(Circulation. 2000;102:793.)
© 2000 American Heart Association, Inc.
Basic Science Reports |
Overexpression of G Protein–Coupled Receptor Kinase-2 in Smooth Muscle Cells Attenuates Mitogenic Signaling via G Protein–Coupled and Platelet-Derived Growth Factor Receptors
From the Department of Medicine (Cardiology), Duke University Medical Center, Durham, NC, and the Department of Immunology, Universitätskliniken, Göttingen, Germany (M.O.).
Correspondence to Neil J. Freedman, MD, Box 3187, Duke University Medical Center, Durham, NC 27710. E-mail neil.freedman{at}duke.edu
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Abstract |
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Background—Neointimal hyperplasia involves activation of smooth muscle cells (SMCs) by several G protein–coupled receptor (GPCR) agonists, including endothelin-1, angiotensin II, thrombin, and thromboxane A2. Signaling of many GPCRs is diminished by GPCR kinase-2 (GRK2). We therefore tested whether overexpression of GRK2 in SMCs could diminish mitogenic signaling elicited by agonists implicated in the pathogenesis of neointimal hyperplasia.
Methods and Results—Overexpression of GRK2 was achieved in primary rabbit aortic SMCs with a recombinant adenovirus. Control SMCs were infected with an empty vector adenovirus. Inositol phosphate responses to endothelin-1, angiotensin II, thrombin agonist peptide, and platelet-derived growth factor (PDGF) were attenuated by 37% to 72% in GRK2-overexpressing cells (P<0.01), but the response to the thromboxane A2 analogue U46619 was unaffected. GRK2 also inhibited SMC [3H]thymidine incorporation stimulated not only by these agonists (by 30% to 60%, P<0.01) but also by 10% FBS (by 35%, P<0.05). However, GRK2 overexpression had no effect on epidermal growth factor–induced [3H]thymidine incorporation. Agonist-induced tyrosine phosphorylation of the PDGF-ß receptor, but not the epidermal growth factor receptor, was reduced in GRK2-overexpressing SMCs. GRK2 overexpression also reduced SMC proliferation in response to endothelin-1, PDGF, and 10% FBS by 62%, 51%, and 29%, respectively (P<0.01), without any effect on SMC apoptosis.
Conclusions—GRK2 overexpression diminishes SMC mitogenic signaling and proliferation stimulated by PDGF or agonists for several GPCRs. Gene transfer of GRK2 may therefore be therapeutically useful for neointimal hyperplasia.
Key Words: muscle, smooth • cells • signal transduction • receptors
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Introduction |
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Vascular injury results in endothelial dysfunction, mechanical stress, and growth factor– or cytokine-mediated activation of medial smooth muscle cells (SMCs), which consequently proliferate and migrate into the subintimal space.1 This neointimal hyperplasia (NH) compromises venous bypass grafts2 and percutaneous coronary interventions, despite intravascular stent implantation.3 Growth factors implicated in SMC activation include several agonists for G protein–coupled receptors (GPCRs): thrombin,4 endothelin-1 (ET-1),5 angiotensin II (Ang II),6 and thromboxane A2 (TXA2).7 Like many GPCRs, receptors for these agonists desensitize rapidly,8 9 in a process that appears to involve GPCR kinases (GRKs), a family of enzymes that phosphorylate agonist-occupied heptahelical receptors.9 GRK2, when overexpressed in model transfected cells, diminishes signaling by the endothelin A and B receptors, the Ang II type 1 receptor, and the thrombin receptor.8
The contribution of GPCR activation to experimental NH has been demonstrated by studies that used antagonists for the type 1 Ang II6 or endothelin receptors.5 However, inhibition of single GPCR or receptor tyrosine kinase10 signaling systems has invariably resulted in only partial inhibition of experimental NH. This incomplete efficacy of individual receptor antagonists suggests the desirability of strategies capable of targeting multiple receptor signaling systems simultaneously.
One candidate molecular strategy for reducing SMC mitogenesis is the cellular overexpression of GRK2, because of its ability to suppress signaling through a number of receptors coupled to phosphoinositide hydrolysis via Gq in model transfected cell systems.8 To test the ability of GRK2 overexpression to suppress mitogenic signaling in vascular SMCs, we used primary SMCs infected with recombinant adenoviruses encoding GRK2 or control constructs. In this model system, we assessed SMC responsiveness to the Gq-coupled receptor agonists ET-1, thrombin, Ang II, and TXA2, and also—because of its apparent role in NH1 —platelet-derived growth factor (PDGF).
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Methods |
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Primary SMC Isolation and Culture
Thoracic aortas were harvested from euthanized male New Zealand White rabbits, and primary SMCs were obtained by the explant outgrowth technique11 in the presence of mycoplasma removal agent (50 µg/mL, ICN Pharmaceuticals, Inc). Immunofluorescence for SMC
-actin confirmed SMC
identity.11 SMCs were passaged in DMEM/10% FBS with
antibiotics,8 split 1:4, and discarded after passage
7.
Adenovirus Production and Infection
The bovine GRK2 cDNA12 was inserted into the
plasmid pSKAC, as described previously.13 The GRK2ct
adenovirus has been described.13
Subconfluent SMCs were infected in batches of 150-mm plates. A single plate was trypsinized and counted; subsequently, plates for infection were washed and exposed (37°C, 30 minutes) to 5 mL of infection medium (DMEM/2% FBS/25 mmol/L HEPES; pH 7.4) with or without virus (multiplicity of infection=100), with gentle agitation. Virus-containing medium was removed, and plates were incubated in fresh infection medium for 24 hours. Cells were then trypsinized and divided into aliquots in various dishes for assays, at either 2.6x104 cells/cm2 (phosphoinositide hydrolysis and [3H]thymidine {[3H]TdR} incorporation) or 1.3x104 cells/cm2 (proliferation, cell cycle, and immunoblotting assays).
Phosphoinositide Hydrolysis
SMCs were rendered quiescent by a 72-hour incubation in
low-mitogen medium (DMEM supplemented with 20 mmol/L HEPES [pH
7.4], fatty acid–free BSA [1 mg/mL], 0.2% FBS, insulin [1.7
µmol/L], transferrin [5.5 µg/mL], sodium selenite [6.7 ng/mL],
and antibiotics8 ). SMCs were labeled in this medium
containing 2 µCi/mL of [3H]inositol for 12 to
18 hours, and then assayed for agonist-stimulated
phosphoinositide (PI) hydrolysis, as described
previously.8
[3H]TdR Incorporation
SMCs were plated in 24-well plates, rendered quiescent (as
above), then challenged with agonist(s) in low-mitogen medium for a
total of 24 hours. After 20 hours, [3H]TdR was
added at 1 µCi/mL (final concentration) for the last 4 hours of the
agonist challenge. After 24 hours, cells were washed 3 times with cold
PBS and solubilized with 0.5 mL/well phenol : 4 mol/L guanidine
thiocyanate (1:1, vol/vol), which was subjected to liquid
scintillation spectrometry to determine [3H]TdR
incorporation (dpm/well) into SMCs. Under the conditions we used, SMCs
incorporated [3H]TdR into acid-precipitable
macromolecules, 
85% of which were digestible with DNAse I (data not
shown).
Immunofluorescence
SMCs on culture slides were fixed for 2 minutes (25°C) in
methanol : acetone (1:1), and then incubated with the indicated
primary IgG (
1 to 10 µg/mL) for 30 minutes (25°C) in PBS with
3% BSA. After 3 washes in PBS, slides were incubated as before, but
with Alexa 488–conjugated anti-mouse IgG (Molecular Probes). After
further washes, slides were imaged by fluorescence microscopy.
To identify GRK2 and SMC -actin simultaneously, SMCs
were incubated sequentially with C5/1 anti-GRK2 IgG,14
Alexa 488–conjugated anti-mouse IgG, and then cyanine 3–conjugated
1A4 anti–SMC -actin (Sigma), with washes between each incubation.
To identify cell nuclei, the blue-fluorescing DNA-binding dye Hoechst
33258 (Molecular Probes) was added to the buffer (5 µg/mL) during the
last antibody incubation.
Immunoprecipitation and Immunoblotting
The PDGF receptor (PDGFR) and the epidermal growth factor
receptor (EGFR) were immunoprecipitated from SMCs as we have described
previously,8 with rabbit IgG specific for cytoplasmic
domains of each receptor (sc-432 and sc-03, Santa Cruz Biotechnology,
Inc). Immunoprecipitates from equal masses of solubilized SMC protein
were electrophoresed on 4% to 20% polyacrylamide gradient
gels and transferred to nitrocellulose.
Immunoblotting was performed as described
previously,8 12 with C5/114 (anti-GRK2),
sc-432 (anti-PDGFR), sc-03 (anti-EGFR), or PY20 (anti-phosphotyrosine,
Transduction Laboratories). To visualize the GRK2 carboxyl terminal
domain polypeptide (GRK2ct, amino acids 495 to 689),13 we
used the monoclonal antibody E23/8 (IgG1/
), raised (by standard
protocols14 ) against a synthetic peptide comprising
residues 658 to 689 of bovine GRK2.
Proliferation Assay
SMC proliferation over 12 days was assessed in response to the
indicated agonists. SMCs were plated in 8-well chamber slides, rendered
quiescent as described above, and treated on day 1 with the indicated
stimulus diluted in low-mitogen medium. On day 6, SMCs were re-treated
as on day 1. On day 12, SMCs were washed, incubated with Hoechst 33258
(5 µg/mL) for 30 minutes (25°C), washed with PBS, and visualized
with a Chroma Blue GFP filter on a Leica fluorescence
microscope. From each slide chamber, 4 images (at x10 magnification)
were captured with Adobe Photoshop. Nuclei were counted
with the UTHSCSA ImageTool program (developed at the University of
Texas Health Science Center at San Antonio and available from the
Internet by anonymous FTP from maxrad6.uthscsa.edu). At least 200 cells
per well of each chamber slide were counted.
Cell Cycle Analysis
SMCs in low-mitogen medium were assayed flow-cytometrically for
DNA content on days 6 and 12 of the proliferation
assays.15 Analyses on days 6 and 12 were
equivalent, so only those from day 12 are presented. To ensure
that apoptotic or necrotic SMCs could be
identified,15 we harvested and pelleted debris or floating
SMCs in the medium and pooled this pellet with that from SMCs removed
from the plate by trypsinization. SMCs were stained with Hoechst 33258,
and 104 cells per cell line were
analyzed. The fraction of apoptotic cells increased 4-
to 6-fold with a 4-hour treatment with 1 µmol/L
staurosporine (Sigma) (data not shown).
Statistical Analysis
To facilitate pooling of results across several independent
experiments, results within each experiment were normalized to values
obtained from SMCs infected with the empty vector adenovirus. With
Prism software (GraphPad), data from uninfected, vector-infected, and
GRK2- or GRK2ct-virus–infected SMCs were analyzed by
repeated-measures (ie, paired within individual experiments) 1-way
ANOVA. Tukey’s multiple comparison test was used to compare the SMC
groups with one another. Data are presented in the text as
±SD, and probability values are 2-tailed.
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Results |
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Overexpression of GRK2 in SMCs
Cells migrating out from de-endothelialized aortic explants were identified as SMCs by immunofluorescent staining for SMC
-actin11 (Figure 1A
). The 90% prevalence of cells
staining for SMC -actin was assessed by comparison of
immunofluorescence for SMC -actin with
fluorescent nuclear staining of all cells (data not shown).
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To achieve overexpression of GRK2 in these primary SMCs, we infected
them with a recombinant adenovirus that effected GRK2 overexpression in
nearly 100% of the SMCs (compare Figure 1C
and 1D), with a
predominantly cytoplasmic distribution of the GRK2 (Figure 1B).
This GRK2 overexpression was 40-fold above endogenous
levels, as assessed by immunoblotting (Figure 2). GRK2 overexpression persisted for
12 days in SMCs maintained in low-mitogen medium.
Immunoblotting of solubilized SMC protein along with a
purified GRK2 standard demonstrated that these
GRK2-adenovirus–infected SMCs expressed 12 to 25 pmol GRK2/mg
total cell protein. Uninfected and empty vector adenovirus–infected
SMCs were indistinguishable with regard to GRK2 expression, and GRK2
overexpression did not alter SMC -actin expression (data not
shown).
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Agonist-Induced SMC PI Hydrolysis and [3H]TdR
Incorporation: Effects of GRK2 Overexpression
To assess SMC second-messenger signaling in response to agonists
implicated in NH, we studied PI hydrolysis mediated by phospholipase C
isoforms ß (stimulated by Gq-coupled,
heptahelical receptors) and 
(stimulated by receptor tyrosine
kinases, like the PDGFR) (Figure 3A).
ET-1, Ang II, thrombin agonist peptide, and PDGF-BB each promoted a
time-dependent cellular accumulation of inositol phosphates, ranging
from 1.5±0.3- to 9±4-fold over basal at 15 minutes.
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Agonist-promoted PI hydrolysis was reduced by 37% to 72% in SMCs
overexpressing GRK2 (Figure 3B). Interestingly, signaling
inhibition by GRK2 appeared to be receptor-specific, because PI
hydrolysis induced by the TXA2 analogue U46619
(3.8±0.3-fold over basal) was unaffected by GRK2 overexpression. Thus,
GRK2 overexpression attenuated second-messenger signaling promoted not
only by several Gq-coupled receptors but also,
surprisingly, by the PDGFRß16 of rabbit vascular
SMCs.
To determine whether the GRK2-mediated reduction of second-messenger
signaling in SMCs could lead to a corresponding reduction in
agonist-promoted DNA synthesis, we assayed SMC
[3H]TdR incorporation evoked by the agonists
used in the preceding experiments (Figure 4). Compared with control cells,
GRK2-overexpressing SMCs incorporated 35% less
[3H]TdR in response to either PDGF or 10% FBS
and 60% less [3H]TdR in response to either
ET-1 or the TXA2 analogue U46619.
Synergistically, the combination of PDGF and ET-1 promoted SMC
[3H]TdR incorporation to an extent 2- to
6-fold greater than any of the foregoing stimuli. Nonetheless, GRK2
overexpression reduced this effect by 30% (P<0.02),
whereas it had no effect on the lesser degree of
[3H]TdR incorporation induced by EGF. Thus, as
we observed with PI hydrolysis, overexpression of GRK2 in SMCs
attenuated [3H]TdR incorporation promoted by
PDGF and various individual Gq-coupled receptor
agonists in a receptor-specific manner. In addition, overexpression of
GRK2 in SMCs reduced [3H]TdR incorporation
promoted by agonist combinations, including 10% FBS, which may more
accurately model stimuli encountered by SMCs in injured arteries or
vein grafts.
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Effects of GRK2 on PDGFR Signaling
Although GRK2 can phosphorylate a wide array of
heptahelical GPCRs, it has yet to be implicated in the regulation of
any receptor tyrosine kinase.9 We therefore found it
surprising that overexpression of GRK2 attenuated both PI hydrolysis
(Figure 3) and [3H]TdR incorporation
(Figure 4) stimulated by the PDGFR(s) in primary SMCs. One
potential explanation for these phenomena could lie in the ability of
the GRK2 carboxyl-terminal domain to bind
phosphatidylinositol-4,5-bisphosphate
(PIP2),9 the preferred substrate for
2 important PDGFR-stimulated effector enzymes: phospholipase C- and
phosphatidylinositol 3'-kinase.17 Sequestration of
PIP2 by overexpressed GRK2 might therefore
inhibit PDGF-stimulated PI hydrolysis as well as downstream events,
such as DNA synthesis.17 Could attenuation of
PDGF-promoted signaling by overexpressed GRK2 involve more than
competition for binding of PIP2? To address this
question, we compared PDGF-promoted signaling in SMCs overexpressing
either GRK2 or a polypeptide encompassing just the carboxyl-terminal
195 amino acids of GRK2 (GRK2ct) (Figure 5). The GRK2ct contains the pleckstrin
homology (PIP2-binding) and G protein
ß–binding domains of GRK2 but lacks the central catalytic and
amino-terminal targeting domains of GRK2.9 Because we
performed these experiments with equivalent levels of GRK2 and GRK2ct
expression (Figure 5A), differences between the effects of GRK2
and the GRK2ct could be ascribed to differences in protein activity,
rather than to differences in intracellular protein concentration.
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, P<0.05 vs control SMCs.
#P<0.05 vs GRK2ct-expressing SMCs.
PDGF-stimulated PI hydrolysis was inhibited by 38% in SMCs
overexpressing GRK2 (Figure 5B). This degree of inhibition was
more than twice that (P<0.05) effected by equivalent
expression of the GRK2ct. Despite these discordant findings in PI
hydrolysis, GRK2 overexpression inhibited PDGF-induced
[3H]TdR incorporation only insignificantly more
than GRK2ct. However, the more robust [3H]TdR
incorporation promoted by the combination of PDGF and ET-1 (Figure 4) was reduced (by 32±3%, P<0.01) only in SMCs
overexpressing GRK2, but not in those overexpressing GRK2ct (data not
shown).
These observations suggest that overexpressed GRK2 may reduce
PDGF-promoted signaling, in part, by binding to the ligands for its
pleckstrin
homology/Gß-binding
carboxyl terminal domain, as the GRK2ct does. However, the larger part
of GRK2-mediated inhibition of PDGF-promoted signaling appears to
operate through either the central catalytic domain, the amino-terminal
targeting domain, or perhaps both of these domains.
If GRK2 interacted via its N-terminal or catalytic domain with the
PDGFR, GRK2 might interfere with agonist-promoted tyrosine
phosphorylation of the PDGFR. To test this possibility,
we assessed the phosphotyrosine content of PDGFRßs immunoprecipitated
from PDGF-challenged SMCs, which either did or did not overexpress GRK2
(Figure 6). GRK2 overexpression reduced
agonist-induced tyrosine phosphorylation of the
PDGFRß by 37±20% (P<0.01). However, GRK2 overexpression
had no effect on agonist-promoted tyrosine
phosphorylation of the SMC EGFR (Figure 6).
Thus, the ability of GRK2 to attenuate agonist-promoted receptor
tyrosine kinase activation appears to be receptor-specific. Moreover,
the specificity of GRK2-mediated receptor tyrosine kinase inhibition is
congruent at the level of receptor activation (Figure 6) and
receptor-stimulated [3H]TdR incorporation
(Figure 4).
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GRK2 Overexpression Reduces SMC Proliferation
Agonist-stimulated [3H]TdR
incorporation may not indicate either an increase in DNA
synthesis18 or ongoing cellular
proliferation.19 We therefore sought to determine whether
the GRK2 overexpression that inhibited agonist-induced
[3H]TdR incorporation would also inhibit SMC
proliferation. Accordingly, aliquots of the same SMCs as those
subjected to assays for [3H]TdR incorporation
were subjected to proliferation studies, presented in Figure 7. In response to either ET-1, PDGF-BB,
or a combination of these agonists, the number of control SMCs
increased 1.4±0.3-, 1.8±0.4-, or 2.0±0.8-fold in 12 days.
The number of SMCs increased 2.7±0.7-fold over 4 days in
response to 10% FBS. Compared with control cells, the proliferation of
GRK2-overexpressing cells was reduced by 50% to 60%
(P<0.01) in response to either ET-1, PDGF-BB, or the 2
agonists together (Figure 6). Likewise, proliferation of
GRK2-overexpressing cells was reduced by 29% (P<0.01) in
response to 10% FBS (Figure 7). This reduction in SMC
proliferation by GRK2 overexpression was achieved without any evidence
of GRK2 adenovirus-induced apoptosis in SMCs maintained in
low-mitogen medium (Table). Thus,
GRK2 overexpression in SMCs attenuates not only agonist-stimulated
[3H]TdR incorporation but also
agonist-stimulated SMC proliferation in vitro.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In primary cultures of vascular SMCs, we have shown that overexpression of GRK2 diminishes signaling elicited via several receptors believed to be important in the pathogenesis of NH, including the PDGFR. GRK2 overexpression suppressed SMC signaling assessed either at the level of the second messenger or considerably downstream from the second messenger, at the level of SMC [3H]TdR incorporation. Moreover, GRK2 overexpression attenuated SMC proliferation without potentiating SMC apoptosis. The possibility that GRK2 overexpression may attenuate NH is suggested by the ability of GRK2 to diminish SMC [3H]TdR incorporation and proliferation promoted by the complex mixture of mitogens in 10% FBS.
GRK2 specifically binds and phosphorylates agonist-bound,
or activated, heptahelical GPCRs.9 Expressed at
physiological levels, GRK2 appears to initiate
desensitization of GPCRs in a manner dependent on GRK2-mediated
phosphorylation of the heptahelical receptor. When
expressed at 40-fold above physiological levels,
however, GRK2-mediated inhibition of receptor signaling involves
primarily agonist-promoted association of the receptor with the GRK (a
process that is both receptor- and GRK-specific).8
Overexpression of just the amino-terminal domain of GRK2 can attenuate
Gq-coupled receptor signaling.8 This
phenomenon may result from binding to either the heptahelical receptor
or the G protein itself, because the GRK2 amino-terminal domain
possesses homology with regulator of G-protein signaling (RGS)
proteins.20
Surprisingly, GRK2 overexpression inhibited signaling not only
via SMC GPCRs but also via the SMC PDGFRß.16 By what
mechanisms could overexpressed GRK2 suppress PDGFR-mediated signaling
in SMCs? PIP2 sequestration has been discussed
above. In addition, overexpressed GRK2 may interfere with PDGFR
signaling by sequestering
Gß subunits. The
heterotrimeric protein Gi has been implicated in
PDGFR-mediated stimulation of p42/p44 mitogen-activated protein
kinases in airway SMCs.21 Gi can
also mediate mitogen-activated protein kinase activation
elicited through heptahelical receptors, and this process is inhibited
by GRK2ct, presumably by sequestration of
Gß
subunits.22 Thus, to the extent that PDGFRs signal via a
Gß-related mechanism
in our vascular SMCs, GRK2ct and GRK2 itself should be expected to
inhibit the signaling. Inhibition of PDGF-evoked mitogenesis through
sequestration of Gß
and PIP2 may, in part, underlie the efficacy of
GRK2ct in attenuating NH in rabbit jugular vein bypass
grafts.23
If the cytoplasmic tail of the PDGFR can mimic heptahelical receptor
cytoplasmic domains by activating
Gi,21 perhaps it can also mimic
heptahelical receptor cytoplasmic domains by activating GRK2, as the
wasp venom peptide mastoparan can.24 If the PDGFR
cytoplasmic domain can activate GRK2, it may also be
phosphorylated by GRK2, with a consequent impairment of
downstream signaling. Indeed, our observation that overexpressed GRK2
reduces PDGFRß tyrosine phosphorylation is
consistent with the possibility of GRK2-mediated PDGFRß
phosphorylation. Recently, casein kinase I2 has been
shown to phosphorylate the PDGFRß on serine(s) and
consequently to reduce PDGF-promoted receptor tyrosine
phosphorylation.25 Intriguingly, sites
phosphorylated by casein kinase I2 are strikingly
similar to those that can be phosphorylated by
GRK2.9 25 Whether GRK2 directly phosphorylates
the PDGFRß remains to be determined.
Conceptually, inhibiting mitogenic signaling at the level of the receptor seems to be strategically advantageous in treating NH. Receptor-mediated signaling involves catalytic cascades that amplify signals as they propagate toward the cell nucleus.26 Such signal amplification may at least partly explain the incomplete effectiveness in treating NH observed with molecular strategies targeting transcriptional or other cell cycle–regulatory proteins.26 With GRK2 overexpression in SMCs, multiple signaling pathways are attenuated simultaneously at the level of cell surface receptors signaling through Gq-coupled and tyrosine kinase pathways. Our data regarding GRK2 overexpression therefore suggest that GRK2 overexpression may be therapeutically useful as a novel, plasma membrane–targeted treatment for NH.
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Acknowledgments |
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This work was supported by NIH grants HL-57432 and HL-03008 (Dr Freedman), an American Heart Association Grant-in-Aid (Dr Peppel), and Deutsche Forschungsgemeinschaft grant Op42/7-1 (Dr Oppermann).
Received February 11, 2000; revision received March 17, 2000; accepted March 22, 2000.
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  G. E. Morris, C. P. Nelson, N. B. Standen, R.A. J. Challiss, and J. M. Willets Endothelin signalling in arterial smooth muscle is tightly regulated by G protein-coupled receptor kinase 2 Cardiovasc Res, October 6, 2009; (2009) cvp310v2. [Abstract] [Full Text] [PDF]
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 D. M. Harris, H. I. Cohn, S. Pesant, R.-H. Zhou, and A. D. Eckhart Vascular smooth muscle Gq signaling is involved in high blood pressure in both induced renal and genetic vascular smooth muscle-derived models of hypertension Am J Physiol Heart Circ Physiol, November 1, 2007; 293(5): H3072 - H3079. [Abstract] [Full Text] [PDF]
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 L. E. Vinge, K. W. Andressen, T. Attramadal, G. O. Andersen, M. S. Ahmed, K. Peppel, W. J. Koch, N. J. Freedman, F. O. Levy, T. Skomedal, et al. Substrate Specificities of G Protein-Coupled Receptor Kinase-2 and -3 at Cardiac Myocyte Receptors Provide Basis for Distinct Roles in Regulation of Myocardial Function Mol. Pharmacol., September 1, 2007; 72(3): 582 - 591. [Abstract] [Full Text] [PDF]
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 J.-H. Wu, R. Goswami, X. Cai, S. T. Exum, X. Huang, L. Zhang, L. Brian, R. T. Premont, K. Peppel, and N. J. Freedman Regulation of the Platelet-derived Growth Factor Receptor-beta by G Protein-coupled Receptor Kinase-5 in Vascular Smooth Muscle Cells Involves the Phosphatase Shp2 J. Biol. Chem., December 8, 2006; 281(49): 37758 - 37772. [Abstract] [Full Text] [PDF]
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J.-H. Wu, R. Goswami, L. K. Kim, W. E. Miller, K. Peppel, and N. J. Freedman The Platelet-derived Growth Factor Receptor-{beta} Phosphorylates and Activates G Protein-coupled Receptor Kinase-2: A MECHANISM FOR FEEDBACK INHIBITION J. Biol. Chem., September 2, 2005; 280(35): 31027 - 31035. [Abstract] [Full Text] [PDF]
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K. Peppel, L. Zhang, E. S. Orman, P.-O. Hagen, A. Amalfitano, L. Brian, and N. J. Freedman Activation of vascular smooth muscle cells by TNF and PDGF: overlapping and complementary signal transduction mechanisms Cardiovasc Res, February 15, 2005; 65(3): 674 - 682. [Abstract] [Full Text] [PDF]
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 L. Zhang, K. Peppel, L. Brian, L. Chien, and N. J. Freedman Vein Graft Neointimal Hyperplasia Is Exacerbated by Tumor Necrosis Factor Receptor-1 Signaling in Graft-Intrinsic Cells Arterioscler Thromb Vasc Biol, December 1, 2004; 24(12): 2277 - 2283. [Abstract] [Full Text] [PDF]
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K. L. Hildreth, J.-H. Wu, L. S. Barak, S. T. Exum, L. K. Kim, K. Peppel, and N. J. Freedman Phosphorylation of the Platelet-derived Growth Factor Receptor-{beta} by G Protein-coupled Receptor Kinase-2 Reduces Receptor Signaling and Interaction with the Na+/H+ Exchanger Regulatory Factor J. Biol. Chem., October 1, 2004; 279(40): 41775 - 41782. [Abstract] [Full Text] [PDF]
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L. Zhang, N. J. Freedman, L. Brian, and K. Peppel Graft-Extrinsic Cells Predominate in Vein Graft Arterialization Arterioscler Thromb Vasc Biol, March 1, 2004; 24(3): 470 - 476. [Abstract] [Full Text]
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A. L. Bookout, A. E. Finney, R. Guo, K. Peppel, W. J. Koch, and Y. Daaka Targeting G{beta}{gamma} Signaling to Inhibit Prostate Tumor Formation and Growth J. Biol. Chem., September 26, 2003; 278(39): 37569 - 37573. [Abstract] [Full Text] [PDF]
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N. J. Freedman, L. K. Kim, J. P. Murray, S. T. Exum, L. Brian, J.-H. Wu, and K. Peppel Phosphorylation of the Platelet-derived Growth Factor Receptor-beta and Epidermal Growth Factor Receptor by G Protein-coupled Receptor Kinase-2. MECHANISMS FOR SELECTIVITY OF DESENSITIZATION J. Biol. Chem., December 6, 2002; 277(50): 48261 - 48269. [Abstract] [Full Text] [PDF]
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 C. Patterson, G. A. Stouffer, N. Madamanchi, and M. S. Runge New Tricks for Old Dogs : Nonthrombotic Effects of Thrombin in Vessel Wall Biology Circ. Res., May 25, 2001; 88(10): 987 - 997. [Abstract] [Full Text] [PDF]
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