it can affect both receptor
desensitization and resensitization (30, 54). It is not yet clear what
the exact role of gravin is in these processes, but it is likely to be
dependent on the ability of gravin to associate with both second
messenger-activated kinases and specific protein phosphatases. The
ability of the
2AR to associate with more than one AKAP protein,
each with distinct yet overlapping sets of associated signaling
molecules, leads to the intriguing possibility that different steps
during receptor regulation are modulated by different AKAPs. These
steps may require the physical separation of signaling components onto
distinct scaffolds, allowing entire complexes to be recruited to the
receptor at specific stages during desensitization and resensitization. AKAP79 is constitutively associated with the
2AR, and thus it may be
required at many stages during receptor regulation (27) including early
events such as the recruitment of GRK2. The association of the receptor
with gravin, however, is regulated by agonist and therefore may be
required for more specific events in the desensitization/resensitization process.
Our studies implicate a role for AKAP79 and PKA in the regulation of
GRK2 recruitment to agonist-occupied 2AR molecules in a manner that
is mediated by the enhanced binding of GRK2 to G
subunits. This
function of a scaffold protein and PKA in the regulation of GRK2
activity represents a novel mechanism by which two kinases activated
during
2AR signaling can influence receptor desensitization.
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ACKNOWLEDGEMENTS |
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We thank Drs. Randy A. Hall, Richard Premont, Audrey Claing for helpful discussion, Millie McAdams and Judy Phelps for DNA sequencing, and Donna Addison and Mary Holben for assistance with the preparation of this manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants HL16037 (to R. J. L) and GM48231 (to J. D. S).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.
§ Present address: Division of Biology, California Inst. of Technology, 12000 E. California Blvd., Pasadena, CA 91125.
¶ Present address: Medical Research Council Laboratory of Molecular Cell Biology, University College London, Gower St., London WC1E 6BT, United Kingdom.
Investigators of the Howard Hughes Medical Institute
** To whom correspondence should be addressed: Howard Hughes Medical Inst., Box 3821, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-2974; Fax: 919-684-8875; E-mail: lefko001@receptor-biol.duke.edu.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M009130200
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ABBREVIATIONS |
---|
The abbreviations used are:
GPCR, G
protein-coupled receptor;
AKAP, A kinase-anchoring protein;
PKA, protein kinase A;
PKC, protein kinase C;
2AR,
2 adrenergic
receptor;
HEK, human embryonic kidney;
GRK, G protein-coupled receptor
kinase;
GST, glutathione S-transferase;
PAGE, polyacrylamide
gel electrophoresis;
Erk1/Erk2, extracellular signal-regulated kinase 1 and/or 2.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Freedman, N. J., and Lefkowitz, R. J. (1996) Recent Prog. Horm. Res. 51, 319-353[Medline] [Order article via Infotrieve] |
2. |
Benovic, J. L.,
Pike, L. J.,
Cerione, R. A.,
Staniszewski, C.,
Yoshimasa, T.,
Codina, J.,
Caron, M. G.,
and Lefkowitz, R. J.
(1985)
J. Biol. Chem.
260,
7094-7101 |
3. |
Bouvier, M.,
Leeb-Lundberg, L. M.,
Benovic, J. L.,
Caron, M. G.,
and Lefkowitz, R. J.
(1987)
J. Biol. Chem.
262,
3106-3113 |
4. | Hisatomi, O., Matsuda, S., Satoh, T., Kotaka, S., Imanishi, Y., and Tokunaga, F. (1998) FEBS Lett. 424, 159-164[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Inglese, J.,
Freedman, N. J.,
Koch, W. J.,
and Lefkowitz, R. J.
(1993)
J. Biol. Chem.
268,
23735-23738 |
6. | Pitcher, J., Lohse, M. J., Codina, J., Caron, M. G., and Lefkowitz, R. J. (1992) Biochemistry 31, 3193-3197[Medline] [Order article via Infotrieve] |
7. | Pitcher, J. A., Freedman, N. J., and Lefkowitz, R. J. (1998) Annu. Rev. Biochem. 67, 653-692[CrossRef][Medline] [Order article via Infotrieve] |
8. | Weiss, E. R., Raman, D., Shirakawa, S., Ducceschi, M. H., Bertram, P. T., Wong, F., Kraft, T. W., and Osawa, S. (1998) Mol. Vis. 4, 27[Medline] [Order article via Infotrieve] |
9. |
Premont, R. T.,
Inglese, J.,
and Lefkowitz, R. J.
(1995)
FASEB J.
9,
175-182 |
10. |
Daaka, Y.,
Pitcher, J. A.,
Richardson, M.,
Stoffel, R. H.,
Robishaw, J. D.,
and Lefkowitz, R. J.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
2180-2185 |
11. |
DebBurman, S. K.,
Ptasienski, J.,
Benovic, J. L.,
and Hosey, M. M.
(1996)
J. Biol. Chem.
271,
22552-22562 |
12. |
DebBurman, S. K.,
Ptasienski, J.,
Boetticher, E.,
Lomasney, J. W.,
Benovic, J. L.,
and Hosey, M. M.
(1995)
J. Biol. Chem.
270,
5742-5747 |
13. |
Koch, W. J.,
Inglese, J.,
Stone, W. C.,
and Lefkowitz, R. J.
(1993)
J. Biol. Chem.
268,
8256-8260 |
14. | Pitcher, J. A., Inglese, J., Higgins, J. B., Arriza, J. L., Casey, P. J., Kim, C., Benovic, J. L., Kwatra, M. M., Caron, M. G., and Lefkowitz, R. J. (1992) Science 257, 1264-1267[Medline] [Order article via Infotrieve] |
15. |
Pitcher, J. A.,
Fredericks, Z. L.,
Stone, W. C.,
Premont, R. T.,
Stoffel, R. H.,
Koch, W. J.,
and Lefkowitz, R. J.
(1996)
J. Biol. Chem.
271,
24907-24913 |
16. |
Pitcher, J. A.,
Touhara, K.,
Payne, E. S.,
and Lefkowitz, R. J.
(1995)
J. Biol. Chem.
270,
11707-11710 |
17. |
Pawson, T.,
and Scott, J. D.
(1997)
Science
278,
2075-2080 |
18. | Colledge, M., and Scott, J. (1999) Trends Cell Biol. 9, 216-221[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Li, Y.,
Ndubuka, C.,
and Rubin, C. S.
(1996)
J. Biol. Chem.
271,
16862-16869 |
20. |
Carr, D. W.,
Stofko-Hahn, R. E.,
Fraser, I. D.,
Cone, R. D.,
and Scott, J. D.
(1992)
J. Biol. Chem.
267,
16816-16823 |
21. |
Fraser, I.,
Tavalin, S.,
Lester, L.,
Langeberg, L.,
Westphal, A.,
Dean, R.,
Marrio, N.,
and Scott, J.
(1998)
EMBO J.
17,
2261-2272 |
22. |
Chen, Q.,
Lin, R. Y.,
and Rubin, C. S.
(1997)
J. Biol. Chem.
272,
15247-15257 |
23. |
Huang, L. J.-S.,
Durick, K.,
Weiner, J. A.,
Chun, J.,
and Taylor, S. S.
(1997)
J. Biol. Chem.
272,
8057-8064 |
24. |
Lin, R.-Y.,
Moss, S. B.,
and Rubin, C. S.
(1995)
J. Biol. Chem.
270,
27804-27811 |
25. |
Takahashi, M.,
Shibata, H.,
Shimakawa, M.,
Miyamoto, M.,
Mukai, H.,
and Ono, Y.
(1999)
J. Biol. Chem.
274,
17267-17274 |
26. |
Huang, L. J.,
Wang, L.,
Ma, Y.,
Durick, K.,
Perkins, G.,
Deerinck, T. J.,
Ellisman, M. H.,
and Taylor, S. S.
(1999)
J. Cell Biol.
145,
951-959 |
27. | Fraser, I. D., Cong, M., Kim, J., Rollins, E. N., Daaka, Y., Lefkowitz, R. J., and Scott, J. D. (2000) Curr. Biol. 10, 409-412[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Westphal, R. S.,
Tavalin, S. J.,
Lin, J. W.,
Alto, N. M.,
Fraser, I. D.,
Langeberg, L. K.,
Sheng, M.,
and Scott, J. D.
(1999)
Science
285,
93-96 |
29. | Ratcliffe, C. F., Qu, Y., McCormick, K. A., Tibbs, V. C., Dixon, J. E., Scheuer, T., and Catterall, W. A. (2000) Nat. Neurosci. 3, 437-444[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Shih, M.,
Lin, F.,
Scott, J.,
Wang, H.,
and Malbon, C.
(1999)
J. Biol. Chem.
274,
1588-1595 |
31. | Gray, P., Scott, J., and Catterall, W. (1998) Curr. Opin. Neurobiol. 8, 330-334[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Gray, P. C.,
Tibbs, V. C.,
Catterall, W. A.,
and Murphy, B. J.
(1997)
J. Biol. Chem.
272,
6297-6302 |
33. | Gray, P., Johnson, B., Westenbroek, R., Hays, L., Yates, J. R., Scheuer, T., Catterall, W., and Murphy, B. (1998) Neuron 20, 1017-1026[Medline] [Order article via Infotrieve] |
34. | Faux, M. C., and Scott, J. D. (1996) Cell 85, 9-12[Medline] [Order article via Infotrieve] |
35. |
Faux, M. C.,
and Scott, J. D.
(1997)
J. Biol. Chem.
272,
17038-17044 |
36. | Coghlan, V., Perrino, B., Howard, M., Langeberg, L., Hicks, J., Gallatin, W., and Scott, J. (1995) Science 267, 108-112[Medline] [Order article via Infotrieve] |
37. | Klauck, T. M., Faux, M. C., Labudda, K., Langeberg, L. K., Jaken, S., and Scott, J. D. (1996) Science 271, 1589-1592[Abstract] |
38. | Kim, C. M., Dion, S. B., Onorato, J. J., and Benovic, J. L. (1993) Receptor 3, 39-55[Medline] [Order article via Infotrieve] |
39. | Papermaster, D., and Dreyer, W. (1974) Biochemistry 13, 2438-2444[Medline] [Order article via Infotrieve] |
40. | Casey, P., Graziano, M., and Gilman, A. (1989) Biochemistry 28, 611-616[Medline] [Order article via Infotrieve] |
41. | Simon, J., Parsons, S., and Salmon, E. (1991) Micron Microsc. Acta 22, 405-412 |
42. |
Oppermann, M.,
Diverse-Pierluissi, M.,
Drazner, M. H.,
Dyer, S. L.,
Freedman, N. J.,
Peppel, K. C.,
and Lefkowitz, R. J.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
7649-7654 |
43. |
Freedman, N. J.,
Liggett, S. B.,
Drachman, D. E.,
Pei, G.,
Caron, M. G.,
and Lefkowitz, R. J.
(1995)
J. Biol. Chem.
270,
17953-17961 |
44. |
Pitcher, J. A.,
Tesmer, J. J.,
Freeman, J. L.,
Capel, W. D.,
Stone, W. C.,
and Lefkowitz, R. J.
(1999)
J. Biol. Chem.
274,
34531-34534 |
45. |
Freedman, N. J.,
Ament, A. S.,
Oppermann, M.,
Stoffel, R. H.,
Exum, S. T.,
and Lefkowitz, R. J.
(1997)
J. Biol. Chem.
272,
17734-17743 |
46. |
Barak, L. S.,
Tiberi, M.,
Freedman, N. J.,
Kwatra, M. M.,
Lefkowitz, R. J.,
and Caron, M. G.
(1994)
J. Biol. Chem.
269,
2790-2795 |
47. |
Pitcher, J. A.,
Hall, R. A.,
Daaka, Y.,
Zhang, J.,
Ferguson, S. S.,
Hester, S.,
Miller, S.,
Caron, M. G.,
Lefkowitz, R. J.,
and Barak, L. S.
(1998)
J. Biol. Chem.
273,
12316-12324 |
48. |
Ferguson, S. S.,
Menard, L.,
Barak, L. S.,
Koch, W. J.,
Colapietro, A. M.,
and Caron, M. G.
(1995)
J. Biol. Chem.
270,
24782-24789 |
49. |
Chuang, T. T.,
LeVine, H., III,
and De Blasi, A.
(1995)
J. Biol. Chem.
270,
18660-18665 |
50. |
Winstel, R.,
Freund, S.,
Krasel, C.,
Hoppe, E.,
and Lohse, M. J.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
2105-2109 |
51. |
Haske, T. N.,
DeBlasi, A.,
and LeVine, H.
(1996)
J. Biol. Chem.
271,
2941-2948 |
52. | Muller, S., Hekman, M., and Lohse, M. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10439-10443[Abstract] |
53. |
Pronin, A. N.,
and Benovic, J. L.
(1997)
J. Biol. Chem.
272,
3806-3812 |
54. |
Lin, F.,
Wang, H.,
and Malbon, C.
(2000)
J. Biol. Chem.
275,
19025-19034 |