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INTRODUCTION |
G protein-coupled receptor kinases
(GRKs)1 phosphorylate the
agonist-activated form of G protein-coupled receptors that in turn
promotes the high affinity binding of arrestins (1). This process
functions to both uncouple the receptor from the G protein and to
promote receptor internalization via clathrin-coated pits. The activity
and cellular localization of GRKs appear to be regulated by a variety
of molecules including activated receptors, G
subunits, phosphatidylinositol 4,5-bisphosphate, PKC, and calmodulin (1). Many of
these interactions are thought to be important largely for their
ability to regulate interaction of GRKs with the plasma membrane where
receptor substrates reside. Recent studies have provided novel
information regarding the function and cellular localization of GRKs.
For example, it was shown that GRK2 can traffic along with
2-adrenergic receptors to the endosome following receptor activation (2). Mayor and co-workers (3) have also demonstrated an association of GRK2 with microsomes that appears to be
mediated via an unidentified GRK2-binding protein. In addition, we and
others (4-6) have recently demonstrated novel interactions between
GRKs and the cytoskeleton. Collectively, these studies suggest that the
function and regulation of GRKs may involve diverse protein/protein interactions.
Caveolae represent distinct cholesterol- and glycosphingolipid-enriched
plasma membrane and vesicular structures in cells that function in a
variety of cellular processes including endothelial transcytosis and
potocytosis (7). Caveolin, a 22-24-kDa integral membrane protein
composed of cytoplasmic N and C termini and a central intramembrane
domain, is thought to be a major structural component of caveolae (7).
A 20-amino acid juxtamembrane region (the scaffolding domain) within
the N-terminal domain has been shown to mediate the association of
caveolin with other proteins (8). Recently, a wide variety of cellular
signaling molecules have been shown to associate with caveolae leading
to the hypothesis that caveolae may serve as cell-surface microdomains
that concentrate and organize cellular signaling pathways (7, 8).
Whereas some of the initial data supporting this hypothesis was derived from cell fractionation methods that may be less specific than originally thought (9-11), more recent studies have demonstrated interactions between signaling molecules and caveolin using a variety
of methods including immunoprecipitation, immunofluorescence microscopy, immunogold electron microscopy, and in vitro
binding. These studies reveal that many proteins involved in mitogenic signaling cascades, including the epidermal growth factor,
platelet-derived growth factor, insulin and Neu (c-ErbB2) receptors,
c-Src, Fyn, Erk-2, and Ras, associate with caveolin (12-19).
Similarly, various G protein-coupled signaling components including
receptors (
2-adrenergic, m2-muscarinic, B2-bradykinin,
cholecystokinin, ETA-endothelin, calcium-sensing and
angiotensin II receptors), G proteins (Gs
, Gi
, Go
, and Gq
) and
various downstream effector molecules (adenylyl cyclase, PKC
, -Y,
-
, and -
, and endothelial and neuronal nitric-oxide synthase)
have been shown to interact with caveolin suggesting a potential role
of caveolae in regulating such pathways (20-31). Since GRKs also play
an important role in regulating G protein-coupled signaling pathways,
we examined the primary sequence of the GRKs for consensus caveolin
binding motifs (32). Indeed, GRK2 and -3 were found to contain a
C-terminal, pleckstrin homology (PH) domain-localized consensus
caveolin binding motif. Here, we investigate the interaction of GRKs
with caveolin both in vitro and in intact cells, and we
demonstrate a previously unappreciated mode of regulation for these kinases.
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EXPERIMENTAL PROCEDURES |
Materials--
Hemagglutinin (HA)-specific polyclonal antibody
was from Babco. GRK1-, GRK5-, and caveolin-1-specific polyclonal
antibodies were from Santa Cruz Biotechnology, and a
caveolin-1-specific monoclonal antibody (2297) was from Transduction
Laboratories, Inc. AP-2
-subunit- and clathrin-specific antibodies
were generously provided by Dr. J. H. Keen. Transferrin
receptor-specific antibody was from Chemicon International.
Maltose-binding protein (MBP) vector, pMAL, and amylose resin were from
New England Biolabs. Most other reagents were from sources previously
described (4).
Protein Expression and Purification--
GRK2 and GRK5 were
overexpressed in and purified from Sf9 insect cells (33, 34),
and purified GRK1 was generously provided by Drs. J. Pitcher and
R. J. Lefkowitz. Purified GST, GST-caveolin-1, GST-GRK2, and
GST-GRK5 fusion proteins, and urea stripped rod outer segments were
prepared as described previously (27, 35, 36). Sf9 expressed and
purified G
1
2 and GRK2 PH domain were generously provided by Dr. S. P. Kennedy. An MBP-caveolin-1 fusion construct containing caveolin residues 1-101
(MBP-caveolin-11-101) was generated by polymerase chain
reaction amplification of base pairs 1-303 of the caveolin-1 cDNA.
The product was then subcloned into the pMal vector in-frame with the
upstream MBP via EcoRI and HindIII restriction
sites in the polylinker region. Purified MBP and MBP-caveolin-1 fusion
proteins were generated as described previously (37).
Gel Electrophoresis and Immunoblotting--
SDS-PAGE was
performed using standard methods (38). Following electrophoresis,
proteins were electroblotted onto nitrocellulose. Immunoblotting was
performed using caveolin-1-, GRK1-, GRK2-, GRK5-, AP-2-, clathrin-,
transferrin receptor-, G
-, and GST-specific primary antibodies,
horseradish peroxidase-conjugated secondary antibody (1:2000 dilution),
and visualization by ECL following the manufacturer's guidelines.
GST-Caveolin and Fusion Binding Assay--
Five µg of purified
GST or GST-caveolin-1 fusion proteins containing either the N-terminal
residues 1-61 (GST-caveolin1-61) or membrane proximal
residues 61-101 (GST-caveolin61-101) immobilized on
glutathione-agarose beads were incubated with 2 µg of purified GRK1,
GRK2, GRK2 PH domain (GRK2 residues 553-670), GRK5 or
G
1
2 in 100 µl of binding buffer (20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 100 mM NaCl and either 0.02% or 1% Triton X-100) at 30 °C
for 60 min. The samples were chilled on ice for 5 min, and the beads
were then pelleted in a microcentrifuge for 10 s, washed three
times with 400 µl of binding buffer, and boiled with SDS sample
buffer. Samples were subjected to 10% SDS-PAGE and immunoblotting
using GRK- or G
-specific antibodies.
MBP-Caveolin Fusion Binding Assay--
One µg of purified MBP
or MBP-caveolin-11-101 immobilized on amylose resin was
incubated with 200 ng of purified GRKs or soluble GST, GST-GRK2
(residues 1-184, 1-147, 1-122, 1-88, 1-63, 70-184, 80-184,
87-184, 94-184, 185-467, or 468-689), or GST-GRK5 (residues 1-200,
1-98, 1-39, 20-49, or 489-590) fusion proteins in 100 µl of
binding buffer (20 mM Tris-HCl, pH 7.5, 5 mM
EDTA, 100 mM NaCl, and 0.2% Triton X-100) at 30 °C for
60 min. Washing, elution, and analysis were identical to GST-caveolin binding experiments with the exception that GST fusion proteins were
immunoblotted with a GST-specific antibody.
Cell Culture and Transfection--
COS-1, A431 and NIH-3T3 cells
were cultured in Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum (or 10% bovine calf serum for NIH-3T3 cells),
100 units/ml penicillin G, and 100 µg/ml streptomycin sulfate at
37 °C in a humidified atmosphere containing 5% CO2.
COS-1 cells grown to 75-95% confluence in a 100-mm dish were
transfected with 20 µg total of pcDNA3, pcDNA3-GRK2, and
pCB7-caveolin-1 DNA using FugeneTM following the
manufacturer's instructions.
Cell Fractionation--
Five 150-mm dishes of A431 or NIH-3T3
cells were grown to confluence, and caveolin-rich fractions were
generated by a detergent-free sodium carbonate method, essentially as
described previously (18). Briefly, dishes were placed on ice for 5 min, rinsed three times with ice-cold phosphate-buffered saline, and
then scraped into ~1.5 ml of 500 mM sodium carbonate, pH
11.0 buffer containing protease inhibitors (1 mM
phenylmethylsulfonyl fluoride, 20 µg/ml benzamidine, and 10 µg/ml
aprotinin). Cells were then subjected to 15 strokes with a Dounce
homogenizer, three 20-s bursts with a Brinkman Polytron (2500 rpm), and
three 30-s bursts with a tip sonicator, all on ice. The extracts were
diluted 1:2 with 90% sucrose in MES-buffered saline, overlaid with 6 ml of 35% sucrose/MES-buffered saline and 3 ml of 5%
sucrose/MES-buffered saline, and centrifuged at 4 °C for ~16 h at
39,000 rpm in a Beckman SW41 rotor. After centrifugation, nine 1.3-ml
fractions were collected, and aliquots of each were subjected to
SDS-PAGE and immunoblotting using either caveolin-1-, GRK2-, AP-2-,
clathrin-, or transferrin receptor-specific antibodies.
Immunoprecipitation--
COS-1 cells, co-transfected with
pcDNA3-GRK2 and pCB7-caveolin-1, were rinsed with ice-cold
phosphate-buffered saline 24 h after transfection and harvested by
addition of 1 ml of extraction buffer (10 mM Tris-HCl, pH
7.4, 1 mM EDTA, 5 mM dithiothreitol, 100 mM NaCl, 1.0% Triton X-100, 60 mM octyl
glucoside, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml
benzamidine, and 10 µg/ml each of leupeptin, pepstatin A and
aprotinin) and rocking at 4 °C for 30 min. The cells were scraped,
vortexed 5 times, and homogenized with two 15-s bursts with a Brinkman
Polytron (2500 rpm). Lysates were centrifuged at 4 °C for 10 min at
maximum speed in a microcentrifuge, and the supernatant was removed.
For immunoprecipitation, 100 µl of supernatant was incubated with
either a GRK2-, caveolin-1, or HA-specific polyclonal antibody or a
transferrin receptor-specific monoclonal antibody for 30 min at 4 °C
followed by addition of 50 µl of 50% protein A-agarose
pre-equilibrated in extraction buffer and an additional 60 min
incubation at 4 °C. Samples were centrifuged for 10 s, pellets
were washed three times for 30 min at 4 °C with extraction buffer,
and bound proteins were eluted with 50 µl of SDS sample buffer and
boiling for 10 min. Samples were subjected to 10% SDS-PAGE and
immunoblotting using GRK2-, caveolin-1-, and transferrin
receptor-specific antibodies.
Phosphorylation Assay--
Rhodopsin phosphorylation reactions
contained, in a total volume of 20 µl, 30 nM GRK1, GRK2,
or GRK5, 400 nM rhodopsin, 100 µM
[
-32P]ATP (5 cpm/fmol), 20 mM Tris-HCl, pH
7.5, 2 mM EDTA, 7.5 mM MgCl2, and
either Me2SO (vehicle) or 0.1-12.5 µM
caveolin peptides (17, 30) in Me2SO. Reactions were
incubated at 37 °C for 5 min, stopped with SDS sample buffer, and
subjected to 10% SDS-PAGE. After autoradiography the
32P-labeled rhodopsin bands were excised and counted.
Peptide phosphorylation reactions were identical except they contained
0.1-10 mM peptide substrate (RRREEEEESAAA) instead of
rhodopsin and 50 nM GRK2 in a total volume of 100 µl.
Reactions were incubated at 30 °C for either 20 or 60 min, blotted
to P81 paper, washed 5 times with 75 mM phosphoric acid,
and counted.
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RESULTS AND DISCUSSION |
In Vitro Interaction of Caveolin and GRKs--
Since many of the
proteins involved in G protein-coupled receptor signaling associate
with caveolae and/or caveolin, we analyzed the primary sequence of GRK2
and found that a consensus caveolin binding motif
(
X
XXXX
,
XXXX
XX
, or
X
XXXX
XX
; where
is an
aromatic residue (32)) exists within the C-terminal PH domain (576WQRRYFYQF584).
To test whether GRK2 can bind to caveolin, purified GRK2 was incubated
with GST, GST-caveolin1-61, and
GST-caveolin61-101 fusion proteins immobilized on
glutathione-agarose. This analysis demonstrated specific binding of
GRK2 (~15% of the total) to GST-caveolin61-101 (Fig.
1), a protein that contains the caveolin
scaffolding domain (residues 81-101) previously implicated in caveolin
interaction with other signaling molecules such as the epidermal growth
factor receptor, c-Src, PKC, and endothelial nitric-oxide synthase (12, 17, 28, 30). Importantly, there was minimal binding to GST alone (data
not shown) or GST-caveolin1-61 (Fig. 1). Moreover, GRK2
binding to GST-caveolin61-101 was modestly enhanced at
higher ionic strength (data not shown), consistent with the binding
being primarily mediated by hydrophobic interactions. Given the
localization of the identified consensus caveolin binding sequence to
the PH domain, we also analyzed the direct binding of purified GRK2 PH
domain and observed significant and specific binding to
GST-caveolin61-101 (data not shown).

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Fig. 1.
In vitro binding of GRKs to
GST-caveolin fusion proteins. Five µg GST-caveolin fusion
proteins containing either the N-terminal (residues 1-61) or membrane
proximal (residues 61-101) regions of caveolin-1 were incubated with 2 µg of purified GRK1, GRK2, GRK5, or G 1 2
as described under "Experimental Procedures." Samples were
subjected to SDS-PAGE and immunoblotting using antibodies specific for
each of the respective GRKs or G . The left lane contains
50 ng of GRK or G 1 2, and the
center and right lanes depict GRK or
G 1 2 binding to
GST-caveolin1-61 or GST-caveolin61-101,
respectively. The gels shown are representative of 4-10 separate
experiments. Std, standard; Cav, caveolin.
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Interestingly, GRK1 and GRK5, which lack a PH domain, also displayed
specific binding to GST-caveolin61-101 (although a low
level of binding to GST-caveolin1-61 was also observed for
GRK5) (Fig. 1). To test further the binding specificity, similar experiments were performed in the presence of 1% Triton X-100 in order
to minimize the role of nonspecific hydrophobic interactions. Whereas
the overall extent of binding was slightly reduced for all of the GRKs
tested, GRK1, GRK2, GRK5, and the GRK2-PH domain, retained specific
binding to GST-caveolin61-101 (data not shown). To
demonstrate further the specificity of GRK/caveolin interactions, we
tested the binding of purified G
1
2 to the
GST-caveolin-1 constructs. Despite modification with an extremely
hydrophobic geranylgeranyl moiety, G
1
2
did not significantly interact with either
GST-caveolin1-61 or GST-caveolin61-101 (Fig. 1). These results demonstrate that the observed GRK/caveolin binding is
not simply due to nonspecific hydrophobic interactions. Overall, these
data suggest that caveolin binding may be a common feature of GRKs and
that a conserved caveolin binding motif may be present.
Analysis of the aromatic residues present in GRKs failed to reveal
conserved sequences that strictly fit the previously identified consensus caveolin binding motifs (32). However, three potential caveolin binding motifs were identified in all GRKs as follows: 63LXXXX
XX
71,
162
XX
XXXXX
XX
XX
177,
and 375
XLXXXX
382.
In order to map caveolin-binding determinants in GRKs, amylose resins
containing either purified MBP or an MBP-caveolin1-101 fusion protein were generated and incubated with either full-length GRKs or various soluble GST-GRK2 or GST-GRK5 fusion proteins. Whereas
full-length GRK1, GRK2, and GRK5 failed to interact with MBP, they each
bound to MBP-caveolin1-101 similar to their demonstrated
binding to GST-caveolin61-101 (data not shown). Analysis
of GST-GRK2 fusion proteins containing the N terminus (residues
1-184), catalytic domain (residues 185-467), and C terminus (residues
468-689) were then tested. This revealed specific binding of
GST-GRK2468-689 (Fig. 2) in
agreement with the observed binding of purified GRK2 PH domain to
GST-caveolin61-101 mentioned above. Interestingly,
GST-GRK21-184 also bound significantly to
MBP-caveolin1-101, whereas
GST-GRK2184-467 exhibited weak binding to both
MBP-caveolin1-101 and MBP, suggesting that this
interaction is not specific (Fig. 2). Importantly, neither GST alone
nor any of the other GST-GRK fusion proteins tested bound to the
MBP-amylose resin under these conditions (data not shown). To map
further the caveolin-binding determinants within the N terminus of
GRK2, GST-GRK21-184 was progressively truncated, and the
resulting fusions were analyzed for caveolin binding.
GST-GRK21-147, GST-GRK21-122, and
GST-GRK21-88 all retained binding to
MBP-caveolin1-101, whereas GST-GRK21-63 failed to bind (Fig. 2). Additional GST-GRK2 fusions
(GST-GRK270-184, GST-GRK280-184,
GST-GRK287-184, and GST-GRK294-184) were also
tested, and all failed to bind MBP-caveolin1-101. This
suggests that the critical binding determinants lie between residues 63 and 70 in GRK2. Indeed, residues 63-71 were initially identified as a
conserved domain with similarity to the consensus sequences for
caveolin binding (Fig. 2C).

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Fig. 2.
In vitro binding of soluble
GST-GRK fusion proteins to a MBP-caveolin fusion protein.
A, 1 µg of MBP-caveolin1-101 was incubated
with 200 ng of GST-GRK2 or GST-GRK5 fusion proteins as described under
"Experimental Procedures." Samples were subjected to SDS-PAGE and
immunoblotting using a GST-specific antibody. Representative blots
demonstrating binding of the indicated GST-GRK2 or GST-GRK5 fusion
protein to MBP-caveolin1-101 are shown. Standards
(Std) containing 10% of the total of either
GST-GRK21-184 or GST-GRK51-200 are shown on
the left. B, a schematic representation of GRK
domains included in the binding assay is shown with binding activity
summarized as either ~10% (+) or no binding ( ) on the
right. C, alignment of GRK sequences in the
region of GRK2 residues 63-71 mapped to contain caveolin-binding
determinants. Conserved aromatic and hydrophobic residues that may be
important for caveolin binding are shaded.
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In order to determine if this N-terminal domain was responsible for the
conserved GRK/caveolin interactions, the binding of several GST-GRK5
fusion proteins to MBP-caveolin1-101 was also tested.
Initial analysis of an N-terminal construct
(GST-GRK51-200) demonstrated significant and specific
binding to MBP-caveolin1-101, whereas a C-terminal
construct (GST-GRK5489-590) failed to bind (Fig. 2). To
map further the N-terminal caveolin-binding site,
GST-GRK51-98, GST-GRK51-39, and
GST-GRK520-49 were analyzed. Although
GST-GRK51-98 retained binding to MBP-caveolin1-101, GST-GRK51-39 and
GST-GRK520-49 failed to bind (Fig. 2) suggesting that the
critical caveolin binding region in GRK5 lies between residues 49 and
98, a region that overlaps the N-terminal caveolin binding region
identified in GRK2. These data suggest that the N-terminal region,
including GRK2 residues 63-71 (LXXXX
XX
)
(Fig. 2C), is important for the conserved GRK/caveolin
binding characteristics. The initial study that proposed caveolin
binding motifs (
X
XXXX
,
XXXX
XX
, or
X
XXXX
XX
) used the caveolin
scaffolding domain to select random peptide ligands from phage display
libraries (32). Interestingly, in that study nearly 10% of the
selected 15-mer peptides contained only two aromatic residues.
Additionally, whereas a generally conserved caveolin consensus motif,
including four aromatic residues (
X
XXXX
XX
) has been
identified in G
subunits, the G
q subunit has
substitutions of valine and leucine for two of these aromatic residues
(32). Despite this substitution, G
q has been shown to
co-immunoprecipitate with caveolin (22). Thus, it seems possible that
caveolin binding requirements may be broader than initially described.
Finally, although the functional significance for the existence of two
caveolin binding regions in GRK2 (residues 63-71 and the PH domain)
remains to be elucidated, it is of interest that the PH
domain-localized motif overlaps with a region that includes
phospholipid-binding determinants (39).
Interaction of GRK2 with Caveolin in Intact Cells--
Two
approaches were used to ascertain whether GRK2 and caveolin associate
in intact cells. The first employed a widely used extraction and
fractionation method that enables the separation of caveolae from other
cellular organelles (18). For these studies, A431 cells were lysed in a
detergent-free sodium carbonate buffer, fractionated on a discontinuous
sucrose gradient, and then analyzed for endogenous caveolin and GRK2 by
SDS-PAGE and immunoblotting. As expected, the 5/35% sucrose interface
(fraction 3) contained the bulk of the cellular caveolin, whereas most
of the cellular protein was found in fractions 8 and 9 (Fig.
3A). Analysis of GRK2 revealed
that while it was primarily present in fractions 8 and 9, a significant
portion also co-fractionated with caveolin (Fig. 3A). A
similar GRK2/caveolin co-fractionation was observed in NIH-3T3 cells
(data not shown). Importantly, a number of molecules known to associate
either peripherally (AP-2 or clathrin) or integrally (transferrin
receptor) with a variety of cellular membranes including the plasma
membrane, endosomes, the endoplasmic reticulum, and the Golgi (40, 41)
were found to be almost exclusively restricted to fractions 8 and 9 (Fig. 3A). Thus, these data confirm that this fractionation
method generates a specific enrichment of certain cellular membranes
including caveolae while excluding most others. However, several recent
studies have further characterized the membranes and associated
proteins found in caveolin-rich fractions generated from these or
similar methods and demonstrated the presence of
non-caveolin-containing membranes (9-11). Thus, although our data are
consistent with caveolar localization of GRK2 in A431 and NIH-3T3
cells, we cannot rule out the possibility that GRK2 might be
associated with one of these other compartments.

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Fig. 3.
Association of GRK2 and caveolin in
intact cells. A, fractionation of endogenous caveolin
and GRK2 from A431 cells. A431 cells were homogenized and fractionated
on a discontinuous sucrose gradient as described under "Experimental
Procedures." Aliquots of each fraction were then subjected to
SDS-PAGE followed by Ponceau-S protein staining or immunoblotting with
caveolin-1- (Cav-1), GRK2-, clathrin-, AP-2-, or transferrin
receptor (Trf-R)-specific antibodies. B,
immunoprecipitation (IP) of caveolin-1 and GRK2 from COS-1
cells. COS-1 cells, co-transfected with pcDNA3-GRK2 and
pCB7-caveolin-1, were harvested 24 h post-transfection, lysed, and
immunoprecipitated with either HA-, caveolin-1-, or GRK2-specific
polyclonal antibodies as described under "Experimental Procedures."
Samples were subjected to SDS-PAGE and immunoblotted using GRK2- and
caveolin-specific monoclonal antibodies.
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In order to demonstrate directly a GRK2/caveolin association in cells,
we used an immunoprecipitation approach. In these studies, COS-1 cells
co-expressing GRK2 and caveolin-1 were lysed in the presence of
detergent (1% Triton X-100 and 60 mM octyl glucoside) in
order to solubilize caveolin. Cleared lysates were then incubated with
either HA-, GRK2-, or caveolin-specific polyclonal antibodies and then
precipitated by addition of protein A-agarose and centrifugation. Bound
proteins were eluted with SDS buffer and subjected to SDS-PAGE and
immunoblotting using GRK2- and caveolin-specific monoclonal antibodies.
As expected, GRK2 and caveolin were effectively precipitated by their
respective specific antibodies whereas HA antibodies failed to
precipitate detectable GRK2 or caveolin. Importantly, when GRK2
immunoprecipitations were probed with monoclonal antibodies for
caveolin-1, a significant amount of caveolin was detected (~3% of
the total) (Fig. 3B). Similarly, caveolin
immunoprecipitations probed with a monoclonal GRK2 antibody revealed a
significant amount of associated GRK2 (~6% of the total) (Fig.
3B). To demonstrate further the specificity of this method,
immunoprecipitation using a transferrin receptor-specific monoclonal
antibody was performed. This antibody significantly precipitated
transferrin receptor but failed to precipitate detectable levels of
either caveolin or GRK2 (data not shown). Thus, these data demonstrate
that a portion of the cellular GRK2 was specifically associated with caveolin in these cells and remained associated during the subsequent solubilization in 1% Triton X-100 and 60 mM octyl
glucoside and immunoprecipitation.
Taken together, the co-fractionation and co-immunoprecipitation of GRK2
and caveolin suggest that GRK/caveolin interactions are likely to occur
in vivo. It is important to note that many studies have
assessed GRK2/3 partitioning to membrane and cytosolic cell fractions
using a variety of methods. In these studies GRK2/3 was found to be
primarily cytoplasmic with only a small fraction (5-15%) associated
with the membrane (2, 42, 43). Thus, the fraction of the total cellular
GRK2 associated with the caveolin-rich fraction (Fig. 3A)
and with caveolin immunoprecipitates (Fig. 3B) likely
represents a large portion of the membrane-associated GRK2. Therefore,
membrane-associated events, such as GRK-mediated receptor
phosphorylation, may be significantly affected by GRK/caveolin interaction.
Functional Significance of GRK/Caveolin Interaction--
In order
to explore the potential functional significance of the GRK/caveolin
interaction, we considered that GRK activity may be regulated by the
caveolin scaffolding domain in a fashion similar to the previously
demonstrated inhibition of the epidermal growth factor receptor (12),
c-Src (17), PKC (28), adenylyl cyclase (29), and endothelial
nitric-oxide synthase (30). In order to test this hypothesis, GRK2
phosphorylation of the receptor substrate rhodopsin was examined in the
presence of either scaffolding domain peptides from caveolin-1, -2, or
-3 or a control peptide, caveolin-153-81 (Fig.
4A). In addition, because the
scaffolding domain peptides are very hydrophobic, we also included a
partially scrambled caveolin-1 scaffolding domain peptide in these
studies (30) (Fig. 4A). Since the amino acid composition of
this peptide is 100% identical to the caveolin-1 scaffolding domain
peptide, it should enable discrimination between sequence-specific binding and nonspecific hydrophobic or ionic interactions. In these
studies caveolin-1 and caveolin-3 scaffolding domain peptides effectively inhibited GRK2 activity in a dose-dependent
manner with IC50 values of ~0.4 and ~0.8
µM, respectively (Fig. 4B). In contrast, the
caveolin-2 scaffolding domain and caveolin-153-81 control
peptides had no effect on GRK2 phosphorylation of rhodopsin. Analysis
of the other GRKs revealed that GRK1 was also effectively inhibited by
caveolin-1 and -3 scaffolding domain peptides with IC50
values of ~2.7 and ~1.8 µM, respectively, as was GRK5
with IC50 values of ~3.0 and ~2.5 µM,
respectively (data not shown). Similarly, GRK1 and GRK5 activity were
unaffected by the caveolin-2 scaffolding domain or
caveolin-153-81 control peptides. Importantly, for all of
the GRKs tested the partially scrambled caveolin-1 peptide was found to
be dramatically less effective at inhibiting receptor phosphorylation
(Fig. 4B). Given that this peptide is 100% identical in
composition and 60% identical in sequence, it is not surprising that
this peptide is partially inhibitory albeit with significantly reduced
potency. Taken together, these results provide good evidence that the
observed inhibition of GRK-mediated receptor phosphorylation by
caveolin scaffolding domain peptides is due to sequence-specific
GRK/caveolin binding. It is worth noting that the average
IC50 for caveolin peptide inhibition of GRK2-mediated
rhodopsin phosphorylation is >4-fold lower than the average
IC50 for GRK1/GRK5 inhibition. This difference may be
related to the presence of the second caveolin binding region in GRK2
located within the PH domain. In fact, the caveolin binding motif
present in the PH domain overlaps a region previously suggested to be
important for binding acidic phospholipids (39). Thus, it is possible
that caveolin could compete with phospholipid binding resulting in
additional inhibition of GRK2-mediated rhodopsin phosphorylation.

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Fig. 4.
Inhibition of GRK activity by caveolin
scaffolding domain peptides. A, sequences of the
caveolin-1, -2, and -3 (CAV-1, -2, and -3) scaffolding domain peptides
are aligned along with a partially scrambled caveolin-1 scaffolding
domain peptide (CAV-1 ). Residues conserved with
caveolin-1 are shaded. The sequence of a contol peptide,
caveolin-153-81 (CAV-1X), is also shown.
B, inhibition of GRK-mediated rhodopsin phosphorylation.
Reactions were performed at 30 °C for 5 min and contained 30 nM GRK2 and 400 nM rhodopsin in the presence of
0-12.5 µM caveolin scaffolding domain peptides, CAV-1
( ), -2 ( ), or -3 ( ) or control peptides CAV-1X ( ) and
CAV-1 ( ) as described under "Experimental Procedures." The
samples were subjected to SDS-PAGE, and the extent of rhodopsin
phosphorylation was determined by excising and counting the
32P-labeled bands. Rhodopsin phosphorylation, expressed as
a fraction of control (i.e. vehicle only), is plotted
against caveolin peptide concentration. C, inhibition of
GRK2-mediated peptide phosphorylation. Peptide phosphorylation
reactions were performed at 30 °C for 60 min and contained 50 nM GRK2, 1 mM RRREEEEESAAA, and 0-12.5
µM caveolin peptides as stated above in B.
Reactions were blotted on to P81 paper, washed in phosphoric acid, and
counted to quantitate extent of phosphorylation. GRK2-mediated
phosphorylation was taken as counts/min above control samples lacking
GRK2, and these data were plotted as above. D, kinetic
analysis of GRK2-mediated peptide phosphorylation. Peptide
phosphorylation reactions were performed at 30 °C for 20 min and
contained 50 nM GRK2, 0.1-10 mM RRREEEEESAAA
and 0 ( ), 1 ( ), or 3 ( ) µM caveolin-1
scaffolding domain peptide. Reactions were stopped as described above,
and the resulting data were used to generate an Eadie-Scatchard plot.
All values are mean ± S.E. from three separate experiments.
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In principle, the inhibition of GRKs might be attributed to (i)
inhibition of the catalytic domain through either direct or allosteric
mechanisms or (ii) inhibition of receptor binding to critical
determinants outside of the catalytic domain. In order to discriminate
between these two possibilities, we tested the effects of the caveolin
scaffolding domain peptides on GRK2 phosphorylation of the peptide
substrate RRREEEEESAAA. In these experiments, all caveolin peptides
demonstrated a similar profile for inhibition of peptide
phosphorylation as with the receptor, with the caveolin-1 and -3 scaffolding domain peptides exhibiting inhibition with IC50
values of ~0.9 and ~0.8 µM, respectively. The
partially scrambled caveolin-1 peptide inhibited GRK2 only at higher
concentrations, with an IC50 greater than 10-fold higher
(~12 µM) than that of the wild type caveolin-1 peptide
(Fig. 4C). These data suggest that the inhibitory properties
of the caveolin peptides are specific and likely to be exerted on the
catalytic domain rather than by perturbing receptor/GRK interactions
outside of the catalytic domain.
In order to discriminate between competitive (direct) and
noncompetitive (allosteric) modes of inhibition, kinetic analysis was
performed by phosphorylating different concentrations of peptide substrate in the presence of 0, 1, or 3 µM caveolin-1
scaffolding domain peptide. An Eadie-Scatchard plot of these data
reveal a profile suggestive of a predominantly
noncompetitive/allosteric mode of inhibition (Fig. 4D).
Similarly, a double-reciprocal plot of the peptide phosphorylation data
yielded Km values of 12.8 and 13.3 mM
peptide substrate for reactions performed in the presence of 0 and 3 µM caveolin peptide, respectively, whereas the
Vmax changed from 19.6 to 8.1 nmol
Pi/min/mg in the presence of 0 and 3 µM
caveolin peptide, respectively (data not shown). Thus, it appears that
an allosteric mode of inhibition is likely to be the predominant
mechanism for caveolin peptide-mediated inhibition of GRKs. This is
consistent with the mapping of caveolin-binding determinants to sites
outside of the catalytic domain. The fact that the GRK1, -2, and -5 are
all inhibited by caveolin suggests that the identified conserved
N-terminal site (Fig. 2) is likely to be most important for mediating
this inhibition. However, caveolin binding to the PH domain of GRK2 may
additionally contribute to its allosteric regulation. Indeed, binding
of acidic phospholipids to this domain of GRK2 has been shown to
mediate allosteric effects on GRK2 activity (44).
Our results suggest that GRKs may be subject to some level of tonic
inhibition in cells that express caveolin-1 or -3. This potential
functional role may be of particular importance in cardiac myocytes
under the pathological condition of congestive heart failure. Sustained
-agonist stimulation of the mouse heart results in characteristic
features of congestive heart failure including left ventricular
hypertrophy and decreased
-adrenergic receptor signaling (45-47).
Such chronic stimulation also results in a 2-3-fold increase in left
ventricular GRK2 and GRK5 levels, an occurrence that may contribute to
the pathological decrease in
-adrenergic receptor signaling in
congestive heart failure (46, 47). Interestingly, a recent study
demonstrated an ~2-fold decrease in myocyte caveolin expression in
hearts from mice subjected to sustained
-agonist stimulation (48).
Thus, it seems plausible that in the failing cardiac myocyte a
concomitant increase in GRK and decrease in caveolin expression could
have combined effects on GRK activity and consequently the
-agonist
responsiveness of the myocyte.
In summary, the above data identify a novel interaction between GRKs
and caveolin that results in potent inhibition of GRK activity. These
interactions are suggested to occur in vivo by the
co-fractionation and co-immunoprecipitation of GRK2 and caveolin in
cells. Although the overall cellular role of this interaction remains
to be elucidated, one possibility is that caveolin serves to suppress
basal GRK activity. Another possibility is that localization of GRKs in
caveolae may direct a distinct set of protein/protein interactions
thereby regulating GRK specificity. Indeed, the cellular localization
and consequently the substrate specificity of protein kinase A is
regulated in such a fashion via its interaction with a family of
scaffolding proteins (49, 50). Thus, future investigation into this
area should include assessment of novel caveolae-dependent protein interactions for GRKs, as well as further elucidation of the
physiological role of GRK/caveolin interaction.