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Originally published In Press as doi:10.1074/jbc.M605756200 on October 3, 2006

J. Biol. Chem., Vol. 281, Issue 49, 37758-37772, December 8, 2006
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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*

Jiao-Hui Wu, Robi Goswami1, Xinjiang Cai, Sabrina T. Exum, Xuewei Huang2, Lisheng Zhang, Leigh Brian, Richard T. Premont, Karsten Peppel3, and Neil J. Freedman4

From the Departments of Medicine (Cardiology) and Cell Biology, Duke University, Medical Center, Durham, North Carolina 27710

Received for publication, June 15, 2006 , and in revised form, September 18, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Smooth muscle cell (SMC) proliferation and migration are substantially controlled by the platelet-derived growth factor receptor-beta (PDGFRbeta), which can be regulated by the Ser/Thr kinase G protein-coupled receptor kinase-2 (GRK2). In mouse aortic SMCs, however, we found that prolonged PDGFRbeta activation engendered down-regulation of GRK5, but not GRK2; moreover, GRK5 and PDGFRbeta were coordinately up-regulated in SMCs from atherosclerotic arteries. With SMCs from GRK5 knock-out and cognate wild type mice (five of each), we found that physiologic expression of GRK5 increased PDGF-promoted PDGFRbeta seryl phosphorylation by 3-fold and reduced PDGFRbeta-promoted phosphoinositide hydrolysis, thymidine incorporation, and overall PDGFRbeta tyrosyl phosphorylation by ~35%. Physiologic SMC GRK5 activity also increased PDGFRbeta association with the phosphatase Shp2 (8-fold), enhanced phosphorylation of PDGFRbeta Tyr1009 (the docking site for Shp2), and reduced phosphorylation of PDGFRbeta Tyr1021. Consistent with having increased PDGFRbeta-associated Shp2 activity, GRK5-expressing SMCs demonstrated greater PDGF-induced Src activation than GRK5-null cells. GRK5-mediated desensitization of PDGFRbeta inositol phosphate signaling was diminished by Shp2 knock-down or impairment of PDGFRbeta/Shp2 association. In contrast to GRK5, physiologic GRK2 activity did not alter PDGFRbeta/Shp2 association. Finally, purified GRK5 effected agonist-dependent seryl phosphorylation of partially purified PDGFRbetas. We conclude that GRK5 mediates the preponderance of PDGF-promoted seryl phosphorylation of the PDGFRbeta in SMCs, and, through mechanisms involving Shp2, desensitizes PDGFRbeta inositol phosphate signaling and enhances PDGFRbeta-triggered Src activation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The pathogenesis of atherosclerosis (1) and neointimal hyperplasia after vascular injury (2) fundamentally involves the platelet-derived growth factor receptor-beta (PDGFRbeta)5 expressed on smooth muscle cells (SMCs) (3). As a receptor protein-tyrosine kinase, the PDGFRbeta autophosphorylates on tyrosyl residues upon binding PDGF. Subsequently, the PDGFRbeta activates intracellular signaling cascades by tyrosine-phosphorylating and/or scaffolding multiple proteins critical for cellular proliferation and migration (4). Until they are destroyed in the lysosome, activated PDGFRbetas appear to continue signaling (5). Thus, regulation of PDGFRbeta signaling prior to receptor degradation attains considerable significance.

Along with PDGFRbeta tyrosyl dephosphorylation (4), an important mechanism for bridling PDGFRbeta signaling involves PDGFRbeta phosphorylation on seryl residues (6, 7). We have recently demonstrated that the PDGFRbeta undergoes agonist-dependent seryl phosphorylation by G protein-coupled receptor kinase-2 (GRK2), a ubiquitously expressed Ser/Thr kinase (7, 8). GRK2-mediated phosphorylation of the PDGFRbeta results in diminished PDGFRbeta signaling, or desensitization, through mechanisms related to reduced PDGFRbeta/Na+/H+ exchanger regulatory factor association (8), reduced PDGFRbeta tyrosyl phosphorylation (7-9), and PDGFRbeta hyperubiquitination (7). This PDGFRbeta desensitization manifests as diminished second messenger signaling (9) and phosphoinositide 3-kinase activation (10) in the short term, and manifests as diminished SMC migration (10), thymidine incorporation (9, 10), and proliferation (9) in the medium to long term.

GRK2 belongs to a seven-member family of Ser/Thr kinases (11, 12), each with a central catalytic domain flanked by amino- and carboxyl-terminal domains that serve membrane-localizing, protein association, and other regulatory functions (11). Allosterically activated by agonist-occupied heptahelical (seven-membrane-spanning) receptors, GRKs characteristically phosphorylate these activated receptors and thereby initiate desensitization that is "receptor-specific" (i.e. that affects only the receptor whose activation prompted GRK activity). Only GRKs 2, 5, and 6 are widely expressed at substantial levels in mammalian tissues. With only 58.6% similarity to GRK2 (13), GRK5 has demonstrated receptor substrate specificity both overlapping with (13) and distinct from (14, 15) GRK2. GRK subtype-specific phosphorylation sites (16) have been shown to result in distinct downstream molecular consequences for receptors phosphorylated by both GRK2 and GRK5 (17-20). In light of the role of GRK2 in regulating PDGFRbeta function in fibroblasts, we initiated this investigation to determine whether the PDGFRbeta can be regulated in SMCs by GRK5 and whether GRK5 and GRK2 employ similar or distinct mechanisms for PDGFRbeta regulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Recombinant Adenoviruses and Plasmids—The cDNA encoding bovine GRK5 was inserted into the plasmid pSKAC, and adenoviruses were produced in 293 cells, purified on CsCl gradients, and titered as we described previously (9). Plasmids encoding the N-terminal FLAGTM-tagged human PDGFRbeta (FPDGFRbeta) (7), bovine GRK2 (21), and bovine GRK5 (21) have been described previously. The Y1009F mutant FPDGFRbeta was created from its WT congener by cassette PCR, using the following primers: 5'-cgcgggccatggcctccgatctcccctggacaccagctccgtcctctttactgccgtgcagcccaatg-3' (underscore denotes the Tyr -> Phe mutation, and italic type denotes the 5' NcoI site) and 5'-cgcggggcggccgcaagcttctacaggaagctatcctctgc-3' (boldface type denotes the stop codon; underscore and italic type denote HindIII and NotI sites, respectively). Subcloning employed pBluescript II KS+ (Stratagene) as a shuttle vector, but both the WT and Y1009F mutant PDGFRbeta constructs were ultimately subcloned into pcDNA I (Invitrogen). PCR fidelity was verified by dideoxy sequencing.

Atherosclerosis Studies in Mice—All animal care conformed to Ref. 59. C57Bl/6J mice without (wild type, WT) or with targeted deletion of the apolipoprotein E gene (apoe-/-; Jackson stock number 002052) were purchased from Jackson Laboratories and fed normal mouse chow. Nine-month-old mice were sacrificed, and the circulatory system was perfused with lactated Ringer solution at 80 mm Hg pressure. The common carotid arteries were excised, embedded in OCT compound, frozen at -150 °C, and sliced at 5 µm on a cryotome to obtain sections of the distal common carotid, just proximal to the carotid bifurcation (a site commonly involved with atherosclerosis in the 9-month-old apoe-/- mouse) (22). Sections were fixed and permeabilized with methanol/acetone (50:50) at room temperature for 2 min, washed twice in Dulbecco's PBS for 1 min, and then incubated (25 °C) for 30 min in "blocking buffer": 3% (w/v) bovine serum albumin in TTBS (0.02% (v/v) Tween 20, 10 mM Tris-Cl, pH 7.4, 140 mM NaCl). Next, sections were incubated (25 °C, 60 min) in blocking buffer containing 1 µg/ml nonimmune rabbit IgG (nonspecific staining) or rabbit IgG specific for GRK5 (sc-565) or the PDGFRbeta (sc-432) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After three washes in TTBS, sections were then incubated (25 °C, 60 min) in blocking buffer containing the DNA-binding dye Hoechst 33342 (10 µg/ml) and 3 µg/ml anti-rabbit IgG labeled with either Alexa 546 (for the PDGFRbeta) or Alexa 488 (for GRK5) (Molecular Probes, Inc., Eugene, OR). After three additional washes in TTBS, sections were mounted in Gel/MountTM (Biomeda Corp.) with a glass coverslip. For specimens stained for {alpha}-SMC actin, we incubated fixed specimens in blocking buffer containing 0.5 µg/ml Cy3-conjugated 1A4 (Sigma) as well as Hoechst 33342 (as above). Fluorescence photomicrographs were taken with ChromaTM narrow band-pass filters and a Spot CCD camera, with a fixed shutter speed for all specimens. Nuclear and protein fluorescence images were obtained from individual microscopic fields by rotating fluorescence filters. Protein fluorescence was normalized to DNA fluorescence within each image, as we described previously (23). Atherosclerotic and nonatherosclerotic specimens were always processed in pairs, so that staining and imaging could be equivalent for both phenotypes.

Cell Culture and Adenovirus Infections—New Zealand White rabbits, grk5-/- and littermate WT mice (14) were sacrificed to make thoracic aortic SMCs, as previously described (9). HEK 293 cells and embryo fibroblasts (MEFs) from grk2-/- and littermate WT mice were grown and transfected as described (8). The grk5-/- mice used in this study were hybrids of the 129/SvJ and C57Bl/6J lines and were generated by mating grk5-/+ mice. Littermate grk5-/- and WT mice were sacrificed simultaneously for SMC production, and all comparisons were made among littermate-derived SMCs. SMCs were used only through passage 7. Infections of SMCs with recombinant adenoviruses were performed at equivalent multiplicities of infection (from 50 to 100), and cells were assayed 48-72 h after infection, as we described previously (9). In GRK5 "add-back" experiments with grk5-/- SMCs, assays were performed 6 h after adenovirus infection (when GRK5 expression levels were equivalent to those obtaining in WT SMCs).

To assay cell surface PDGFRbeta expression levels in transfected HEK cells, we used cell surface immunofluorescence and flow cytometry, as we described previously (7). Cell surface PDGFRbeta expression levels of all cell lines were within 30% of control cell values; cell lines outside of this range were not used to generate data. Compared with HEK cells co-transfected with empty vector plasmid, HEK cells overexpressing either GRK2 or GRK5 demonstrated equivalent (~35%) reductions in PDGF-promoted phosphoinositide hydrolysis; GRK expression was 20-40-fold over endogenous levels (data not shown).

SMC Migration and [3H]Thymidine Incorporation—SMC migration was assayed with a protocol modified from one we reported previously (10). SMCs were serum-deprived for 72 h after adenovirus infection, trypsinized, transiently (<5 min) treated with 8% fetal bovine serum (to neutralize the trypsin), and washed with low mitogen medium. Next, SMCs were plated onto TranswellTM membranes (8-µm pores; Costar) in 24-well dishes and allowed to attach for 4 h. PDGF-BB or vehicle was then delivered outside of the TranswellTM membranes, and SMCs were allowed to migrate for 16 h before fixation with methanol. (Pilot studies demonstrated that originally quiescent SMCs do not divide during this time period with PDGF stimulation (data not shown).) SMC nuclei were stained with Hoechst 33342, imaged by fluorescence microscopy, and counted electronically, as we reported previously (10). SMC [3H]thymidine incorporation was assessed during the final 4 h of a 24-h agonist stimulation, as we reported previously (9). Parallel aliquots of SMCs were subjected to lysis and IB and demonstrated that control and GRK5-overexpressing SMCs expressed equivalent levels of the PDGFRbeta (data not shown).

Immunoblotting and Immunoprecipitations—These assays used antibodies and procedures described previously (7-9). Immunoprecipitation (IP) of endogenous PDGFRbetas used either rabbit or goat anti-PDGFRbeta IgG (Santa Cruz Biotechnology), whereas IP of transfected (N-terminal epitope-tagged) PDGFRbeta constructs (WT and Y1009F) used anti-FLAG M2-agarose (Sigma). IB employed goat anti-PDGFRbeta phospho-Tyr1009, -Tyr1021, and -Tyr740 as well as murine or rabbit anti-Shp2 (Src homology 2 domain-containing protein-tyrosine phosphatase-2) (all from Santa Cruz Biotechnology), anti-c-Src and anti-c-Src phospho-Tyr416 (anti-activated c-Src) (Calbiochem), and anti-PDGFRbeta phospho-Tyr579 (GeneTex, Inc.). Phosphorylated PDGFRbeta and co-immunoprecipitated band densities were normalized to cognate PDGFRbeta band densities, as described previously (8). PDGFRbeta tyrosyl and seryl phosphorylation were equivalent after 5 or 10 min of PDGF stimulation (data not shown). The efficiency of PDGFRbeta IP was greater than 95%, as assessed by PDGFRbeta IB performed simultaneously on post-IP supernatants and serially diluted pre-IP SMC lysate aliquots (data not shown).

Phosphoinositide Hydrolysis—SMCs were metabolically labeled with [3H]inositol and stimulated as indicated to provoke phosphoinositide hydrolysis as we described previously (9, 24), except that we used HEPES-buffered saline (pH 7.4) (20 mM HEPES, 20 mM LiCl, 110 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2) during SMC stimulation and LiCl preincubation. Fluoroaluminate (Al(F4)-) was used as described (24), and we calculated the percent conversion of [3H]inositol into inositol phosphates as we described previously (24). In assays with mouse SMCs, 2 nM PDGF-AA evoked only 14 ± 2% as much phosphoinositide hydrolysis as 2 nM PDGF-BB (n = 4 SMC lines); thus, 83 ± 2% of our PDGF-BB-dependent mouse SMC phosphoinositide hydrolysis resulted from SMC PDGFRbeta activity (4). We therefore elected not to down-regulate the SMC PDGFR{alpha} before stimulating the SMC PDGFRbeta in phosphoinositide hydrolysis experiments.

SMC Proliferation—SMC proliferation was quantitated by enzyme-linked immunosorbent assay (ELISA) for the nuclear scaffolding protein lamin (25). SMCs were plated at 5 x 103/well in low mitogen medium (9) on day 0 in replicate 96-well plates. On day 1, one plate was washed with PBS, fixed, and permeabilized with methanol/acetone (1:1) for 2 min, washed with PBS, and frozen at -80 °C. SMCs on the second plate were refed with low mitogen medium lacking ("basal") or containing agonists at the indicated concentrations. SMCs were refed on day 6 and then harvested, washed, permeabilized, and frozen as above on day 12. Replicate frozen plates were thawed and incubated (25 °C) for 30 min in "blocking buffer": 3% bovine serum albumin in PBS. Next, wells were incubated for 1 h (25 °C) in blocking buffer with 1 µg/ml of either an irrelevant murine IgG2b (nonspecific signal) or an anti-lamin A/C monoclonal IgG2b (Santa Cruz Biotechnology) (total signal). Wells were washed with PBS twice and then incubated for 1 h with anti-mouse/horseradish peroxidase in blocking buffer. After two PBS washes, wells were incubated in 50 µl/well substrate solution: 0.1 mg of o-phenylenediamine (Sigma)/ml of 0.03% H2O2. Reactions were terminated when adequate color development was achieved (~30 min), with 50 µl of 1 M H2SO4. The color of each well was read at 490 nm. Specific A490 was calculated as total - nonspecific; nonspecific signal constituted 20-25% of total signal. Assays were performed in triplicate. The number of SMCs in agonist-stimulated wells was normalized to that in basal wells on day 12 (which, in turn, was ~10% above the number measured on day 1).

In Vitro Phosphorylation with Purified GRK5—Recombinant bovine GRK5 was synthesized in baculovirus-infected Sf9 insect cells, as we have previously reported (13). GRK5 was purified as before (13), except that elution from the heparin-Sepharose column was with a 100-ml linear gradient of NaCl that was 150-1200 mM in buffer A (20 mM HEPES, 2 mM EDTA, pH 7.2) and included 0.02% (v/v) Triton X-100. Fractions at ~800 mM NaCl were pooled and diluted with buffer A to reduce the [NaCl] to <150 mM (buffer B). Subsequently, the diluted, purified GRK5 was concentrated by ultrafiltration and stored at 0.5 mg/ml in 50:50 (v/v) glycerol/buffer B at -20 °C. By Coomassie Blue staining of SDS-polyacrylamide gels, the GRK5 preparation was ~95% pure.

Phosphorylation reactions were carried out with 300 nM GRK5 exactly as we described previously for GRK2 (7), except that the source of PDGFRbeta was grk5-/- SMCs. After PDGF (or vehicle) challenge for 5 min (37 °C), SMCs were solubilized and PDGFRbetas were immunoprecipitated. The PDGFRbeta for each reaction was immunoprecipitated from a confluent 100-mm plate.

RNA Interference—Small interfering RNAs were chemically synthesized for Shp2 (5'-cccaaaaagaguuacauugcc-3', residues 1080-1100 of the murine sequence) (26), GRK2 (5'-aagaaauaugagaagcuggag-3', residues 172-291 of the murine sequence) (19), and GRK5 (5'-ccccugcaaagaactcttc-3', residues 408-427 of the murine sequence) (19) (Dharmacon, Inc.). Mouse SMCs were grown in 12-well dishes and transfected with mRNA-specific or negative control siRNA (5'-aauucuccgaacgugucacgu-3'; Dharmacon) at a concentration of 100 nM, using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions, except for the following: immediately before transfection, SMC growth medium was changed to serum-free OPTI-MEM I; after adding the solution of siRNA and Lipofectamine 2000 to the SMCs, we incubated SMCs at 37 °C in a CO2 incubator for only 4 h, after which time we added 1 volume of 2x growth medium (without antibiotics) and continued incubation for an additional 48 h. Growth medium was then replaced with serum-free medium (Dulbecco's modified Eagle's medium, 20 mM HEPES, pH 7.4, 0.1% (w/v) bovine serum albumin), containing (phosphoinositide hydrolysis) or lacking (IB) 1 µCi of [3H]inositol/ml for a further 20 h. Assays (phosphoinositide hydrolysis and IB) were performed 72 h after siRNA transfection. Flow cytometry of SMCs transfected with fluorescein isothiocyanate-labeled and unlabeled siRNA (Dharmacon) demonstrated the efficiency of transfection under these conditions to be 90-95% (data not shown).

Statistical Analyses—Results from multiple experiments were averaged for independent groups but analyzed pairwise, within experiments, by repeated measures analysis of variance and Tukey's post hoc test for multiple comparisons (Prism 2TM Software, GraphPad, Inc.). Data are presented in the text as mean ± S.D. and in the figures as mean ± S.E.


Figure 1
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  		FIGURE 1.
Prolonged PDGFRbeta stimulation down-regulates GRK5, but not GRK2. Quiescent WT mouse SMCs were incubated without (control) or with 2 nM PDGF-BB for the indicated times, and then lysed. Twelve µg of SMC protein were subjected to SDS-PAGE and IB (sequentially) for GRK5, GRK2, and actin. A, blots from a single experiment, representative of two performed in duplicate with independent SMC lines. Probing parallel blots with nonimmune IgG yielded no bands in the areas of interest. B, GRK band densities were divided by the cognate actin band densities, and these ratios were normalized to those obtained in control samples at each time point. The means ± S.E. of two independent SMC lines are displayed. C,20 µg of protein from MEFs or SMCs of the indicated genotype were subjected to SDS-PAGE and serial IB for the indicated GRK and actin. Results are representative of three independent experiments with three cell lines of each genotype.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In cell systems in which GRK2 mediates most of the PDGF-induced seryl phosphorylation of the PDGFRbeta, persistent PDGFRbeta signaling results in down-regulation of both GRK2 (27) and the PDGFRbeta (7). To determine which widely expressed GRK (GRK2 or GRK5) mediates most of the PDGFRbeta regulation in SMCs, we first tested whether the expression level of either of these GRKs was regulated coordinately with the PDGFRbeta. Indeed, with prolonged PDGF stimulation that down-regulates the PDGFRbeta (7), we observed down-regulation of GRK5, but not GRK2 (Fig. 1). To resolve the apparent paradox between these results and our data showing that endogenous GRK2 regulates PDGFRbetas in fibroblasts (8), we compared GRK expression levels in SMCs and fibroblasts. Consistent with a GRK5-dominant mechanism for PDGFRbeta regulation in SMCs, SMCs expressed 3 ± 1-fold more GRK5 than fibroblasts, and only 0.5 ± 0.2-fold as much GRK2 (Fig. 1C).


Figure 2
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  		FIGURE 2.
GRK5 and the PDGFRbeta are coordinately up-regulated in SMCs within atherosclerotic lesions. The distal carotid arteries from 9-month-old C57Bl/6J WT and congenic apoe-/- (atherogenic) mice were harvested, embedded, and sectioned as described under "Materials and Methods." Serial sections were stained immunofluorescently for either {alpha}-SMC actin, GRK5, or the PDGFRbeta, as indicated; all sections were counterstained with Hoechst 33342 for nuclear DNA. Scale bar, 50 µm; original magnification was x1,100. Shown are samples from two specimens stained in parallel, representative of three such specimen pairs. Specimens stained with nonimmune primary IgG yielded only DNA fluorescence (data not shown).

 
If vascular SMC GRK5 expression is regulated coordinately with the PDGFRbeta in a physiologically meaningful way, then we should expect GRK5 to be up-regulated under pathological conditions that promote PDGFRbeta up-regulation too. To test this expectation, we examined GRK5 and PDGFRbeta expression in atherosclerosis, a pathologic process involving SMC proliferation and migration in response to myriad cytokines and growth factors (28). The "fibrous cap" of atherosclerotic lesions in the mouse comprises largely SMCs, identified in Fig. 2 by staining for {alpha}-SMC actin. Although atherosclerotic and normal arteries demonstrate equivalent {alpha}-SMC actin expression per cell, atherosclerotic arteries demonstrate substantially more PDGFRbeta and GRK5 expression per {alpha}-SMC actin-expressing cell (Fig. 2). Interestingly, unlike GRK5, GRK2 was not up-regulated in these atheroma SMCs (data not shown). The coordinate up-regulation of GRK5 and the PDGFRbeta in SMCs of atherosclerotic arteries, along with coordinate down-regulation of GRK5 and the PDGFRbeta in cultured SMCs, suggests that GRK5 and the PDGFRbeta in SMCs may be functionally related.

To determine possible effects of GRK5 on PDGFRbeta activity in SMCs, we began by overexpressing GRK5 in primary rabbit SMCs with a recombinant adenovirus. The prevalence of GRK overexpression was assessed by immunofluorescence microscopy to be 90-100%, as described previously (9). In GRK5-overexpressing SMCs, GRK5 was ~20-30-fold overexpressed, relative to endogenous GRK5, and PDGFRbeta expression was 100 ± 15% of that seen in control SMCs (Fig. 3A and data not shown).

GRK5 Desensitizes Heptahelical and PDGF Receptors—Phosphoinositide hydrolysis elicited through SMC heptahelical receptors was clearly desensitized by GRK5 overexpression (Fig. 3B). As we have observed in HEK cells (24), GRK5 inhibited phosphoinositide hydrolysis elicited by endothelin and PAR1 (protease-activated receptor-1). However, contrary to results in HEK cells overexpressing GRK5 (29) or SMCs overexpressing GRK2 (9), GRK5 overexpression also blunted phosphoinositide hydrolysis evoked through thromboxane A2 receptors. This inhibition of Gq-coupled receptor signaling could have been mediated at the level of G{alpha}q/11 subunits or by the GRK5 RGS (regulator of G protein signaling) domain (29, 30). Supporting this hypothesis, GRK5 overexpression inhibited thromboxane-evoked phosphoinositide hydrolysis to a degree equivalent to that observed with fluoroaluminate (~40%), which activates G proteins independently of receptors (31). In contrast, GRK5 overexpression inhibited endothelin- and PAR1-evoked phosphoinositide hydrolysis to a greater extent (~70%, p < 0.05). Thus, these levels of GRK5 overexpression appeared to reduce signaling with both receptor-specific and G protein-related mechanisms.

Overexpression of GRK5, like GRK2 (9), also inhibited phosphoinositide hydrolysis effected by the PDGFRbeta (the only PDGFR expressed in rabbit aortic SMCs) (32) by 60% (Fig. 3B). Thus, the ability to desensitize both PDGF and heptahelical receptors appears to extend across GRK subtypes. Importantly, this inhibition of PDGFRbeta-evoked phosphoinositide hydrolysis did not involve heterotrimeric G proteins. In rabbit SMCs, we found that the PDGFRbeta activates G{alpha}i, but not G{alpha}q/11 (7). Although Gbeta{gamma}i subunits can activate PLC-beta (33), we found no evidence of such activation by the PDGFRbeta in our SMCs. Treatment of SMCs with pertussis toxin (to inactivate Gi/o) failed to affect PDGF-induced phosphoinositide hydrolysis, but eliminated Gi/o-dependent (34) activation of extracellular signal-regulated kinase (ERK) by lysophosphatidic acid (data not shown). In light of these data, it seemed that overexpression of GRK5 inhibited PDGFRbeta-mediated activation of PLC-{gamma}, a tyrosine kinase-dependent event (4).

PDGF-promoted SMC Migration and Proliferation Are Attenuated by GRK5—We tested whether overexpression of GRK5 would inhibit PDGF-induced SMC migration, since this process involves not only PLC-{gamma} but also phosphoinositide 3-kinase (10), p125 focal adhesion kinase (32), reactive oxygen species (35), and small G proteins (36, 37). PDGF-promoted migration was 2.2 ± 0.6-fold/basal in vector-infected SMCs, and basal SMC migration was indistinguishable among vector- and GRK5 adenovirus-infected SMC groups (Fig. 3C). However, PDGF-promoted migration was halved by overexpression of GRK5 (Fig. 3C), in a manner congruent with GRK2-overexpressing SMCs (10).

GRK5 Diminishes PDGFRbeta-evoked SMC Thymidine Incorporation and Proliferation—Although it results from PDGFRbeta signaling distinct from that required for migration (4, 10, 38-40), PDGFRbeta-evoked SMC thymidine incorporation was also diminished 65-70% in GRK5-overexpressing SMCs, in response to PDGF alone or in synergistic combination with Gq-coupled receptors (Fig. 4A), just as we observed with GRK2 (9, 10). GRK5, like GRK2 (9), also blunted thymidine incorporation induced by the myriad agonists in fetal bovine serum (in which PDGF plays a critical role) (41). Whereas GRK5 overexpression substantially attenuated thymidine incorporation evoked by the combination of PDGF and Gq-coupled receptor agonists, it failed to affect comparable thymidine incorporation evoked by PDGF plus EGF. Thus, GRK5-mediated desensitization demonstrated substrate specificity for receptor proteintyrosine kinases in a manner very similar to that observed with GRK2 (10).


Figure 3
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  		FIGURE 3.
SMC phosphoinositide hydrolysis and migration are reduced by GRK5 overexpression. SMCs were infected with adenoviruses encoding GRK5 or no protein (control, vector, or -), and assayed or solubilized for IB 3 days later. A, IB were performed on 10µg of cell lysate, with nonimmune (Neg) or anti-GRK5/6 IgG (58) recognizing the 68-kDa GRK5. In parallel, 35 µg of SMC lysate was immunoblotted with nonimmune IgG or serially for PDGFRbeta and actin. Results are representative of ≥10 blots, performed with each assay. B, SMCs were metabolically labeled and exposed (37 °C) to low-mitogen medium (9) containing vehicle (basal), 100 nM endothelin-1 (ET-1), 0.4 nM human PDGF-BB, 10 µM thromboxane A2 analogue (U46619), 100 µM thrombin agonist peptide (SFLLRN) (each for 15 min), or fluoroaluminate (30 min). Resultant inositol phosphates were divided by SMC [3H]inositol uptake to obtain percent conversion (% conversion), as described (24), and plotted as the mean ± S.E. of at least three experiments. Basal inositol phosphates for vector- and GRK5-infected SMCs were 4 ± 1.4 and 1.9 ± 0.8 (percent conversion units), respectively. *, p < 0.05 compared with control. C, migration in response to medium lacking (basal) or containing 0.4 nM PDGF-BB. At the left appear fluorescence micrographs of Hoechst 33342-stained SMC nuclei located on the bottom of TranswellTM membranes. PDGF-promoted SMC migration was calculated as (number of SMCs migrated with PDGF) - (number of SMCs migrated without PDGF), and then normalized to values obtained with vector-infected (control) SMCs, to obtain the percentage of control (% of control), plotted as the mean ± S.E. of four independent experiments. *, p < 0.05 compared with control. Basal migration was as follows: 243 ± 50, 282 ± 166, and 261 ± 64 cells for control, uninfected, and GRK5-infected SMCs, respectively.

 


Figure 4
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  		FIGURE 4.
GRK5 overexpression attenuates PDGF-promoted SMC proliferation in a receptor-specific manner. A, SMC thymidine incorporation. Quiescent SMCs infected with the indicated adenovirus were exposed to low-mitogen medium containing vehicle (basal), the indicated agonists at concentrations specified above, 5% fetal bovine serum (FBS), or PDGF plus 0.17 nM EGF. [3H]Thymidine incorporation after 24 h of agonist exposure is plotted as the mean ± S.E., from at least four experiments performed in triplicate. *, p < 0.05 compared with vector-infected SMCs. Uninfected and vector-infected SMCs showed indistinguishable stimulus-induced thymidine incorporation (data not shown). B, lamin ELISA. The indicated number of quiescent SMCs were plated in low mitogen medium and subjected the next day to lamin ELISA. Shown are the means ± S.D. of a single experiment performed in triplicate, representative of eight performed. For A490 versus SMC number, R2 = 0.957. C, SMC proliferation. Quiescent, adenovirus-infected SMCs (5 x 103/well) were exposed to low mitogen medium containing vehicle (basal), 1.5 nM fibroblast growth factor-2 (FGF), or other agonists as specified in Fig. 3. SMC proliferation after 12 days of agonist exposure was assessed by lamin ELISA and plotted as 100 x ((stimulated/basal) - 1), means ± S.E. from at least three independent experiments performed in quadruplicate. *, p < 0.05 compared with vector-infected SMCs (by repeated measures analysis of variance).

 


Figure 5
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  		FIGURE 5.
GRK5 overexpression reduces PDGFRbeta tyrosyl phosphorylation and enhances PDGFRbeta/Shp2 association. A, SMCs infected with the indicated adenovirus were rendered quiescent and then stimulated with 2 nM PDGF-BB (37 °C, 10 min) and subjected to IP with anti-PDGFRbeta or nonimmune IgG (-). Divided IP samples were subjected to parallel SDS-PAGE and IB, for either phosphotyrosine (pY) or PDGFRbeta. Shown are data from a single experiment, representative of three performed. B, band densities for phosphotyrosine in A were normalized to cognate PDGFRbeta band densities; each ratio was normalized to that obtained from IPs of PDGF-stimulated SMCs infected with the vector adenovirus (control), to obtain the percentage of control (% of control). Data (mean ± S.E.) are from three independent experiments. *, p < 0.05 compared with control. C, PDGFRbeta IPs performed as in A were probed sequentially with anti-PDGFRbeta and anti-Shp2 antibodies. Cognate cell lysates (20 µg/lane) were immunoblotted for Shp2. Results are representative of three independent experiments.

 
To determine whether the same levels of GRK overexpression that inhibited signaling, migration, and thymidine incorporation would also inhibit SMC proliferation, aliquots of the SMC lines used for the former assays were subjected to 12-day proliferation studies with an ELISA for the nuclear scaffolding protein lamin, which we used as a surrogate for counting nuclei. (We found an excellent correlation between SMC number and results from the lamin ELISA (Fig. 4B).) GRK5 inhibited PDGF-promoted proliferation by ~35% (p < 0.05) and inhibited Gq-coupled receptor-promoted proliferation less consistently (Fig. 4C). Importantly, in inhibiting PDGFRbeta-evoked SMC proliferation, GRK5 also demonstrated specificity for regulating receptor protein-tyrosine kinases, in that GRK5 overexpression failed to reduce SMC proliferation elicited through SMC fibroblast growth factor receptors (Fig. 4C).


Figure 6
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  		FIGURE 6.
Physiologic expression of GRK5 diminishes PDGFRbeta-evoked phosphoinositide hydrolysis, thymidine incorporation, and PDGFRbeta tyrosyl phosphorylation. A, aortic SMC lines from individual GRK5-deficient (KO) and littermate WT mice were solubilized, and 20 µg were immunoblotted with (a) murine anti-GRK5/6 (58) (top) or anti-PLC-{gamma}; (b) rabbit anti-GRK2 or -PDGFRbeta; or (c) cognate nonimmune IgG from mouse (bottom) or rabbit (not shown). IBs are representative of three performed for each protein in five SMC lines of each genotype; nonimmune blots revealed no bands corresponding to proteins of interest. B, SMCs were metabolically labeled and stimulated as in Fig. 3. Inositol phosphates expressed as stimulated/basal were averaged among three independent SMC lines of each genotype (≥2 experiments/SMC line); means ± S.E. are plotted. Basal inositol phosphate values were 0.98 ± 0.43 and 0.72 ± 0.42 (percentage of conversion units (24)) for WT and KO SMCs, respectively. C, quiescent SMCs were stimulated (or not; basal) with 2 nM PDGF-BB or 1.7 nM EGF and subjected to [3H]thymidine incorporation assay, as in Fig. 4. Shown are the means ± S.E. from four experiments performed with two independent pairs of WT and KO SMC lines. Basal [3H]thymidine incorporation values were 2 ± 1 x 103 cpm for both WT and KO SMCs. *, p < 0.05 compared with WT (paired analysis). D, SMCs exposed for 5 min (37 °C) to low mitogen medium containing vehicle or 2 nM PDGF-BB were solubilized, and anti-PDGFRbeta or cognate nonimmune IgG (-) was used for IP. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted serially for the PDGFRbeta and then phosphotyrosine (pY). Relative densities of phosphotyrosine bands were quantitated as in Fig. 5B and plotted as means ± S.E. from three independent experiments. Shown are blots from a single experiment, representative of three performed with at least two WT and at least two KO SMC lines each. *, p < 0.05 compared with GRK5 KO.

 
GRK5 Reduces PDGFRbeta Tyrosyl Phosphorylation—To understand the receptor specificity of GRK5 for PDGFRbeta desensitization, we sought to determine whether GRK5 affected activation of the PDGFRbeta itself, assessed by PDGFRbeta tyrosyl phosphorylation. Indeed, total PDGFRbeta tyrosyl phosphorylation was reduced by ~30% in SMCs overexpressing GRK5 (p < 0.05; Fig. 5, A and B). This reduction in PDGFRbeta activation by GRK5 thus strikingly resembled that achieved by GRK2 (7, 9). However, GRK5-mediated deactivation of the PDGFRbeta appeared to employ mechanisms distinct from those employed by GRK2. SMC overexpression of GRK5, but not GRK2, enhanced (by 2.5 ± 0.7-fold) the association of the PDGFRbeta with the Src homology 2 domain-containing protein-tyrosine phosphatase-2 (Shp2), which dephosphorylates the PDGFRbeta (4) (Fig. 5C) (data not shown).

Physiologically Expressed GRK5 Regulates the PDGFRbeta—To investigate PDGFRbeta regulation by physiologic levels of GRK5 in SMCs, we used grk5-/- and WT SMC lines. By immunoblotting SMC extracts, we found comparable expression of PDGFRbeta, GRK2, GRK6, and PLC-{gamma}1 among SMC lines isolated independently from five grk5-/- and five littermate WT mice (Fig. 6A) (data not shown).

In these SMC lines, the importance of GRK5 in regulating the SMC PDGFRbeta manifested itself clearly. Endogenous GRK5 reduced PDGFRbeta-evoked phosphoinositide hydrolysis by 35% (p < 0.05), but had no effect on fluoroaluminate-induced (G protein-mediated) phosphoinositide hydrolysis (Fig. 6B). In addition, endogenous GRK5 diminished thymidine incorporation evoked by the PDGFRbeta (by 56%), but not the EGF receptor (Fig. 6C). Finally, physiologically expressed GRK5 also reduced PDGFRbeta tyrosyl phosphorylation, by 35 ± 10% (Fig. 6D). Thus, physiologically expressed GRK5 mediated receptor-specific PDGFRbeta desensitization at the level of PDGFRbeta autophosphorylation/activation, second messenger signaling, and signaling further downstream from the receptor, and all in a manner congruent with that observed by comparing GRK5-expressing with GRK5-overexpressing SMCs (Figs. 3, 4, 5).


Figure 7
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  		FIGURE 7.
GRK5 serine-phosphorylates the PDGFRbeta in intact SMCs and in purified protein preparations. A, phosphorylation in intact SMCs. SMCs were stimulated and processed for PDGFRbeta IP and IB just as in Fig. 6D, except that serial IB was performed for the PDGFRbeta and then phosphoserine (pSer), not phosphotyrosine. Shown are blots from a single experiment, representative of three performed with at least two WT and at least two KO SMC lines each. Relative densities of pSer bands were quantitated as in Fig. 5B and plotted as means ± S.E. from three independent experiments. *, p < 0.05 compared with GRK5 KO. B, PDGFRbeta phosphorylation with purified GRK5. PDGFRbetas were immunoprecipitated from grk5-/- SMCs that had been stimulated and processed just as in A. After IP, purified GRK5 was added to PDGFRbeta immune complexes, and phosphorylation proceeded (35 °C) for 30 min. Immune complexes were then pelleted; supernatant GRK5 and pelleted PDGFRbetas were resolved by separate SDS-PAGE procedures and subjected to IB. The PDGFRbeta sample was divided and probed in parallel for PDGFRbeta and then pSer (sequentially) or phosphotyrosine. Shown are blots from a single experiment, representative of four performed with two grk5-/- SMC lines. Relative densities of phosphoserine bands were normalized to cognate PDGFRbeta band densities ("arbitrary units"), averaged across four independent experiments, and plotted as means ± S.E. *, p < 0.02 compared with PDGFRbetas from cells treated without PDGF. #, p < 0.01 compared with PDGFRbetas incubated without purified GRK5.

 
For heptahelical receptors, receptor-specific desensitization by GRK5 corresponds to GRK5-mediated phosphorylation of the receptors on Ser or Thr residues (11). To test whether GRK5-mediated PDGFRbeta regulation in SMCs followed this paradigm, we assessed PDGF-induced PDGFRbeta seryl phosphorylation in WT and grk5-/- SMCs. Indeed, physiologic expression of GRK5 did augment PDGF-induced seryl phosphorylation of the PDGFRbeta, by 2.9 ± 0.9-fold (Fig. 7A).

To ascertain that GRK5 itself was responsible for the excess PDGFRbeta seryl phosphorylation we observed in GRK5-expressing SMCs, we used purified GRK5 to phosphorylate the partially purified PDGFRbeta in vitro (Fig. 7B). In the absence of purified GRK5, we found some PDGF-dependent PDGFRbeta seryl phosphorylation (Fig. 7B, lane 2). This PDGFRbeta seryl phosphorylation, however, could be attributed to intracellular Ser/Thr kinases, acting before PDGFRbeta IP (as in grk5-/- SMCs, in Fig. 7A). As a result of purified GRK5 activity in vitro, this agonist-dependent PDGFRbeta seryl phosphorylation increased ~2-fold (Fig. 7B). Purified GRK5 activity showed an even larger relative increase in seryl phosphorylation with PDGFRbetas obtained from unstimulated SMCs (~4-fold). However, it should be noted that this "agonist-independent" effect was not independent of PDGFRbeta activation. IgG in our immune complex kinase assay dimerizes immunoprecipitated PDGFRbetas, and thereby promotes PDGFRbeta autophosphorylation/activation (Fig. 7B), which is a prerequisite for GRK-mediated PDGFRbeta phosphorylation (7).

In these experiments with vascular SMCs, physiologically expressed GRK5 phosphorylated and desensitized the PDGFRbeta in a manner resembling that of GRK2 expressed physiologically in fibroblasts (8). Indeed, although GRK2 appeared to mediate most of the agonist-induced PDGFRbeta Ser phosphorylation in fibroblasts (8), GRK5 mediated most of the agonist-induced PDGFRbeta Ser phosphorylation in SMCs. Furthermore, GRK5 augmented PDGFRbeta seryl phosphorylation as it does with heptahelical receptors (12), in an agonist-dependent manner and on a rapid time scale congruent with desensitization of second messenger production (seen in Fig. 6B).


Figure 8
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  		FIGURE 8.
The PDGFRbeta is regulated in SMCs predominantly by GRK5 and not GRK2. WT SMCs were treated with siRNA that targeted the mRNA for no known protein (Control), for GRK2, or for GRK5. Subsequently, the SMCs were metabolically labeled with [3H]inositol and, 72 h after siRNA treatment, stimulated as in Fig. 6 or processed for IB. A, inositol phosphates expressed as stimulated/basal were averaged among two independent SMC lines (with each siRNA-treated group assayed in triplicate); means ± S.E. are plotted. *, p < 0.05 compared with control SMCs. Basal inositol phosphate values were 2.3 ± 0.8, 2.6 ± 0.4, and 2.3 ± 0.3 (percent conversion units (24)) for SMCs treated with control, GRK2, and GRK5 siRNA, respectively. B, extracts (35 µgof protein) from SMCs treated with the indicated siRNA were subjected to SDS-PAGE and IB. Blots were probed serially for GRK2, GRK5, and actin. Shown are the results of a single experiment, representative of three performed. Serial IB for PDGFRbeta showed equivalent PDGFRbeta expression in all SMC groups (not shown). C, densitometry of GRK bands was normalized to corresponding actin bands on each blot, and these ratios were normalized to cognate ratios obtained from SMCs transfected with control siRNA, to obtain the percentage of control. Shown are the mean ± S.E. of three experiments. *, p < 0.05 compared with control cells.

 
Does the predominance of SMC GRK5 in PDGFRbeta seryl phosphorylation correlate with a predominance of GRK5 in PDGFRbeta desensitization? To address this question, we sought to determine the relative contributions of GRK5 and GRK2 to PDGFRbeta regulation in SMCs. To that end, we used siRNA to reduce SMC expression of either GRK2 or GRK5, and assessed the effect of GRK knock-down on phosphoinositide hydrolysis. As we observed with GRK5-null and WT SMCs (Fig. 6B), G protein (fluoroaluminate)-evoked phosphoinositide hydrolysis was unaffected by changes in GRK expression (Fig. 8A). In contrast, PDGF-evoked phosphoinositide hydrolysis was enhanced (~35%) by reduction in the expression of just GRK5, and not GRK2 (Fig. 8A). Moreover, this GRK-specific difference obtained even though the siRNA-mediated knock-down of GRK2 was somewhat more efficacious than that for GRK5 (Fig. 8, B and C). Consequently, the GRK isoform that regulates PDGFRbeta signaling in SMCs predominantly is GRK5, and not GRK2.

GRK5-mediated PDGFRbeta Desensitization Involves Shp2— To understand how GRK5-mediated seryl phosphorylation of the PDGFRbeta could diminish receptor Tyr phosphorylation (Fig. 7B), we tested whether physiologically expressed GRK5 augmented the association of the PDGFRbeta with the phosphatase Shp2, as overexpressed GRK5 did (Fig. 5C). Compared with GRK5-null SMCs, GRK5-expressing SMCs evinced 8 ± 5-fold more PDGF-induced Shp2/PDGFRbeta association (range 2-14-fold, in five pairs of WT and KO SMC lines; p < 0.05) (Fig. 9A). Thus, even expressed at physiologic levels, GRK5 serine-phosphorylated the PDGFRbeta in a manner that correlated with augmented recruitment of Shp2 to the receptor. Shp2, of course, could serve as an "effector" of GRK5-initiated PDGFRbeta desensitization by mediating PDGFRbeta dephosphorylation.

How could GRK5-mediated Ser phosphorylation of the PDGFRbeta augment Shp2 recruitment to the PDGFRbeta? To address this question, we asked whether GRK5 activity affected phosphorylation of PDGFRbeta Tyr1009, since phospho-Tyr1009 is the primary PDGFRbeta docking site for Shp2 (4). With IgG specific for the Tyr1009-phosphorylated PDGFRbeta, we found that Tyr1009 was hyperphosphorylated in GRK5-expressing as compared with GRK5-null SMCs (Fig. 9B). Thus, there was a greater prevalence of Tyr1009-phosphorylated PDGFRbetas in GRK5-expressing SMCs, and consequently a greater prevalence of PDGFRbetas capable of recruiting Shp2.

This finding demonstrates that the GRK5-mediated reduction in overall PDGFRbeta tyrosyl phosphorylation is site-specific. Indeed, although GRK5 activity enhanced phosphorylation of PDGFRbeta Tyr1009, it substantially diminished phosphorylation of PDGFRbeta Tyr1021 (Fig. 9C). PDGFRbetas from GRK5-expressing SMCs demonstrated 9 ± 3-fold less phospho-Tyr1021 than PDGFRbetas from GRK5-null SMCs (p < 0.05). This GRK5-associated reduction in PDGFRbeta phospho-Tyr1021 would be expected to reduce PLC-{gamma}1/PDGFRbeta association and consequent PLC-{gamma}1-mediated phosphoinositide hydrolysis (4), just as we observed with these SMCs in Fig. 6B. Such site-specific reduction in PDGFRbeta tyrosyl phosphorylation probably explains why overall PDGFRbeta tyrosyl phosphorylation is only modestly reduced in GRK5-expressing (as compared with GRK5-null) SMCs (Fig. 6D).


Figure 9
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  		FIGURE 9.
GRK5-mediated phosphorylation of the PDGFRbeta enhances Shp2/PDGFRbeta association and phosphorylation of PDGFRbeta Tyr1009, but reduces phosphorylation of PDGFRbeta Tyr1021. WT or GRK5 KO SMCs were stimulated and subjected to PDGFRbeta IP and IB as in Fig. 7A. Blots were probed sequentially for Shp2 and PDGFRbeta (A), in parallel for the PDGFRbeta phosphorylated at Tyr1009 (pY-1009) (B), or sequentially for the PDGFRbeta and the PDGFRbeta phosphorylated at Tyr1021 (pY-1021) (C). Cell lysates (20 µg) were immunoblotted for Shp2 (A). IBs are from single experiments, representative of at least three performed of each type, with at least pairs of WT and KO cells.

 
To confirm that physiologic GRK5 expression was responsible for the differences in PDGFRbeta phosphorylation and signaling we observed between GRK5-expressing and -null SMCs, we took two approaches. First, as described above, we obtained congruent results from five pairs of WT and cognate GRK5-null SMC lines. Second, we used our GRK5 adenovirus to express GRK5 at 104 ± 8% of WT levels in GRK5-null SMCs, to test whether "rescuing" GRK5 expression would convert a GRK5-null to a WT SMC phenotype (Fig. 10). For this purpose, we assayed PDGF-induced phosphorylation of the PDGFRbeta Tyr1021 and found that GRK5 "rescue" expression in GRK5-null SMCs reduced phosphorylation of PDGFRbeta Tyr1021 by 7 ± 3-fold (p < 0.05) (Fig. 10), and enhanced PDGFRbeta/Shp2 association by ~3-fold (Fig. 10). These results were remarkably congruent, of course, with those obtained by comparing WT and grk5-/- SMCs in Fig. 9. Thus, whether expressed endogenously or heterologously, physiologic levels of GRK5 expression mediate PDGFRbeta desensitization.


Figure 10
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  		FIGURE 10.
PDGFRbeta desensitization is rescued in GRK5-null SMCs by physiologic expression levels of GRK5. A, GRK5 KO SMCs were infected with the indicated adenovirus and serum-starved for 5 h, and then stimulated with 2 nM PDGF-BB (or not) for 10 min, lysed, and subjected to IP as in Fig. 9. Divided IP samples were subjected to parallel SDS-PAGE and IB for either the PDGFRbeta phosphorylated at Tyr1021 (pY-1021) or the PDGFRbeta itself; PDGFRbeta blots were subsequently reprobed for Shp2. B, SMC lysates (20 µg) were subjected to SDS-PAGE and IB for GRK5. Images are from individual experiments, representative of at least three performed, with two independent lines of GRK5 KO SMCs.

 
Thus far, we have correlated GRK5-mediated seryl phosphorylation of the PDGFRbeta with diminished PDGFRbeta tyrosyl phosphorylation, desensitization of PDGFRbeta-evoked SMC signaling, and enhancement of PDGFRbeta/Shp2 association. To demonstrate more directly that GRK5-mediated PDGFRbeta regulation involves Shp2, we compared GRK5-mediated desensitization of the WT PDGFRbeta and Y1009F mutant PDGFRbeta, which recruits Shp2 poorly (42) (Fig. 11, A and B). To compare these PDGFRbetas under conditions of comparable GRK5 levels (Fig. 11D), we used HEK cells (which lack endogenous PDGFRbetas) (27). We used phosphorylation of PDGFRbeta Tyr1021 and Tyr740 as read-outs for PDGFRbeta activation. Correlating again with enhancement of Shp2/PDGFRbeta association (Fig. 11B), increased cellular GRK5 activity substantially reduced phosphorylation of Tyr1021 in the WT PDGFRbeta, but not in the Y1009F PDGFRbeta (Fig. 11, A and C). Likewise, increased cellular GRK5 activity approximately halved phosphorylation of Tyr740 in the WT, but not in the Y1009F PDGFRbeta (Fig. 11, A and C). Thus, GRK5-mediated desensitization of the PDGFRbeta appears to require the PDGFRbeta (a) to recruit Shp2 normally and (b) to enhance this recruitment consequent to GRK5-mediated PDGFRbeta phosphorylation.


Figure 11
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  		FIGURE 11.
GRK5-mediated desensitization of the PDGFRbeta requires intact Shp2/PDGFRbeta association. HEK 293 cells were transfected with plasmids encoding a human N-terminal FLAG-tagged PDGFRbeta construct (WT or Y1009F), GRK5 ("high" GRK5 level), or no protein (Vector, "native" GRK5 level). Cells were exposed to medium containing vehicle (-) or 2 nM PDGF-BB (+) for 5 min (37 °C), and then lysed and subjected to IP of the indicated PDGFRbeta construct. Divided IP samples were subjected to parallel SDS-PAGE and sequential IB (with intervening membrane stripping) for the PDGFRbeta and then either the PDGFRbeta phosphorylated at Tyr1021 (pY-1021), the PDGFRbeta phosphorylated at Tyr740 (pY-740), or Shp2. A, blots from a single experiment are displayed and represent three experiments performed with similar results. HEK cells transfected without a PDGFRbeta construct yielded no signals on these blots, and all cell lines expressed equivalent levels of Shp2 (data not shown). B, quantitation of Shp2/PDGFRbeta association. Shp2 band densities were normalized to cognate PDGFRbeta band densities; each ratio was normalized to that obtained from IPs of PDGF-stimulated HEK cells transfected with the WT PDGFRbeta and empty vector ("control" cells) to obtain the "percentage of control." *, p < 0.05 compared with cognate cells expressing native GRK5 levels. C, quantitation of PDGFRbeta pY-1021 and pY-740 data. Band densities for pY-1021 and pY-740 were normalized to cognate PDGFRbeta band densities, and data (mean ± S.E. from three independent experiments) were processed as in B.*, p < 0.05 compared with cognate cells expressing native GRK5 levels. D, WT and Y1009F PDGFRbeta cells express equivalent levels of GRK5. Lysates from control (40 µg of protein) and GRK5-overexpressing cells (10 µg of protein) were immunoblotted with A16/17 anti-GRK5 or nonimmune mouse IgG1 (non) (58).

 
If Shp2 is required for GRK5-promoted desensitization of the PDGFRbeta, then deficiency of Shp2 should diminish differences observed between GRK5-expressing and -null SMCs. To test this expectation, we reduced SMC Shp2 expression with RNA interference and assessed PDGF-induced phosphoinositide hydrolysis (Fig. 12). The reduction in Shp2 expression achieved with siRNA in these experiments was only ~40% (at least in part because of the long half-life of Shp2 (43)) (Fig. 12B). Nonetheless, by augmenting PDGF-induced phosphoinositide hydrolysis in WT SMCs, Shp2 knock-down did diminish the difference in PDGFRbeta signaling between WT and grk5-/- SMCs (Fig. 12C), and thereby attenuated GRK5-mediated PDGFRbeta desensitization. Thus, Shp2 does appear to be an effector of GRK5-promoted PDGFRbeta desensitization.

PDGFRbeta/Shp2 Association Is Augmented by the Activity of GRK5, but Not GRK2—To determine whether an Shp2-based mechanism for PDGFRbeta desensitization was specific for GRK5, we tested whether physiologic expression of GRK2 also augments recruitment of Shp2 to the PDGFRbeta. For this purpose, we used GRK2-null MEFs, stably transfected to express physiologic levels of (or no) GRK2 (8). We found that the association of the PDGFRbeta and Shp2 was indistinguishable in the absence and presence of GRK2 activity (Fig. 13A), despite the fact that GRK2 activity effected a ~25% reduction in overall PDGFRbeta tyrosyl phosphorylation and a ~50% reduction in PDGF-induced phosphoinositide hydrolysis (8) (data not shown). To compare GRK2 with GRK5 activity in the same cellular milieu, we overexpressed GRK2 or GRK5 in HEK cells expressing equivalent levels of PDGFRbetas. Although GRK5 enhanced PDGFRbeta/Shp2 association by 180 ± 60%, GRK2 reduced this association by 60 ± 20% (p < 0.05 for each) (Fig. 13B). In this same system, GRK2 overexpression diminished the association of the PDGFRbeta with the Na+/H+ exchanger regulatory factor, as we observed before (8); however, despite its effect on PDGFRbeta/Shp2 association, GRK5 overexpression had no effect on PDGFRbeta/Na+/H+ exchanger regulatory factor association (data not shown). Together, our results in SMCs, MEFs, and 293 cells suggest that GRK5- and GRK2-mediated PDGFRbeta desensitization result from discrete molecular mechanisms.


Figure 12
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  		FIGURE 12.
Reduction of Shp2 expression diminishes GRK5-mediated desensitization of the PDGFRbeta in SMCs. WT and grk5-/- (KO) SMCs were treated with siRNA that targeted either no known mRNA (control or CTL), or the mRNA for Shp2. Subsequently, SMCs were metabolically labeled with [3H]inositol and, 72 h after siRNA treatment, stimulated as in Fig. 6 or processed for IB. A, extracts (30 µg of protein) from SMCs treated with the indicated siRNA were subjected to SDS-PAGE and IB. Blots were probed serially for Shp2 and tubulin. Shown are the results of a single experiment, representative of three performed. Subsequent PDGFRbeta IB demonstrated that Shp2 siRNA did not affect PDGFRbeta expression (not shown). B, densitometry of Shp2 bands was normalized to corresponding tubulin bands on each blot, and these ratios were normalized to cognate ratios obtained from WT SMCs transfected with control siRNA, to obtain the "percentage of WT control." Shown are the mean ± S.E. of three experiments. *, p < 0.05 compared with WT control cells. C, inositol phosphates obtained from stimulated SMCs were divided by inositol phosphates obtained from corresponding unstimulated SMCs (-fold/basal); these ratios were normalized within each experiment to the cognate -fold/basal value obtained for PDGF-stimulated WT SMCs treated with control siRNA (6 ± 2-fold/basal), to obtain the "percentage of WT control." Plotted are means ± S.E. from six independent experiments (two with each of the three WT/KO SMC pairs). *, p < 0.05 compared with cognate control siRNA-treated SMCs. Basal inositol phosphate values were 5 ± 3, 5 ± 2, 7 ± 1, and 7 ± 2 (percentage of conversion units (24)) for WT and KO SMCs treated with control and Shp2 siRNA, respectively.

 


Figure 13
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  		FIGURE 13.
GRK2 activity fails to enhance Shp2/PDGFRbeta association. A, GRK2 KO MEFs stably transfected with vector (KO) or GRK2 plasmid expressing physiologic levels of GRK2 (WT) were processed as in Fig. 9 for co-IP of Shp2 with the PDGFRbeta. B, HEK 293 cells transiently transfected with the indicated plasmids were processed as in A, and IP blots were probed sequentially for Shp2 and PDGFRbeta. All transfected HEK cell lines expressed equivalent levels of Shp2 (data not shown). Results in A and B are from single experiments, representing at least three performed with independent cell lines.

 
GRK5 Potentiates PDGFRbeta-induced Activation of Src, but Not ERK—The association of Shp2 with the PDGFRbeta is believed to activate Shp2 (4). Consequently, since GRK5 activity in SMCs augments Shp2/PDGFRbeta association, we expected to observe not only reduced PDGFRbeta tyrosyl phosphorylation but also other evidence of enhanced PDGF-induced Shp2 activity. To test this expectation, we examined PDGF-promoted ERK1/2 and Src activation, which can be mediated by Shp2 (4, 44). Despite large differences in PDGFRbeta/Shp2 association (Fig. 9), GRK5-expressing and -null SMCs demonstrated equivalent activation of ERK-1 and -2 within 5 min of PDGF stimulation (data not shown). This finding is consonant with data from Shp2-deficient fibroblasts. Even the absence of Shp2 does not diminish PDGFRbeta-promoted ERK activation within 5 min of PDGF stimulation (44). However, GRK5-expressing SMCs demonstrated 1.9 ± 0.3-fold more Src activation than GRK5-null SMCs (Fig. 14, A and B), even though GRK5 activity did not affect PDGFRbeta autophosphorylation on its docking site for Src (Tyr579) (Fig. 14C). Thus, GRK5-mediated seryl phosphorylation of the PDGFRbeta desensitizes signaling selectively. Although PLC-{gamma}1 and perhaps other pathways promoting SMC migration and proliferation are desensitized, Src signaling is augmented. In this way, GRK5-mediated phosphorylation of the PDGFRbeta mirrors GRK-mediated phosphorylation of heptahelical receptors, a process which desensitizes signaling through G proteins but activates signaling through ERKs (11, 19, 20), and possibly Src (45, 46). A provisional model for GRK5-mediated regulation of the PDGFRbeta is presented in Fig. 15.


Figure 14
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  		FIGURE 14.
GRK5 activity augments PDGF-evoked activation of Src. A, quiescent GRK5-null and WT SMCs were stimulated (or not) with 2 nM PDGF-BB for 10 min (37 °C) and lysed; 20 µg of protein from each cell group underwent parallel SDS-PAGE and IB for either activated Src, phosphorylated on Tyr416 (pY-416), or total Src (bottom). B, Src pY-416 band density was normalized to cognate Src band density, and ratios were normalized to those obtained for PDGF-stimulated GRK5-null SMCs to obtain the "percentage of GRK5 KO." Shown are means ± S.E. from eight experiments performed with four paired KO and WT SMC lines. *, p < 0.05 compared with GRK5-null. C,WT and GRK5 KO SMCs were stimulated (or not) with PDGF-BB and subjected to PDGFRbeta IP, SDS-PAGE, and IB as in Fig. 9. Blots were probed serially for the PDGFRbeta and then the PDGFRbeta phosphorylated on Tyr579 (one of two Src docking sites (4)). Shown are the results of a single experiment, representative of three performed.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study demonstrates for the first time that the PDGFRbeta is phosphorylated and desensitized by GRK5, a widely expressed kinase previously known only to regulate a multitude of heptahelical receptors (11). Moreover, GRK5 mediates the preponderance of PDGF-induced seryl phosphorylation and desensitization of the PDGFRbeta in SMCs, and reduces overall PDGFRbeta tyrosyl phosphorylation in a manner that is highly site-specific. Whether assessed as second messenger production, migration, thymidine incorporation, or proliferation, PDGFRbeta-promoted SMC activity was reduced in a receptor-specific manner by GRK5 activity. Although GRK5-mediated PDGFRbeta desensitization critically involves the PDGFRbeta phosphatase Shp2, GRK2-mediated PDGFRbeta desensitization does not.


Figure 15
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  		FIGURE 15.
Proposed scheme for GRK5-mediated regulation of the PDGFRbeta. A, the agonist-activated, dimerized, and autophosphorylated PDGFRbeta is schematically depicted in a caveola, into which it appears to migrate after activation (51). The PDGFRbeta, which can activate heterotrimeric Gi (7), allosterically activates GRK5, which may be localized to caveolae by its binding to caveolins-1 (49). Once activated, the GRK5 phosphorylates the activated PDGFRbeta on seryl residues. B, by as yet unknown mechanisms, GRK5-mediated phosphorylation of PDGFRbeta seryl residues leads to enhanced phosphorylation of PDGFRbeta Tyr1009, the docking site for Shp2 on the PDGFRbeta; consequently, recruitment of Shp2 to the PDGFRbeta is enhanced. Shp2 then dephosphorylates selected phosphotyrosyl residues on the PDGFRbeta (like phospho-Tyr1021 and phospho-Tyr740 (Figs. 9, 10, 11)); the resulting decrease in PDGFRbeta activity is symbolized by removal of the asterisk from R*. C, because GRK5 activity on the PDGFRbeta augments Shp2/PDGFRbeta association and Shp2 activation without affecting the ability of PDGFRbeta to recruit Src (Fig. 14C), PDGF-induced Src activation is enhanced in GRK5-expressing SMCs. With increased levels of activated Shp2 recruited to the PDGFRbeta, there is greater Shp2-mediated dephosphorylation of Src at its (autoinhibitory) phospho-Tyr527, and consequently greater Src activation (4). Thus, overall, GRK5 activity desensitizes PDGFRbeta signaling through phospholipase C{gamma}-1 and phosphatidylinositol 3-kinase (but not ERK), and promotes PDGFRbeta signaling via Src. Activated molecules are indicated by an asterisk and/or by shading; pS, phosphoserine; pY, phosphotyrosine.

 
As a regulator of PDGFRbeta activity, GRK5 might seem unusual. With rare exception (47), substrates for GRK5 are all heptahelical receptors that activate heterotrimeric G proteins. However, we have shown that, like many heptahelical receptors, the PDGFRbeta itself activates G{alpha}i in SMC membranes, even in the absence of ATP (7) (an approach that precludes Gi-coupled sphingosine-1-phosphate receptor activation by PDGFRbeta-activated sphingosine kinase) (48). As a result, the PDGFRbeta cytoplasmic tail conformation that activates Gi might reasonably be expected to activate GRK5 allosterically, too. Moreover, the PDGFRbeta and GRK5 could be expected to share subcellular localization in quiescent cells. GRK5 resides primarily on membranes (11) and binds to caveolin-1 (49), whereas the PDGFRbeta resides in caveolae (50), at least transiently (51).

A role for GRK5 in regulating the PDGFRbeta might also seem improbable, however, from the perspective of studies that employed purified proteins to examine inhibition of GRK5 activity. Consequent to PDGFRbeta-mediated PLC-{gamma}1 activation, intracellular [Ca2+] rises, and protein kinase C isoforms are activated (4). In preparations with purified proteins, Ca2+/calmodulin binding and protein kinase C-mediated phosphorylation of GRK5 have been shown to inhibit the ability of GRK5 to associate with membrane-bound substrates (11). Moreover, whereas protein kinase C-mediated GRK2 phosphorylation relieves Ca2+/calmodulin-mediated inhibition of GRK2 (52), it actually inhibits GRK5 catalytic activity (11). These mechanisms may underlie the apparent inability of GRK5 to regulate angiotensin II AT1 receptor signaling in mouse myocardium (53). Nevertheless, in intact SMCs, our results indicate that despite possible attenuation by Ca2+/calmodulin- and protein kinase C-mediated inhibition, the net activity of physiologically expressed GRK5 is clearly sufficient to achieve agonist-dependent PDGFRbeta phosphorylation and desensitization.

By what mechanism might GRK5-mediated seryl phosphorylation of the PDGFRbeta enhance phosphorylation of the PDGFRbeta Tyr1009, and thereby PDGFRbeta/Shp2 association? Although the Ser/Thr kinase GRK5 could not plausibly phosphorylate PDGFRbeta Tyr1009 directly, GRK5-mediated Ser phosphorylation could enhance Tyr1009 phosphorylation indirectly. The PDGFRbeta sequence (54) surrounding Tyr1009 includes DTS1005SVLY1009, where Ser1005 and Ser1006 are highly plausible GRK phosphorylation sites (11). If GRK5 were to phosphorylate either Ser1005, Ser1006, or both, the resulting phosphoseryl residue(s) would increase the negative charge N-terminal to Tyr1009, and thereby possibly enhance phosphorylation of Tyr1009 by the PDGFRbeta Tyr kinase (55). (The mouse sequence is identical to human over a 19-amino acid stretch surrounding Tyr1009, but the mouse tyrosyl residue is numbered 1008.) In light of this proposed scheme, it is of interest to note that metabolically labeled endothelial cells phosphorylate PDGFRbetas on seryl residues distinct from (and C-terminal to) Ser1005 and Ser1006 (39). However, these endothelial cell data may not be relevant to cells (like SMCs) in which GRK5 is the dominant GRK. GRK2 appears to be the dominant GRK in endothelial cells (56), and we have shown that GRK2 phosphorylates the PDGFRbeta on Ser1104, perhaps among other sites (8). The PDGFRbeta residue(s) phosphorylated by GRK5 are as yet unidentified.

Although GRK5-mediated PDGFRbeta seryl phosphorylation reduces autophosphorylation at PDGFRbeta Tyr1021 (the docking site for PLC-{gamma}1 (4)) by ~9-fold (Fig. 9), the mechanism by which GRK5 reduces this site-specific Tyr phosphorylation remains somewhat uncertain. We have correlated reduction in PDGFRbeta Tyr1021 phosphorylation with the GRK5-mediated increase in PDGFRbeta/Shp2 association. However, the ability of Shp2 to dephosphorylate PDGFRbeta Tyr1021 is relatively poor in studies performed with purified proteins (57). Could this apparent site-specific relative "deficiency" in Shp2-mediated PDGFRbeta dephosphorylation result from the absence of key Shp2 or PDGFRbeta binding partners in the purified protein assay? Such a possibility is suggested by our data in SMCs. It remains to be determined whether accessory proteins facilitate direct Shp2-mediated dephosphorylation of PDGFRbeta Tyr1021 or whether Shp2 dephosphorylates PDGFRbeta Tyr1021 indirectly, through one or more of these accessory proteins.

The discovery that GRK5 regulates the PDGFRbeta in SMCs suggests that GRK5, like the PDGFRbeta itself (1, 3), may play a role in the pathogenesis of atherosclerosis. The up-regulation of GRK5 in SMC-like atheroma cells supports this concept (Fig. 2). From what we have learned with GRK5-deficient and WT SMCs in this study, we would expect that GRK5 activity should attenuate atherosclerosis. Whether this possibility obtains in vivo, of course, remains to be determined.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grants HL77185, HL63288, and HL73005 (to N. J. F.), HL64744 (to K. P.), and GM59989 and DA16347 (to R. T. P.) as well as an American Heart Association grant-in-aid (to N. J. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported by a Glenn/AFAR medical student scholarship. Back

2 Supported by a Eugene Stead medical student scholarship. Back

3 To whom correspondence may be addressed: Jefferson Medical College 1025 Walnut St., Rm. 311 Philadelphia, PA 19107. E-mail: karsten.peppel{at}jefferson.edu. 4 To whom correspondence may be addressed: Box 3187 Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-6873; Fax: 919-684-6870. E-mail: neil.freedman{at}duke.edu.

5 The abbreviations used are: PDGFRbeta, PDGF receptor-beta; PDGF, platelet-derived growth factor; SMC, vascular smooth muscle cell; GRK, heterotrimeric G protein-coupled receptor kinase; IP, immunoprecipitation or immunoprecipitate; IB, immunoblot(s), immunoblotting; KO, knockout; EGF, epidermal growth factor; PLC, phospholipase C; siRNA, small interfering RNA; MEF, mouse embryo fibroblast; WT, wild type; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; ERK, extracellular signal-regulated kinase. Back


   ACKNOWLEDGMENTS
 
We thank W. Darrell Capel for expert assistance in purifying GRK5.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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