From the Biology Department, Siena College,
Loudonville, New York 12211, the ¶ Department of Chemistry and
Biochemistry, Institute for Cellular and Molecular Biology, The
University of Texas at Austin, Austin, Texas 78712, the
Department of Microbiology and Immunology, Kimmel Cancer Center,
Thomas Jefferson University, Philadelphia, Pennsylvania 19107, and
** Senomyx, Inc., La Jolla, California 92037
Received for publication, August 27, 2002, and in revised form, October 30, 2002
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ABSTRACT |
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G protein-coupled receptors (GPCRs)
transduce cellular signals from hormones, neurotransmitters, light, and
odorants by activating heterotrimeric guanine nucleotide-binding
(G) proteins. For many GPCRs, short term regulation is initiated by
agonist-dependent phosphorylation by GPCR kinases (GRKs),
such as GRK2, resulting in G protein/receptor uncoupling. GRK2 also
regulates signaling by binding G G protein-coupled receptors
(GPCRs)1 are a large family
of integral membrane proteins that form seven transmembrane helices and
couple to heterotrimeric guanine nucleotide (G)-binding proteins on
their cytoplasmic surface. They transmit the signals from light and
odorant receptors as well as the signals initiated by numerous hormones
and neurotransmitters. In their inactive state, heterotrimeric G
proteins are complexes of three polypeptide chains (G GRKs are found in metazoans and, in mammals, the GRK family has seven
members (7, 12). GRKs are serine/threonine kinases with a tripartite
modular structure. A central ~350 amino acid kinase domain is closely
related by sequence identity to those of cAMP-dependent
protein kinases, protein kinase C, and ribosomal S6 kinases (13). At
the carboxyl terminus of the catalytic core (14) homology to
cAMP-dependent protein kinase predicts a putative "nucleotide gate" (15). The catalytic domain is flanked by an amino-terminal domain of 178 residues and a carboxyl-terminal domain
that varies in structure among members of the family. Using distinct
mechanisms, the carboxyl-terminal domains of GRKs direct the membrane
association of these kinases (16, 17).
The amino-terminal domains of all GRK family members are homologous to
the regulator of G-protein signaling (RGS) family of proteins (18). RGS
proteins are a multifunctional family of proteins of variable length
that share a ~120-amino acid "RGS domain." In this paper, we
refer to this domain as the RGS homology (RH) domain. RGS proteins act
as GTPase activating proteins (GAPs) for the G Other families of proteins have RH domains but their roles in
regulating heterotrimeric G protein signaling are either distinct from
RGS proteins, not well characterized, or do not regulate heterotrimeric
G protein signaling. Axin plays a role in the wnt/embryonic development
signaling pathway (26) and shares ~30% amino acid identity with RGS
proteins of other subfamilies. This RH domain has never been
demonstrated to bind or GAP a G The RH domain of GRK2 is most closely related to that of axin (26%
amino acid identity) and RGS12 (24% amino acid identity) and binds to
G Materials
Human embryonic kidney cells (HEK293) and African Green monkey
kidney cells (COS-1) were from the American Tissue Culture Collection.
G Preparation and Mutagenesis of pGEX-GRK2-(45-178) Constructs
Nucleotides encoding residues 45-178 of bovine GRK2 cDNA
were amplified by the polymerase chain reaction using primers that incorporated BamHI and EcoRI restriction sites at
the 5' and 3' ends of the coding region, respectively. The resulting
PCR fragment was subcloned into BamHI and EcoRI
sites of the glutathione S-transferase fusion protein
vector, pGEX-2T (Amersham Biosciences) to generate pGEX-GRK2-(45-178). Sequential PCR (41) was used to produce the E78K,
V83A, and D160K derivatives of pGEX-GRK2-(45-178), and Quik-Change
mutagenesis (Stratagene) was used to generate all other mutations. The
GRK2 portion of each construct was sequenced to verify that only the
intended mutation had occurred.
Purification of GST-GRK2 Fusion Proteins
GST-GRK2 fusion proteins were expressed and purified by
modifications of the procedures of Smith and Johnson (42) and Frangioni and Neel (43). Briefly, 40-ml cultures in Luria broth containing 5 µg/ml carbenicillin were grown at 37 °C to an optical density of
0.5, fusion protein expression was induced by the addition of
isopropyl-1-thio- GST-GRK2 Pull-down Assays with Bovine Brain
G Bovine brain extract was used as a source of
G Rhodopsin Phosphorylation
COS-1 cells were grown at 37 °C to 50-90% confluence on
10-cm dishes in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. The cells were transfected with 10 µg of total DNA (pcDNA3 alone,
pcDNA3-GRK2, or pcDNA3-mutant GRK2) using FuGENE 6 (Roche
Molecular Biochemicals) following the manufacturer's recommendations.
The cells were harvested after 48 h, washed twice in ice-cold
phosphate-buffered saline, and lysed in 1 ml of buffer (20 mM HEPES, pH 7.2, 150 mM NaCl, 10 mM EDTA, 0.02% Triton X-100, 0.5 mM
phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, and 100 µg/ml
benzamidine) by Polytron homogenization (two 15-s bursts at 2500 rpm).
Lysates were centrifuged for 10 min at 40,000 × g to
remove particulate matter and supernatants were then assayed.
To test for GRK activity, lysates containing wild type (WT) or mutant
GRK2 protein were assayed for their ability to phosphorylate light-activated rhodopsin. Two microliters of COS-1 cell lysate were
incubated with 20 mM Tris-HCl, pH 7.5, 2 mM
EDTA, 5 mM MgCl2, 100 µM ATP,
~1 µCi of [ Homology Modeling
A homology model of the RH domain of GRK2 (residues 42-178) was
based on the structure of the RH domain of axin (PDB code 1EMU), which
is the closest homolog based on a BLAST (44) search of the protein data
bank (26% identity within residues 64-174 of GRK2). The GRK2 model
was built manually using the program O (45) by choosing appropriate and
reasonable rotamers for nonidentical residues (46). In regions that
appeared to have higher sequence identity with other RH domains of
known structure, the GRK2 model was adjusted locally according to those
models. The In Vivo Inositol Phosphate Determination
GRK2 Mutants--
To measure in vivo synthesized
inositol phosphate (IP), 3.3 × 105 COS-1 cells were
plated on 6-cm dishes in DMEM (Mediatech, Herndon, VA) containing
penicillin (100 units/ml), streptomycin (100 µg/ml), and 10% fetal
bovine serum. After 24 h, cells were transfected using FuGENE 6 (Roche Molecular Biochemicals) with 1 µg of total DNA at a ratio of
3:1 pcDNA3-HA-G G Confocal Microscopy--
HEK293 cells were transfected in 6-well
plates with the indicated amounts of expression plasmids for
GRK2-(45-178)-GFP, RGS2-GFP, and/or G Identification of GRK2 RGS Domain Mutants That Are Defective in
Binding to G
The three-dimensional structures of several RH domains have been
determined by x-ray crystallography (RGS4, RGS9, axin, PDZRhoGEF, and
p115RhoGEF) (23, 24, 52-54), and solution NMR (GAIP and RGS4) (55,
56). Together these studies show that these RH domains share a very
similar fold (two four-helix bundles, see Fig.
1, B and C). The
x-ray structures of two of these proteins, RGS4 and RGS9, were
determined both alone and in complex with their G
The first residues of GRK2 targeted for mutagenesis were those that are
identical or similar to residues in RGS4 known to contact
G
Binding of axin to its RH domain ligand, an
We then compiled a structural alignment to compare the RH domain of
GRKs to RH domains whose structures have been solved (Fig. 1A) and used the known three-dimensional structures of RH
domains that had the highest sequence identity to the GRK2 RH domain
(axin and GAIP) to model residues 42-175. The model was then used to predict residues that might be close to Arg106 and
Asp110 in three-dimensional space. Substitutions F109I,
M114A, and E116A exhibited the greatest diminution in the binding to
G GRK Mutants with G GRK Mutants with G G GRK2 Binds RGS-resistant G
We first looked at the ability of the RGS-resistant mutant of
G
We next looked at the ability of GRK2 to attenuate signaling by the
RGS-resistant mutant. HEK293 cells were co-transfected with
G Through extensive mutational analysis, we have identified eight
residues in the GRK2 RH domain (Arg106, Phe109,
Asp110, Met114, Lys115,
Glu116, Leu117, and Val137) that,
when mutated, alter binding to G G One mutant, K115A, has a particularly interesting phenotype. WT GRK2
prefers binding to the transition state over the GDP-bound form of
G Switch regions I, II, and III constitute most of the buried surfaces on
G Of the eight residues whose alterations cause G Residues in the RGS It has recently been shown that LARG, in addition to binding
G In summary, extensive mutational analysis of GRK2 shows that
Gq/ll and inhibiting
G
q stimulation of the effector phospholipase C
. The
binding site for G
q/ll resides within the amino-terminal
domain of GRK2, which is homologous to the regulator of G protein
signaling (RGS) family of proteins. To map the G
q/ll binding site on GRK2, we carried out site-directed mutagenesis of the
RGS homology (RH) domain and identified eight residues, which when
mutated, alter binding to G
q/ll. These mutations do not
alter the ability of full-length GRK2 to phosphorylate rhodopsin, an
activity that also requires the amino-terminal domain. Mutations causing G
q/ll binding defects impair recruitment to the
plasma membrane by activated G
q and regulation of
G
q-stimulated phospholipase C
activity when
introduced into full-length GRK2. Two different protein interaction
sites have previously been identified on RH domains. The G
binding
sites on RGS4 and RGS9, called the "A" site, is localized to the
loops between helices
3 and
4,
5 and
6, and
7 and
8.
The adenomatous polyposis coli (APC) binding site of axin involves
residues on
helices 3, 4, and 5 (the "B" site) of its RH
domain. We demonstrate that the G
q/ll binding site on
the GRK2 RH domain is distinct from the "A" and "B" sites and
maps primarily to the COOH terminus of its
5 helix. We suggest that
this novel protein interaction site on an RH domain be designated the
"C" site.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
). Upon activation, GPCRs catalyze the exchange of GTP for GDP on
the G
subunit resulting in dissociation of the GTP-bound G
subunit from the G
dimer (1). G
and G
are then free to
regulate effectors such as adenylyl cyclase, phospholipase C
(PLC
), cGMP phosphodiesterase, ion channels, Rho family
guanine-nucleotide exchange factors (RhoGEF), and activate
mitogen-activated protein kinase signal transduction pathways (2-5).
One common feature of GPCR signaling is the rapid loss of cellular
sensitivity even in the presence of a stimulus. Insensitivity to the
extracellular stimulus reflects intracellular events: receptor/G
protein uncoupling, G protein inactivation, and receptor sequestration
(and receptor degradation), which together act to regulate the duration
and/or magnitude of the signaling event (6). One mode of receptor desensitization is initiated by phosphorylation of the activated receptor by a kinase of the G protein-coupled receptor kinase (GRK)
family (7). Phosphorylation then promotes binding of the GPCR to a
family of proteins called arrestins (8). This occludes G
interaction with receptor and, in some nonvisual cells, leads to
sequestration of the receptor away from the plasma membrane into
endocytic vesicles (8-11).
i/o
(including G
t) and G
q family of G
subunits (19-21) and as antagonists of G
/effector interaction (19,
22). In general, these proteins bind preferentially to the GDP-aluminum fluoride (GDP·AlF
S
GDP-bound form of G
(21). The crystal structures of the
RGS4·G
i1 complex and the
RGS9·G
t/i chimera·cGMP phosphodiesterase
complex
show that the RGS proteins contact switch regions I, II, and III of G
, which are polypeptide loops that undergo conformational changes in the transformation between the GDP-bound (inactive) and the GTP-bound (active) states of the G protein. In these examples, the
binding of the RGS protein appears to stabilize the three switch
regions in a conformation that preferentially binds the transition
state for GTP hydrolysis (23, 24). RH domains can be grouped into five
subfamilies based on their evolutionary relatedness: R4, R7, R12, RZ,
and RA (axin) families (21). Members of the R4, R7, R12, and RZ
families are negative regulators of G protein signaling as described
above. The newly described G
s-specific RGS, RGS-PX1
(25), likely defines another RGS subfamily as this protein is similarly
related to all 5 RGS subfamilies (~24% amino acid identity).
subunit (27). Instead the RH domain
of axin binds the tumor suppressor protein, APC (28), a downstream
target in the wnt signaling pathway. The APC binding site of axin is
distinct from the G
binding site of RGS proteins. A family of
guanine nucleotide exchange factors for the monomeric G protein Rho
(RhoGEFs) also has RH domains that share <20% identity to the RGS
family. p115RhoGEF, PDZRhoGEF, and LARG (leukemia-associated RhoGEF),
bind and in some cases serve as GAPs for G
12,
G
13, and G
q, via their RH domains yet they are also downstream effectors of G
12 and
G
13 (29-32). D-AKAP2, dual specificity A
kinase anchoring protein 2, binds the regulatory subunit of
cAMP-dependent protein kinase and has 2 RH domains. However, no G protein interaction has been reported for this protein (33).
q and G
11 in an
AlF
s, G
i, or
G
12/13 (34-36). Whereas all GRKs have putative
amino-terminal RH domains, G
interaction has only been observed for
GRK2 and GRK3. Unlike other G
q-binding RGS proteins such
as RGS2 (37), RGS3 (38), RGS4 (22), and RGS18 (39), the GRK2 RH domain
does not stimulate the GTPase activity of G
q in a single
turnover GAP assay and only weakly stimulates GTPase when
G
q is reconstituted with M1 muscarinic receptor and assayed in an agonist-induced steady-state GTPase assay (34). Because
the GRK2 RH domain inhibits G
q-stimulated PLC
activity both in vivo and in vitro yet lacks
significant GAP activity in vitro, it has been postulated
that GRK2 RGS acts by sequestration of G
q. It is also
possible that GRK2 is an effector of G
q. In this
scenario, activation of G
q would recruit GRK2 to the
site of an activated receptor. To investigate the role of
GRK2/G
q interaction in the regulation of Gq
signaling, we have created mutations in the RH domain of GRK2 that
result in altered binding to G
q/11. Surprisingly, we
found that the surface of GRK2 used to bind G
q/11 is
distinct from the interaction site utilized by other RGS proteins to
bind G
subunits and from the site used by axin to bind APC.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
q/11-specific polyclonal antibodies were generously provided by Dr. D. Manning or purchased from Santa Cruz Antibodies, and
EE-specific monoclonal antibody was provided by Dr. H. Bourne. RGS2-GFP
(40) and GRK2-(45-178)-GFP were expressed from the plasmid
pEGFP (Clontech, Palo Alto, CA) and were generously
provided by Dr. S. Heximer and Dr. R. Penn, respectively.
myo-[3H]Inositol was from Amersham
Biosciences, Dowex AG1-X8 resin was from Bio-Rad, and scintillation
fluid was from Packard. Molecular biologicals were from Roche Molecular
Biochemicals unless otherwise indicated, immunoblotting detection
reagents were from Pierce, and all other biochemicals were from Sigma
or Fisher.
-D-galactopyranoside to 0.5 mM, and incubation was continued for 3 h at 25 °C.
Cells were pelleted, washed in STE (20 mM Tris, 150 mM NaCl, 1 mM EDTA, pH 8), and frozen at
70 °C. Pellets were resuspended on ice in STE containing 100 µg/ml lysozyme and incubated on ice for 15 min before the addition of
-mercaptoethanol to 10 mM, phenylmethylsulfonyl fluoride
to 100 µM, leupeptin to 1 µg/ml, benzamidine to 20 µg/ml, and Sarkosyl to 1.5%. Lysates were sonicated in 10-s bursts
followed by 15-s rest periods to reduce viscosity. Insoluble protein
was removed by centrifugation at 12,000 rpm for 10 min at 4 °C and
Triton X-100 was added to a final concentration of 2%. The lysate was adjusted to 25 mg/ml protein as determined by the Bradford assay using
-globulin as a standard (Bio-Rad) and fusion proteins were bound to
glutathione-agarose beads (3 ml of packed beads/100 mg of protein) by
mixing for 1 h at 4 °C. The beads were washed once with STE,
1.5% Sarkosyl, 2% Triton X-100, three times with STE, and stored in
STE, 25% glycerol, 10 mM
-mercaptoethanol at
20 °C. To determine the amount of GRK2 associated with
glutathione-agarose beads, fusion proteins were eluted in 50 mM Tris-HCl, pH 8, 10 mM glutathione, 10 mM
-mercaptoethanol at room temperature for 1 h.
Bradford assays were then carried out on the eluates.
q/11
q/11 for in vitro binding assays. For 1-ml
binding assays, 8 µg of fusion protein was incubated overnight at
4 °C with 200 µg of brain extract protein, prepared as described
by Carman et al. (34), in 20 mM Tris-HCl, pH 8, 2 mM MgSO4, 6 mM
-mercaptoethanol, 100 mM NaCl, 0.05%
C12E10, 5% glycerol, 100 µM GDP
in the absence or presence of 30 µM aluminum chloride and
5 mM sodium fluoride
(AlF
q/11-specific polyclonal antibodies,
incubated with peroxidase-conjugated secondary antibody, and
G
q/11 was visualized by chemiluminescence using
SuperSignal West Pico (Pierce).
-32P]ATP, and ~3.5 µM
rhodopsin for 10 min at 30 °C in room light. Reactions were quenched
by addition of SDS sample buffer followed by 30 min incubation at room
temperature. Rhodopsin was separated by electrophoresis on a 10%
SDS-polyacrylamide gel, and gels were fixed in 0.7 M
trichloroacetic acid, 0.14 M 5'-sulfosalicylic acid for 10 min to remove unincorporated radionucleotide, washed twice in 50%
ethanol, 16% acetic acid for 10 min, dried, and then subjected to
autoradiography. Rhodopsin bands were excised and counted in a liquid
scintillation counter. Repeated measures analysis of variance was used
to test the statistical significance.
5/
6 loop of the GRK2 RH domain has no obvious
sequence homology to RH domains of known structure and was ultimately
modeled based on the axin structure because the fit of the side chains
appeared to be reasonable and because the
5/
6 loops of GRK2 and
axin are identical in length (the loop is one amino acid shorter in the
RGS family of proteins). The overall model was refined in O to idealize
its stereochemistry.
q-R183C:pcDNA3-GRK2 (or mutant derivatives of GRK2). Following a 24-h incubation, transfected cells
were replated (~7 × 104 cell/well) in triplicate on
24-well plates and incubated in complete DMEM. The media was removed
and cells were labeled with myo-[3H]inositol
(Amersham Biosciences) for 13-18 h in DMEM, without sodium pyruvate,
with high glucose, with L-glutamine, and with pyridoxine
hydrochloride. In early experiments, labeling was carried out in
inositol-free DMEM (Invitrogen), whereas later experiments utilized complete DMEM. Cells were washed in the same media lacking radiolabel but containing 5 mM LiCl for 1 h at
37 °C. The media was removed and cells were lysed with 0.75 ml of 20 mM formic acid for 30 min at 4 °C before 0.1 ml of 3%
ammonium hydroxide was added. Inositol was separated from IP by
sequential elution from 1-ml Dowex AG1-X8 (100-200 mesh) columns. The
inositol fraction was eluted with 0.18% ammonium hydroxide, whereas
IPs were eluted with 4 M ammonium formate, 0.2 M formic acid. The inositol and IP fractions were mixed
with Ultima Gold and Ultima Flo AF scintillation fluid (Packard),
respectively, and subjected to scintillation counting. To compare
experiments with differing levels of
myo-[3H]inositol incorporation, IP
production was determined as a fraction, IP/(IP + inositol), and
plotted relative to the control G
q-R183C-stimulated IP
production. Statistical significance was assessed using repeated measures analysis of variance with a Dunnett's post-test.
q-G188S Mutant--
IP accumulation experiments
shown in Fig. 7 were carried out as described above except that HEK293
cells were utilized. In addition, 250 ng of plasmids encoding
G
q-R183C or G
q-R183C/G188S were
transfected with increasing amounts of pcDNA3-GRK2,
pcDNA3-RGS2, and pB6-GAIP plasmids as indicated in the figure.
pcDNA3 vector was used as carrier DNA such that 1 µg of DNA was
transfected in each well of the 6-well plate. An unpaired t
test was used to assess statistical significance.
q using FuGENE 6 reagent (Roche Molecular Biochemicals). After 24 h, transfected
cells were replated onto glass coverslips and grown for an additional
24 h before fixing in 3.7% formaldehyde for 20 min. Cells were
washed with phosphate-buffered saline and then incubated in blocking
buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1%
Triton X-100, and 2.5% nonfat milk). Coverslips were then incubated in
blocking buffer containing a 1:100 dilution of anti-G
q
polyclonal antibody (Santa Cruz) for 1 h. Following washes with
blocking buffer, cells were incubated in a 1:100 dilution of Alexa
Fluor 594-conjugated goat anti-rabbit secondary antibody (Molecular
Probes) for 30 min. The coverslips were washed and mounted on glass
slides with Prolong Antifade reagent. Representative images were
recorded by confocal microscopy at the Kimmel Cancer Center Bioimaging
Facility using a Bio-Rad MRC-600 laser scanning confocal microscope
running CoMos 7.0a software and interfaced to a Zeiss Axiovert 100 microscope with Zeiss Plan-Apo 63× 1.40 NA oil immersion objective.
Dual-labeled samples were analyzed using simultaneous excitation at 488 and 568 nm. Images of "x-y" sections through
the middle of a cell were recorded.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
q/11--
For a growing list of
Gq-coupled receptors, it has been reported that
desensitization can occur in a GRK2-dependent but
phosphorylation-independent fashion. For example, hormone-mediated
PLC
activation via the metabotropic glutamate receptor (mGluR1a)
(47), the parathyroid hormone receptor (48), the thromboxane
A2 receptor (34), the endothelin receptor (49), and the
angiotensin II-1A receptor (50), is inhibited by overexpression of
kinase-deficient GRK2-K220R. The parathyroid receptor and mGluR1a
interact with full-length GRK2 (47, 48) and the RH domain of GRK2
co-immunoprecipitates with mGluR1a (51). For the mGluR1a (51), the
endothelin receptor (49), and the thromboxane A2 receptor
(34), overexpression of the RH domain of GRK2 inhibits
Gq-stimulated phosphoinositide hydrolysis. Thus,
phosphorylation-independent regulation of these receptors may be due
either to GRK2/receptor interaction or to GRK2·G
q
complex formation (or both). As a first step to determine the extent to
which G
q binding by GRK2 regulates
Gq-coupled receptor signaling, we used site-directed
mutagenesis to create GRK2 mutants that are defective in
G
q binding.
ligands. The
structures demonstrate that the RGS protein contacts all three switch
regions of the G
subunit and that the RH domain does not undergo a
large conformational change in tertiary structure upon binding the G
subunit. Crystal structures in combination with mutational analyses
have identified amino acids in the R4 family that contact the G
subunit (23, 24, 57, 58). For RGS4 and RGS9, G
contact sites are
primarily localized to the loops between
helices,
3 and
4,
5 and
6, and
7 and
8 (see Fig. 1, A and
B, for details). This has been designated the "A" site
(59). G
i residues important for RGS4 interaction are
also conserved in other G
subunits including G
q.
Furthermore, mutation of an RGS4 residue in the
3/
4 loop critical
to the RGS4/G
i1 interaction (E87K) prevents
RGS4/G
q interaction (60). Therefore, it is presumed that
the RGS4/G
q interface mimics the RGS4/G
i1 interaction.
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Fig. 1.
Protein binding surfaces of RH
domains. A, structural alignment of the RH domain of
GRKs with RGS, axin, and p115RhoGEF family members. An initial multiple
sequence alignment of the RH domains was generated by ClustalW (72).
Subsequently, RH domains of known structure were superimposed within
the program O (73) and were used to verify and adjust the sequence
alignment. Sequences with a known crystal or NMR structure are labeled
with an asterisk. Boundaries of helices within the
domains are depicted as blue boxes above the alignment. In
p115RhoGEF and presumably LARG, the lengths of helices 7 and 8 differ
from other RGS proteins and their locations are indicated by the
orange helices below the alignment. Residues that were
mutated (usually to alanine, see text) in GRK2 but do not alter binding
to G
q-GDP·AlF
q-GDP·AlF
q-GDP are colored white on
top of a purple background (this work, see text). Residues of RGS4
and RGS9 that contact the switch regions of G
i1 and the
G
i/t chimera, respectively, are yellow (23,
24), whereas contacts of RGS9 with the
helical domain of the
G
i/t chimera are teal (24). Axin contacts
with the APC peptide are green (52). Residues in RGS2 that,
when converted to their equivalents in RGS4, enhance GAP activity
toward G
i (74) are indigo. Mutations in
p115RhoGEF that decrease G
13 GAP activity are hot
pink (53). Dashed boxes indicate regions that are
structurally heterogeneous among the known structures. All sequences
are human unless otherwise indicated. Accession numbers for the
sequences used in the alignment are Q15835 (GRK1), P21146 (GRK2),
P26818 (GRK3), P32298 (GRK4), P34947 (GRK5), P43250 (GRK6), NP_631948
(GRK7), P41220 (RGS2), P49799 (RGS4), O46469 (RGS9), NP_005864
(RGS19/GAIP), AAC51624 (axin), BAA20834 (PDZRhoGEF), NP_004697
(P115RhoGEF), and NP_056128 (LARG). B, structure of the RH
domain from RGS4. Residues that contact G
i are drawn
with yellow carbons atoms. The nine
-helices of the
canonical RH domain are labeled
1-
9 (23). Nitrogen atoms are
colored blue and oxygen atoms red. C,
structure of axin bound to the APC peptide (52). Residues that contact
the APC peptide are drawn with green carbon atoms. The APC
peptide is drawn as a black coil. D, homology
model of the RH domain of GRK2. The model was built using the
structures of axin and GAIP as a guide (see "Experimental
Procedures"). The gold and purple color scheme
described above applies to parts B-D of this figure.
i1 (GRK2 residues Asp160 and
Lys164 corresponding to RGS4 residues Asp163
and Arg167, respectively) (Fig. 1A). A construct
that encodes a GST fusion protein bearing the RH domain of GRK2 (amino
acids 45-178) was used as a template for mutagenesis. Purified WT and
mutant GST fusion proteins on glutathione-agarose beads were incubated
with bovine brain lysates in the presence of GDP or
GDP·AlF
q/11 was assessed by immunoblotting with
G
q/11-specific antibody. WT, D160K, and K164A bound
similarly to G
q/11 in an
AlF
3 and
4 and between
7 and
8 (H75A/L76A, E77A, E78K,
K80A, V83A, and N156A) was carried out without identifying any residues
important for G
q/11 binding. Furthermore, double mutants
such as E78K/D160K, V83A/D160K, and D160K/K164A retained the ability to
bind G
q/11 in the presence of
AlF
i and G
t,
respectively, residues in the
3/
4 and
7/
8 loops did not appear to be critical for G
q/11 binding by GRK2.
helix from APC,
utilizes residues within the
3,
4, and
5 helices (52). This
region, which has been designated the "B" site (59), was targeted
in the next round of mutagenesis. Whereas mutations in
4 (E84A,
E87A, and K90A) and a double mutant in
5 (V103A/C104A) had no effect
on binding,
5 substitutions R106A and D110A resulted in diminished
binding to G
q/11 (Fig.
2).
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Fig. 2.
Identification of eight GRK2 RH domain
mutants with altered binding to G q/11. Upper
panel, glutathione-agarose beads bearing GST fusion proteins,
either WT (GST-GRK2-(45-178)) or GST-GRK2-(45-178) substituted at one
of eight single amino acid positions, were incubated with bovine brain
extract in the presence (+) or absence (
) of aluminum fluoride
(AlF
q/11 was
visualized by immunoblotting. Lower panel, GST fusion
proteins used in the GST pull-down assay above were separated by
SDS-PAGE and visualized by Coomassie staining.
q/11 in the presence of
GDP·AlF
q/11. Interestingly, K115A showed no reproducible decrease in
GDP·AlF
q/11. Mutations with the greatest effect map to the
5 helix and the beginning of the
5/
6 loop (and to the
6/
7 loop to a much lesser extent) and form a continuous surface
(assuming that the axin-based model is a good representation of the
GRK2 RH domain).
q Binding Defects Are Not Impaired
in in Vitro Receptor Phosphorylation--
All amino acids selected for
mutagenesis were predicted by our homology model to be exposed at the
surface of the RH domain. However, it is possible that some of these
residues are in fact buried and substitution to alanine might cause
alteration in the tertiary structure. To test whether single amino acid
mutations in the RH domain altered another function of the
NH2-terminal domain, we looked at the ability of
G
q-binding mutants to phosphorylate activated rhodopsin.
The NH2-terminal domain of GRKs is thought to be involved
in the activation of the kinase domain by agonist-stimulated receptors.
An antibody directed at residues 17-34 of GRK1 blocks rhodopsin, but
not peptide, phosphorylation (61). Likewise, E7A-GRK1 and E5A-GRK2
mutants are defective in rhodopsin, but not peptide, phosphorylation
(62). To test the integrity of the NH2-terminal domain of
G
q-binding mutants, the R106A, D110A, M114A, K115A, and
V137A mutations were introduced into an expression vector encoding
full-length GRK2 (pcDNA3-GRK2). Lysates from COS-1 cells
transfected with WT or mutant GRK2 were assayed in a rhodopsin phosphorylation assay. We did not observe any statistically significant defects in the ability of G
q-binding mutants of GRK2 to
phosphorylate light-activated rhodopsin (Fig.
3).
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Fig. 3.
G q/11 binding-defective
mutants phosphorylate rhodopsin. Upper panel, lysates were
prepared from COS-1 cells transfected with full-length WT or mutant
GRK2 constructs and equal volumes were used in a kinase assay with
light-activated rhodopsin as a substrate. An autoradiograph
representative of three separate experiments is shown. Middle
panel, levels of WT or mutant GRK2 present in each lysate were
compared by immunoblotting of equal volumes of lysate. Lower
panel, the kinase activity in WT and mutant GRK2 lysates was
quantified and mean ± S.E. for the three separate experiments are
displayed.
q Binding Defects Are Not
Recruited to Plasma Membrane by Activated G
q--
A
constitutively active mutant of G
q,
G
q-Q209L, is able to promote plasma membrane recruitment
of a GFP fusion protein containing the RH domain of GRK2,
GRK2-(45-178)-GFP.2 The
ability to induce plasma membrane localization of GRK2-(45-178)-GFP is
specific to G
q because other activated G
subunits
fail to recruit GRK2-(45-178)-GFP to plasma membranes.2 We
thus used this assay to examine interactions between
G
q-Q209L and a G
q binding-defective
mutant of GRK2 in cultured cells (Fig. 4). When expressed in HEK293 cells, the
GFP-tagged RH domain of GRK2 is localized in the nucleus and throughout
the cytoplasm (Fig. 4A). Likewise, GRK2-(45-178)-GFP
bearing the D110A mutation is diffusely localized throughout the
nucleus and cytoplasm when expressed alone (Fig. 4D).
Co-expression of G
q-Q209L and GRK2-(45-178)-GFP results
in their co-localization at cellular plasma membranes (Fig. 4,
B and C). However, D110A-GRK2-(45-178)-GFP
remains in the nucleus and cytoplasm when co-expressed with
G
q-Q209L (Fig. 4, E and F). This
defect in G
q-Q209L-induced plasma membrane recruitment
of D110A-GRK2-(45-178)-GFP parallels the failure of this mutant to
bind G
q/11 in the in vitro binding assay.
When R106A-GRK2-(45-178)-GFP was co-expressed with
G
q-Q209L in similar experiments, this mutant
demonstrated consistent but weak plasma membrane localization (data not
shown).
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Fig. 4.
G q-Q209L induces plasma
membrane recruitment of GRK-(45-178)-GFP but not
D110A-GRK2-(45-178)-GFP. HEK293 cells were transfected with 0.02 µg of WT GRK2-(45-178)-GFP (A-C),
D110A-GRK2-(45-178)-GFP (D-F), along with 1 µg of pcDNA3 (A and D) or 1 µg of
pcDNA3 containing EE epitope-tagged G
q-Q209L
(B, C, E, and F). Subcellular
localization was determined by confocal microscopy. GRK2-(45-178)-GFP
(A and B) and D110A-GRK2-(45-178)-GFP
(D and E) were visualized by GFP fluorescence,
whereas G
q- Q209L (C and
F) was visualized using an anti-
q polyclonal
antibody followed by an Alexa 594-conjugated anti-rabbit antibody.
Representative micrographs are shown. More than 100 cells were examined
in at least five separate experiments. Bar, 10 µm.
q Binding Mutants of GRK2 Are Defective in the
Regulation of G
q-R183C-stimulated PLC
Activity in
Vivo--
To determine whether the binding defects are manifested in
full-length GRK2, we tested the ability of GRK2 mutants to regulate G
q-R183C activation of PLC
in vivo. COS-1
cells were transfected with G
q-R183C alone, or
co-transfected with G
q-R183C and WT or mutant GRK2, and
inositol phosphate production was determined. WT GRK2 reduced
G
q-R183C-stimulated inositol phosphate production by
~48% (p < 0.01, Fig.
5). Likewise, the K115A mutant, which binds both the GDP- and GDP·AlF
q, was equally effective (53%
reduction) as WT GRK2 at preventing PLC
activation
(p < 0.01). In contrast, the R106A, D110A, and M114A
derivatives had no statistically significant effect on
G
q-R183C-stimulated PLC activity. The V137A mutant, which exhibited only a modest binding defect in the in vitro
binding assay, only inhibited PLC
activation by 27%
(p < 0.05). In general, the ability of full-length WT
or mutant GRK2 to inhibit G
q-R183C-stimulated inositol
phosphate production in vivo reflects the propensity of the
analogous GST-GRK2-(45-178) fusion protein to bind brain G
q/11 in vitro.
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Fig. 5.
Inhibition of
G q-R183C-stimulated inositol phosphate accumulation by
WT but not mutant GRK2. COS-1 cells, transfected with
G
q-R183C alone or in combination with full-length GRK2
or mutant derivative, were labeled with
myo-[3H]inositol for 18 h. Inositol was
separated from inositol phosphates as described under "Experimental
Procedures" and quantified by scintillation counting. Shown are
mean ± S.E. for four to 10 experiments performed in triplicate.
Differences between G
q-R183C alone and
G
q-R183C + GRK2-WT, GRK2-K115A, or GRK2-V137A were
statistically significant (p < 0.05). Differences
between G
q-R183C alone and G
q-R183C + GRK2-R106A, GRK2-D110A or GRK2-M114A were not statistically
significant.
q-G188S
Mutant--
Because the RH domain of GRK2 preferentially binds to the
transition state of G
q/11, at least one of the three
switch regions is expected to be involved in their interaction.
However, because G
q binds a surface of the GRK2 RH
domain that is distinct from RGS4/G
interface, the G
q
contribution to the GRK2/G
q interface is likely to be
distinct as well. To test this hypothesis, we evaluated the ability of
the RGS-resistant mutant, G
q-G188S, to interact with
GRK2. An RGS-resistant G
mutant was first described for the G
subunit of the Saccharomyces cerevisiae trimeric G protein,
Gpa1p (63). gpa1sst mutants are supersensitive to the
mating pheromone,
-factor, because of a failure to bind the RGS
protein, Sst2p. The binding defect is because of a Gly
Ser
substitution in switch I of the G
subunit. Gly
Ser substitutions
in mammalian G
subunits G
i, G
o, and
G
q also result in resistance to RGS proteins of the R4
and R7 subfamilies (63, 64).
q to recruit GRK2-GFP to the plasma membrane. HEK293
cells were transiently transfected with G
q,
G
q-R183C, or G
q-R183C/G188S and either
GRK2-(45-178)-GFP or RGS2-GFP. A previous report demonstrated that
co-expression of an activated mutant of G
q promoted the plasma membrane localization of RGS2-GFP, whereas expression of RGS2-GFP alone resulted in nuclear and cytoplasmic localization, with
very weak plasma membrane localization (40). When co-transfected with
G
q, GRK2-GFP was found throughout the cell, and RGS2-GFP displayed prominent nuclear staining with some cytoplasmic and faint
plasma membrane localization (Fig. 6). In
contrast, when transfected with the R183C GTPase mutant of
G
q, GFP-GRK2 was partially recruited to the plasma
membrane (Fig. 6), as also observed with expression of
G
q-Q209L (Fig. 4B), and GFP-RGS2 was strongly recruited to the plasma membrane (Fig. 6). However, GFP-RGS2 and GFP-GRK2 differed when assayed for plasma membrane recruitment upon
co-expression with the RGS-resistant mutant of G
q. When co-expressed with G
q-R183C/G188S, GRK2-GFP was recruited
to the plasma membrane to the same extent as when co-expressed with
G
q-R183C. In contrast, RGS2-GFP was only faintly
detected at the plasma membrane, and instead, was localized to the
nucleus and cytoplasm (Fig. 6). Thus, the G188S mutation of
G
q prevents RGS2 but not GRK2 binding.
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Fig. 6.
G q-R183C/G188S recruits GRK2
RH domain, but not RGS2, to the plasma membrane. HEK293 cells were
co-transfected with 0.25 µg of pcDNA3 containing
hemagglutinin-tagged versions of G
q,
G
q-R183C, or G
q-R183C/G188S, and either
0.025 µg of GRK2 (45-178)-GFP (left panel) or 0.25 µg
of RGS2-GFP (right panel) as indicated. Either 0.725 or 0.5 µg of pcDNA3 was included to adjust total transfected DNA to 1 µg. Cells on coverslips were fixed in formaldehyde and mounted on
glass slides as described under "Experimental Procedures." The
localization of GFP-tagged proteins was visualized by confocal
microscopy. Only cells expressing plasma membrane localized
G
q, G
q-R183C, or
G
q-R183C/G188S (not shown), as determined by using an
anti-
q polyclonal antibody followed by an Alexa
594-conjugated anti-rabbit antibody, were chosen to identify
subcellular localization of the GFP-tagged proteins. Representative
micrographs are shown. More than 100 cells were examined in at least
five experiments. Bar, 10 µm.
q-R183C or G
q-R183C/G188S and increasing
amounts of RGS2, GAIP, or GRK2 (full-length) cDNAs, and inositol
phosphate production was measured. GRK2, RGS2, and GAIP each inhibited
the G
q-stimulated PLC
activity in a
dose-dependent fashion with the inhibition being 89, 71, and 54%, respectively, at the highest level of DNA transfected (Fig.
7). Immunoblotting suggested that
G
q expression was similar in all experiments and that
increasing the level of GRK2, RGS2, and GAIP cDNA increased the
level of expression (data not shown). Whereas GAIP inhibited
G
q-R183C-stimulated PLC
activity by 54%, it was much
less effective at inhibiting G
q-R183C/G188S-stimulated IP production (19% decrease, p < 0.05). Likewise,
RGS2 showed diminished ability to regulate IP production stimulated by
the RGS-resistant mutant (31% decrease) relative to its ability to decrease the G
q-R183C-stimulated PLC
activity (71%,
p < 0.05). In stark contrast, GRK2 inhibited PLC
activity stimulated by both G
q-R183C (89% decrease) and
G
q-R183C/G188S (95% decrease) to a similar extent.
Thus, unlike RGS proteins RGS2 and GAIP, GRK2 can tolerate substitution
of Ser for the conserved Gly in the switch I region.
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Fig. 7.
G q-R183C/G188S-stimulated
inositol phosphate production is sensitive to GRK2 but resistant to
RGS2 and GAIP. HEK293 cells were transfected with
G
q-R183C (A) or G
q-R183C/G188S
(B) alone or with increasing amounts of full-length GRK2
(
), GAIP (
), or RGS2 (
). Cells were labeled with
myo-[3H]inositol and inositol was separated
from inositol phosphates as described under "Experimental
Procedures" and quantified by scintillation counting. The experiment
shown is representative of three independent experiments and displayed
as the average of triplicates ± S.D.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
q/11. The binding
defects attributed to these mutations are not likely to be due to
misfolding of the RH domain because we were unable to detect any
differences between the ability of WT and GRK2 mutants to carry out
receptor phosphorylation, a function that requires an intact
NH2-terminal domain. With the exception of K115A, all of
the mutants exhibit diminished binding to
GDP·AlF
q/11.
Mutations with the most dramatic effects map to the COOH-terminal half
of the
5 helix and the NH2-terminal region of the
5/
6 loop (Fig. 1, A and D). The GRK2
binding surface is distinct from both the binding interface used by
RGS4 and RGS9 to bind G
subunits (
3/
4,
5/
6, and
7/
8 loops) and the surface used by axin to bind APC helical
peptide (
helices 3, 4, and 5). Comparison of the G
binding site
on GRK2 with the G
contact sites on RGS4/RGS9 suggests that these
sites do not overlap (Fig. 1, B-D). Although RGS4 and RGS9
also utilize the
5/
6 loop, the GRK2 contact sites are in the
amino terminus of that loop whereas the RGS4/RGS9 contact sites are
concentrated toward the carboxyl terminus. Therefore, our experiments
identify a novel G
binding site on the RH domain. Consistent with
the nomenclature of Zhong and Neubig (59) we propose that this surface
be termed the "C" site.
q binding-defective mutants not only fail to bind
G
q/11 in an in vitro pull-down assay, but
they are also defective in cell-based assays. First, unlike the WT GRK2
RH domain fusion protein GRK2-(45-178)-GFP that co-localizes with
activated G
q at the plasma membrane, the
G
q binding-defective mutant D110A-GRK2-(45-178)-GFP fails to be recruited to the plasma membrane and instead remains in the
nucleus and cytoplasm. Likewise, whereas full-length WT GRK2 inhibits
G
q-R183C-stimulated PLC
activity by ~50%,
G
q binding-defective mutants GRK2-R106A, GRK2-D110A, and
GRK2-M114A have no affect on PLC
activity. Thus, the failure to bind
to G
q in vitro has the expected in
vivo ramifications: these GRK2 mutants fail to bind to activated
G
q at the plasma membrane and fail to regulate the
activity of this G
subunit.
q (Fig. 1) (34). Whereas undiminished in its capacity to bind
G
q/11-GDP·AlF
q/11-GDP.
One hypothesis is that binding by the K115A mutant traps
G
q/11-GDP in a
G
q/11-GDP·AlF
q/11. In this scenario, the Lys
Ala substitution
would relieve that inhibition. An alternative hypothesis is that the
K115A mutation allows recognition of some feature of the GDP-bound
state that is not accessible to the WT protein.
subunits in the RGS4/G
i1 and RGS9/G
t
interfaces (23, 24). Because the RH domain interface in the
GRK2/G
q interaction is novel, we propose that some
aspect of the G
q interaction surface is distinct from
the surfaces utilized by G
i1 and G
t in
their RGS4 and RGS9 interactions. In support of this hypothesis, we
found that the RGS-resistant G188S mutant of G
q is not
refractory to the GRK2 RH domain. The G
q-R183C/G188S
mutant recruits GFP-GRK2-(45-178) to the plasma membrane and
full-length GRK2 effectively blocks G
q-R183C/G188S-stimulated PLC
activity. The analogous
residue in G
i1, Gly183, sits in close
proximity to RGS4 Glu83 in the RGS4/G
i1
co-crystal and is surrounded by other switch I residues that make
hydrogen bonds and ionic contacts with RGS4. Substitution of the
Gly183 with almost any residue would be predicted to alter
the tertiary structure and decrease complementarity to the RGS4
interface. Because G
q-R183C/G188S does not inhibit GRK2
interaction, we speculate either that Gly188 is not located
in the GRK2 interface or that minor changes in the G
q
and/or GRK2 tertiary structure can accommodate the G
q Gly
Ser substitution within the interface.
q binding
defects, six are strictly conserved in GRK3 but not in other members of
the GRK family (Fig. 1). This clearly explains why GRK2 and GRK3, but
not GRK1, GRK4, GRK5, nor GRK6, bind G
q. Surprisingly, four residues that make important contributions to G
q
binding by GRK2 are conserved in other RGS proteins. For example, the Arg106 position is a basic residue in RGS2, RGS3, RGS4,
RGS5, RGS8, RGS13, RGS16, RGS17, RGS18, RGS19, and RGS20.
Phe109, a residue that is exposed to the solvent in RGS4,
is Phe or Tyr in most RGS proteins. Asp110 is an asparagine
or glutamate in RGS4, RGS5, RGS8, RGS16, RGS17, RGS18, GAIP, and RGS20.
Finally, Glu116 is glutamate, aspartate, or glutamine in
RGS2, RGS3, RGS4, RGS5, RGS8, RGS16, and RGS18. Thus, four of the
presumed GRK2 contact residues are each conserved in RGS2, RGS4, RGS5,
RGS8, RGS16, and RGS18. RGS proteins are modular and the family members
are sometimes categorized based on presence or absence of protein interaction domains in the full-length polypeptide (20). RGS2, RGS4,
RGS5, RGS8, RGS16, and RGS18 are all members of the "small" RGS
subfamily and contain only short sequences outside the RGS domain.
Because RGS2, RGS4, and RGS16 can bind G
q and because the GRK2/G
q interface is contiguous with the
RGS4/G
i1 and RGS9/G
t interface, perhaps
residues in the
5 helix and NH2-terminal portion of the
5/
6 loop may play a role in binding of G
q by other
RGS family members.
5 helix may be conserved because they bind other
regulatory ligands. Indeed, RGS4 binds phosphatidylinositol 3,4,5-trisphosphate (PIP3) but a double mutant K112E/K113E
lacks this capability (65). In our alignment (Fig. 1A), RGS4
Lys112 corresponds to GRK2 Arg106; therefore, a
G
q contact site in GRK2 corresponds to a
PIP3 contact site in RGS4. PIP3 inhibits the
GAP activity toward G
i1 of RGS1, RGS10, and GAIP, but
not that of RGS16. Furthermore, many RGS proteins have putative
calmodulin (CaM) binding sites that map to a region spanning the COOH
terminus of
4 helix and the NH2-terminal half of the
5 helix. CaM binds to RGS1, RGS2, RGS4, RGS10, RGS16, and GAIP in a
calcium-dependent fashion, but does not alter the GAP
activity of RGS4 (65). However, CaM does reverse the inhibitory effect
of PIP3 on GAP activity. Lys112 and
Lys113 of RGS4 represent the COOH terminus of the consensus
CaM binding site, yet the K112E/K113E double mutant does not alter CaM
binding to RGS4. Because of the proximity of the putative
PIP3- and CaM-binding sites to the C site of the
GRK2 RH domain, it would be interesting to test whether
PIP3 (or other phosphatidylinositides) or CaM affect
binding to G
q by RGS family proteins. Incidentally, GRK2 binds to phosphatidylinositides and phosphatidylserine (66-70) and to
CaM with low affinity (71), but none of the these ligand-binding sites
are located within its RH domain.
12 and G
13, can also bind
G
q, a characteristic that distinguishes it from its
close relatives, p115RhoGEF and PDZRhoGEF (32). The RH domain of RhoGEF
family members shares only a small number of residues implicated in
G
interaction by RGS4 and RGS9. Likewise the
5,
5/
6 loop
region of LARG does not bear similarity to the G
q
binding region of GRK2 (see alignment in Fig. 1A). Thus, the
G
q interaction site on this RH domain-containing protein appears distinct from the C site.
q/11 binding to the RH domain occurs at a novel G
binding site that we have called a C site. This, in combination with
the inability of the switch I mutant G
q-G188S to affect
GRK2 RH domain association, suggests that GRK2 binding occurs on a
distinct surface of G
q/11 as well. Interestingly,
several mutations in the
5 helix that inhibit G
q/11
binding do not impair rhodopsin phosphorylation, suggesting that the
region of the GRK2 NH2-terminal domain necessary for
receptor phosphorylation is distinct from the C site of the RH domain.
Further studies are necessary to map residues of the GRK2
NH2 terminus that are necessary for receptor
phosphorylation and to define features of G
q that are
required for GRK2 interaction.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Dave Manning and Henry Bourne
for providing Gq/11-specific and EE antibodies,
respectively, and Drs. Scott Heximer, Ray Penn, and Chris Carman for
providing RGS2-GFP, GRK2-GFP, and GST-GRK2(RH)-K164A constructs.
R. S. M. thanks Dr. Richard Neubig for helpful suggestions,
Dr. Ray Penn for statistical advice, and Siena undergraduates Erin
Twiss, Carlos Gonzalez, Billy Robinson, Jay Kubik, and Mike Ragusa
for their contributions to this work. R. S. M. also thanks
Dr. Ken Helm for sharing equipment and reagents, Betsey Harvey for
support, and Drs. Jim Angstadt, Tom Coohill, and Doug Fraser for
encouragement. Finally, we thank the reviewer for helpful comments.
![]() |
FOOTNOTES |
---|
* This work was supported by National Science Foundation Grant MCB9728179 and an American Heart Association Southeastern Pennsylvania Affiliate Beginning grant-in-aid (to R. S. M.), American Heart Association Texas Affiliate Beginning Grant-in-aid 0060118Y and a Welch Foundation Chemical Research Grant F-1487 (to J. J. G. T.), a fellowship from the American Heart Association Pennsylvania-Delaware Affiliate (to P. W. D.), National Institutes of Health Grants GM44944 and GM47417 (to J. L. B.) and GM56444 and GM628884, and a grant from the Pew Scholars Program in the Biomedical Sciences (to P. B. W.).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: Siena College, Biology Dept., 123 Morrell Science Center, 515 Loudon Rd., Loudonville, NY 12211. Tel.: 518-783-2462; Fax: 518-783-2986; E-mail: sternemarr@siena.edu.
Published, JBC Papers in Press, November 8, 2002, DOI 10.1074/jbc.M208787200
2 P. W. Day and P. B. Wedegaertner, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
GPCR, G
protein-coupled receptor;
PLC, phospholipase C
;
GRK, GPCR kinase;
RGS, regulator of G protein signaling;
GAP, GTPase activating protein;
RH, RGS homology;
AlF
S, guanosine
5'-3-O-(thio)triphosphate.
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REFERENCES |
---|
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