©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Convergence of Angiotensin II and Platelet-derived Growth Factor Receptor Signaling Cascades in Vascular Smooth Muscle Cells (*)

Daniel A. Linseman , Christopher W. Benjamin , David A. Jones (§)

From the (1) From Cardiovascular Pharmacology, Upjohn Laboratories, Kalamazoo, Michigan 49001

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Signaling cascades elicited by angiotensin II resemble those characteristic of growth factor stimulation. In this report, we demonstrate that angiotensin II converges with platelet-derived growth factor (PDGF) -receptor signaling cascades, independent of PDGF. Stimulation of smooth muscle cells with angiotensin II resulted in tyrosine phosphorylation on Shc proteins and subsequent complex formation between Shc and growth factor receptor binding protein-2 (GRB2). A 180-kDa protein co-precipitating with ShcGRB2 complexes also demonstrated increased phosphorylation in response to angiotensin II. Immunoblot analyses and proteolytic digests failed to distinguish this 180-kDa protein from authentic PDGF -receptors. Corresponding with Shc and PDGF receptor phosphorylation induced by angiotensin II was the recruitment and phosphorylation of c-Src. Autocrine release of platelet-derived growth factor failed to account for Shc complex formation at the PDGF receptor following angiotensin II treatment, and a specific angiotensin II type I receptor antagonist, losartan, abolished the response. These results support a novel model for cross-talk between the G-protein-linked angiotensin II receptor and the PDGF receptor tyrosine kinase in vascular smooth muscle cells. Communication with the PDGF receptor may account for the ability of angiotensin II to elicit responses typical of growth factor signal transduction.


INTRODUCTION

Angiotensin II elicits a hypertrophic phenotype in cultures of smooth muscle cells and may be a critical mediator of vascular hypertrophy and neointimal hyperplasia (1, 2, 3, 4) . Recent cloning of the vascular type I angiotensin II receptor provided evidence for including it in the G-protein-coupled receptor superfamily (5, 6) . Interestingly, certain aspects of signal transduction characteristic of angiotensin II stimulation resemble those evoked by platelet-derived growth factor. Activation of phospholipase C- (7) , tyrosine kinases (8, 9) , mitogen-activated protein kinases (10, 11) , and expression of early growth response genes (12) exemplify phenomena common to angiotensin II and platelet-derived growth factor signal transduction. Since the angiotensin II type I receptor is G-protein linked, the mechanism by which it activates processes characteristic of growth factor signaling is unclear.

Induction of mitogen-activated protein kinase activity by platelet-derived and other growth factors appears subsequent to activation of p21. Proteins upstream of p21 have recently been identified that sense growth factor receptor activation and stimulate guanine nucleotide exchange on p21(13-18). Growth factor receptor binding protein-2 (GRB2)() senses activated receptors by binding specific, phosphorylated tyrosine residues via its SH2 domain. In addition to binding to phosphorylated tyrosine kinase receptors, GRB2 also recognizes phosphorylated tyrosine residues on Shc proteins. A number of growth factors, including PDGF and angiotensin II, can stimulate tyrosine phosphorylation on Shc (19-23). Shc can bind directly to PDGF -receptors in stimulated cells and may, through recruitment of GRB2, relay receptor activation to downstream signaling proteins (20, 21) . Salcini et al. (24) have recently demonstrated the requirement for ShcGRB2 complexes in the transformation of fibroblasts by overexpression of Shc proteins. The SH3 domains of GRB2 facilitate stable complex formation with the nucleotide exchange factor SOS. This ShcGRB2SOS complex, thereby, transmits receptor ligand binding to p21(25) .

In an effort to understand smooth muscle cell responses to vascular growth factors, we examined signaling proteins upstream of p21 in cultures of isolated, rat aortic smooth muscle cells (RASMC) exposed to angiotensin II. In this report, we demonstrate that angiotensin II induces phosphorylation on Shc and recruitment of GRB2. Furthermore, ShcGRB2 immune complexes contained phosphorylated PDGF -receptors and c-Src. Assembly of this complex in response to angiotensin II was independent of autocrine PDGF release. These observations illustrate an unexpected link between angiotensin II type I receptor and PDGF -receptor signal transduction in vascular smooth muscle cells and suggest a mechanism by which angiotensin II elicits signaling events characteristic of mitogenic stimuli.


EXPERIMENTAL PROCEDURES

Materials

Recombinant human BB-PDGF and antibodies specific for phosphotyrosine, Shc, GRB2, PDGF -receptors and PDGF were from Upstate Biotechnology Incorporated, Transduction Laboratories or Santa Cruz Biotechnology. A radioimmunoassay for human PDGF was obtained fom Amersham Corp. Assays were performed on media as directed and values were corrected for rat PDGF cross-reactivity. Cell culture reagents and Protein G-agarose were from Life Technologies, Inc. Secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA). ECL reagents were from Amersham.

Cell Culture

Rat aortic smooth muscle cells were isolated as described (26) and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 15% fetal calf serum. Cells were split at confluence and fed every third day. Cultures were used between passages 5 and 10. A10 smooth muscle cells were obtained from the American Type Culture Collection and cultured identically to the RASMC.

Immunoprecipitations

Cells were grown to confluence and serum starved in DMEM lacking fetal calf serum for 18 h. Following stimulation, cell medium was removed, and cells were rinsed twice with ice-cold phosphate-buffered saline (Ca, Mg-free). Cells were scraped into lysis buffer A (20 mM HEPES, pH 7.4, 1% Triton X-100, 50 mM sodium chloride, 1 mM EGTA, 5 mM -glycerophosphate, 30 mM sodium pyrophosphate, 100 µM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) (27) . Cell debris was removed by centrifugation at 12,000 g for 10 min. Supernatants containing approximately 350 µg of protein were transferred to new tubes containing 2 µg of indicated antibody and incubated at 4 °C for 2.5 h with mixing. Protein G-agarose was added and lysates incubated for an additional 30 min. Precipitates were washed three times in ice-cold lysis buffer A and resuspended finally in SDS-PAGE (28) sample buffer. Complexes were boiled for 5 min and electrophoresed through 10% SDS-PAGE gels. Proteins were transferred to PVDF membranes (Immobilon, Millipore Corp., Bedford, MA) and processed for immunoblot analysis.

Immunoblot Analysis

Nonspecific binding sites were blocked in PBS containing 0.1% Tween 20 (PBS-T) and 1% bovine serum albumin for 1 h at 20 °C. Primary antibodies were diluted in blocking solution and incubated with the membranes for 1 h at room temperature. Excess primary antibody was removed by washing the membranes four times in PBS-T. The blots were incubated with appropriate secondary antibodies in PBS-T containing 5% milk diluent (Kirkegaard and Perry Laboratories, Inc.) for 1 h. Membranes were washed as before and processed for ECL. In certain experiments the filters were reprobed after stripping in 0.1 M Tris-HCl, pH 8.0, 2% SDS, and 100 mM -mercaptoethanol for 30 min at 52 °C. Filters were rinsed briefly in PBS-T and processed as above with a different primary antibody.

V8 Digests

Serum-starved RASMC cultures were labeled with 5 mCi/ml [P]orthophosphate in phosphate-free DMEM. Cells were washed five times with ice-cold PBS to remove unincorporated label, then stimulated with PDGF at 30 ng/ml or angiotensin II at 1 µM for 15 min. Shc or PDGF receptors were precipitated as described above and complexes resolved by SDS-PAGE. Following exposure to film, gel fragments containing the labeled 180-kDa protein were excised and transferred to a fresh gel. SDS-PAGE sample buffer containing 2 µg of purified V8 protease was added to indicated lanes and samples electrophoresed into a 4% stacking gel (29). Electrophoresis was stopped for 1 h to allow digestion of the proteins. Protein fragments were resolved through a 10% gel and exposed to film for autoradiography.

Media Transfer Experiments

To examine PDGF activity, media from RASMC exposed to angiotensin II at various concentrations was transferred onto serum-starved A10 cells and incubated at 37 °C for 10 min. Cells were lysed and 200 µg of protein analyzed for phosphotyrosine incorporation by immunoblotting. In certain experiments, media from RASMC treated with angiotensin II was saturated with an antibody capable of neutralizing PDGF for 15 min at 37 °C. Neutralized media was transferred onto fresh RASMC and incubated for 10 min prior to analysis for Shc and receptor phosphorylation. To examine autocrine release of factors other than PDGF, media from RASMC treated with 10 nM angiotensin II was saturated with 10 µM losartan and transferred onto fresh RASMC that had been preincubated with 10 µM losartan. Cells were incubated for 15 min at 37 °C and Shc protein complexes analyzed as above.


RESULTS

We first measured tyrosine phosphorylation on Shc and subsequent recruitment of GRB2 in response to angiotensin II. Shc was immunoprecipitated from quiescent cultures of RASMC exposed to various concentrations of angiotensin II for 10 min. Analysis of tyrosine-phosphorylated proteins in Shc immunoprecipitates revealed phosphorylation of 46-, 52-, and 66-kDa Shc isoforms (Fig. 1A). Further analysis illustrated the recruitment of GRB2 to Shc immune complexes in parallel with Shc phosphorylation (Fig. 1B). GRB2 association with Shc correlated quantitatively with angiotensin II. Complex formation was detectable at 1 nM angiotensin II and maximal by 100 nM. In similar experiments, complex formation was detected within 5 min and plateaued by 10 min following angiotensin II exposure. The complex was still prominent at 120 min (data not shown). A similar pattern of Shc phosphorylation (Fig. 1A) and GRB2 association (Fig. 1B) was observed following treatment with PDGF. Analysis of the blots with an antibody directed against Shc confirmed the identity of these phosphorylated bands and assured equivalent precipitation efficiency (Fig. 1C).


Figure 1: Angiotensin II stimulates Shc phosphorylation and association with GRB2 and additional proteins in vascular smooth muscle cells. Quiescent, confluent monolayers of RASMC were stimulated with various concentrations of angiotensin II (lanes 1-12) or PDGF (lane 13) for 10 min. A, Shc immunoprecipitates were analyzed for phosphotyrosine containing proteins and GRB2 (B). The blot in A was stripped and reprobed with an antibody specific for Shc (C).



Additional tyrosine-phosphorylated proteins appeared in Shc immune complexes in response to angiotensin II with a profile strikingly similar to that observed from cells treated with PDGF (Fig. 1A). Two such proteins at 60 and 180 kDa are similar in size to the protein tyrosine kinase, Src, and the PDGF -receptor, respectively. Tyrosine phosphorylation on these proteins correlated with ShcGRB2 complex formation both temporally and with respect to angiotensin II concentration. This warranted additional investigation because Src and PDGF -receptors are putative activators of Shc (20, 30) . Analysis of the 180 kDa region with an antibody specific for the PDGF -receptor demonstrated cross-reactivity with this protein and suggested that the receptor, Shc, and GRB2 occur in a single complex in response to angiotensin II (Fig. 2A). In agreement with the presence of the PDGF receptor, a c-Src antibody recognized the phosphorylated 60-kDa protein that co-precipitated with Shc in angiotensin II- and PDGF-stimulated cells. This demonstrated recruitment of c-Src to the complex in response to angiotensin II or PDGF (Fig. 2B).


Figure 2: Association of PDGF receptors and c-Src with Shc immune complexes isolated from cells treated with angiotensin II or PDGF. Serum-deprived RASMC were stimulated with vehicle, 1 µM angiotensin II, or 30 ng PDGF/ml for 10 min. Shc immunoprecipitates were analyzed with antibodies directed against the PDGF -receptor (A) and c-Src (B).



To explore the possibility that PDGF -receptors contribute to angiotensin II signaling, we next examined receptor phosphorylation in immune complexes precipitated by two additional antibodies. Fig. 3A (upper exposure) illustrates that antibodies directed against Shc, GRB2, or phosphotyrosine co-precipitated the 180-kDa protein and confirmed that tyrosine phosphorylation of this protein was enhanced by angiotensin II or PDGF. Again, identity of the 180-kDa protein as the PDGF -receptor was suggested by cross-reactivity with a PDGF -receptor-specific antibody (Fig. 3A, lower exposure). Phosphorylation of the 180-kDa protein was further demonstrated by incorporation of radiolabeled phosphate. PDGF -receptors and Shc were immunoprecipitated from angiotensin II- or PDGF-stimulated RASMC that had been labeled with [P]orthophosphate. Label incorporation into precipitated PDGF -receptors and the 180-kDa protein increased approximately 2-fold in PDGF and angiotensin II-stimulated cells and confirmed agonist-dependent phosphorylation (Fig. 3B).


Figure 3: Phosphorylation of a 180-kDa protein co-precipitating with Shc from angiotensin II- or PDGF-stimulated cells. Serum-deprived, confluent monolayers of RASMC were stimulated with 1 µM angiotensin II or 30 ng/ml PDGF. A. tyrosine-phosphorylated PDGF receptors were co-precipitated from RASMC treated with angiotensin II or PDGF using antibodies directed against Shc (lanes 1-3), GRB2 (lanes 4-6), and phosphotyrosine (lanes 7-9). The membranes were stripped and analyzed with an antibody specific for the PDGF -receptor (lower panel). B, RASMC were incubated in phosphate-free DMEM supplemented with 5 mCi/ml [P]orthophosphate. Cultures were then stimulated with vehicle (lane 1) PDGF, (lanes 2 and 3), or angiotensin II (lanes 4 and 5). PDGF -receptor (lanes 1, 2, and 4) or Shc (lanes 3 and 5) complexes were immunoprecipitated. Precipitated proteins were fractionated by SDS-PAGE and exposed for autoradiography.



Successful incorporation of radiolabel into the 180-kDa protein following angiotensin II stimulation allowed comparison of proteolytic maps of the candidate band to authentic PDGF -receptor digestions. Radiolabeled protein co-migrating with authentic PDGF -receptor (Fig. 4A) was excised, transferred to a new gel and digested with V8 protease. Fig. 4B typifies the digestion patterns of authentic PDGF -receptors and the 180-kDa protein. Numerous protease-dependent fragments characterized authentic PDGF -receptor digests and mirrored the pattern liberated by digestion of the 180-kDa protein. Proteolytic maps in conjunction with antibody cross-reactivity confirmed the 180-kDa protein present in Shc immune precipitations as the PDGF -receptor.


Figure 4: Angiotensin II stimulates the incorporation of radiolabeled phosphate into PDGF receptors in vascular smooth muscle cells. RASMC were incubated in phosphate-free DMEM supplemented with 5 mCi/ml [P]orthophosphate. A, cultures were then stimulated with PDGF (lanes 1-3) or angiotensin II (lane 4). PDGF -receptor (lanes 1 and 2) or Shc (lanes 3 and 4) complexes were immunoprecipitated. Precipitated proteins were fractionated by SDS-PAGE and exposed for autoradiography. B, the 180-kDa proteins labeled in A were excised, transferred to a fresh gel, and treated with buffer (lane 1) or buffer containing 2 µg of V8 protease (lanes 2-4). Lanes 1 and 2 illustrate patterns of undigested and digested PDGF - receptors, respectively. Digestion patterns of the labeled 180-kDa protein that co-precipitates with Shc in response to PDGF or angiotensin II are presented in lanes 3 and 4, respectively.



We next examined whether the link between angiotensin II type I receptor stimulation and assembly of Shc signaling complexes at the PDGF receptor was induced by autocrine release of PDGF. We have observed previously that the rat A10 smooth muscle cell line is non-responsive to angiotensin II with respect to eliciting tyrosine phosphorylation, but fully responsive to PDGF in this regard. This cell line, therefore, provided a sensitive system for detection of PDGF release by RASMC responding to angiotensin II. Stimulation of PDGF receptor tyrosine kinase activity in A10 cell cultures is evident by immunoblotting following exposure to as little as 100 pg of PDGF/ml. When media from RASMC conditioned with angiotensin II was transferred onto A10 cell cultures, however, tyrosine phosphorylation on PDGF receptors was undetectable (Fig. 5A). Fig. 5B provides additional evidence for the lack of PDGF activity in media from RASMC conditioned with angiotensin II. In this approach, media from RASMC treated with angiotensin II was saturated with an antibody that neutralizes PDGF and then transferred onto fresh cultures. Control experiments demonstrated that the antibody could neutralize at least 10 ng of PDGF/ml. Consistent with the A10 cell assay, activity in the media from angiotensin II-stimulated cells was fully effective in eliciting Shc and receptor phosphorylation. Finally, media from cells exposed to angiotensin II contained undetectable levels of PDGF as determined by a sensitive radioimmunoassay (data not shown).


Figure 5: Autocrine release of PDGF fails to account for angiotensin II stimulation of PDGF receptor signaling cascades. A, media from angiotensin II conditioned RASMC was transferred to cultures of rat A10 vascular smooth muscle cells. PDGF receptor phosphorylation in cell lysates was determined by immunoblot analysis. B, Media from angiotensin II-conditioned RASMC was saturated with a PDGF-neutralizing antibody and transferred onto fresh cells. Shc complexes were isolated and analyzed as in Fig. 1.



Although PDGF release failed to account for activation of the signaling pathway in response to angiotensin II, this did not rule out autocrine release of a factor different from PDGF. To examine this possibility, we tested whether media transferred from RASMC stimulated with 10 nM angiotensin II could activate the signaling cascade in the presence of losartan. Fig. 6A illustrates that losartan pretreatment of the cells abolished complex formation following direct addition of 10 nM angiotensin II. In contrast, angiotensin II-conditioned media contained an activity that was not fully blocked by the angiotensin II receptor antagonist (Fig. 6B), suggesting liberation of a factor other than PDGF.


Figure 6: Losartan inhibits Shc phosphorylation following direct addition of angiotensin II to RASMC, but fails to inhibit activity in angiotensin II-conditioned media. A, RASMC were pretreated with or without 10 µM losartan then stimulated with 10 nM angiotensin II. B, media from primary RASMC exposed to angiotensin II was transferred to fresh cells, pretreated with 10 µM losartan. In each experiment, Shc was immunoprecipitated and proteins analyzed for phosphotyrosine.




DISCUSSION

Angiotensin II elicits signaling responses that resemble PDGF signaling in vascular smooth muscle cells. Our work substantiates these observations by demonstrating that angiotensin II stimulates the association of Shc and GRB2 in rat aortic vascular smooth muscle. In addition to this phenomenon, we present a novel link between the angiotensin II G-protein-coupled receptor and PDGF receptor signaling. In this model, the PDGF receptor serves as a docking site for known upstream activators of p21-ras in smooth muscle cells responding to angiotensin II. Evidence supporting this hypothesis include: (i) Shc immune complexes from angiotensin II and PDGF-treated cells contained a 180-kDa tyrosine phosphorylated protein that co-migrates with the PDGF -receptor; (ii) an antibody specific for the PDGF -receptor cross-reacted with the 180-kDa protein that co-precipitates with Shc in response to angiotensin II; (iii) proteolytic maps of the 180-kDa protein are indistinguishable from authentic PDGF receptors; and (iv) autocrine release of PDGF failed to account for Shc complex formation in response to angiotensin II.

Tyrosine phosphorylation on Shc and subsequent complex formation with GRB2 appear central to upstream signaling strategies elicited by a wide variety of growth factors and cytokines. ShcGRB2 complex formation is critical to activation of p21 in cells responding to insulin (24) , EGF (23) , and in the transformation of cells by over expression of Shc (24) . Our observation that Shc and GRB2 associate in response to angiotensin II implicates Shc and GRB2 as important mediators in the angiotensin II signaling response. This is in agreement with work by Schorb et al.(22) demonstrating the phosphorylation of Shc in cardiac fibroblasts following exposure to angiotensin II. Although they did not evaluate GRB2 recruitment to Shc, it is likely that both cardiac fibroblasts and vascular smooth muscle cells utilize this pathway to transduce angiotensin II signals. Since ShcGRB2 complex formation contributes to PDGF signal transduction in vascular smooth muscle cells, activation of these molecules may provide a mechanism through which angiotensin II activates mitogen-activated protein kinases.

Our results suggest that the similarity between the upstream signaling events elicited by angiotensin II and PDGF extend beyond ShcGRB2 complex formation. To this end, we have detected PDGF receptors and c-Src in Shc immune complexes isolated from angiotensin II-treated vascular smooth muscle cells (21) . Both of these proteins co-precipitate with Shc from cells responding to PDGF. The appearance of PDGF receptors in Shc immune complexes is consistent with recent results demonstrating the association of Shc and PDGF receptors in unstimulated vascular smooth muscle cells. Support for this hypothesis is provided by Fig. 2. Identification of the 180-kDa protein as the PDGF receptor by antibody cross-reactivity also shows little change in the amount of receptor co-precipitated following angiotensin II stimulation. The recruitment of GRB2 and c-Src to the complex, however, is clearly increased and agonist dependent. Fig. 3A adds further support to this hypothesis. Precipitations performed with antibodies specific for GRB2 and phosphotyrosine contain increased amounts of PDGF receptor in response to angiotensin II and PDGF that correspond with phosphorylation on the receptor. In contrast, precipitation with antibodies to Shc showed constant receptor levels even though phosphorylation on the receptor increased. Since Shc can associate with the receptor in unstimulated cells and angiotensin II causes tyrosine phosphorylation on Shc, one might expect the complex to assemble in response to angiotensin II by using the PDGF receptor as a docking site. Whether the receptor is activated in this complex, as might by suggested by enhanced tyrosine phosphorylation, requires further investigation.

The appearance of c-Src in Shc immune complexes isolated from angiotensin II-stimulated cells is notable in that Src is a putative activator of Shc. This was established by work demonstrating that v-Src transformed rat-1 cells contain constitutively phosphorylated Shc (30) . Further c-Src can associate directly with activated PDGF receptors via its SH2 domain (29) , and c-Src has been detected in Shc immune complexes isolated from PDGF-treated A10 smooth muscle cells (21) . Activation of Src by angiotensin II may represent the key convergence point between the angiotensin II and PDGF signaling pathways. Catalytically active Src, in close association with Shc and the receptor, may account for the increased tyrosine phosphorylation observed on Shc and the PDGF receptor in response to angiotensin II. It is interesting that a pivotal role for Src family tyrosine kinases has been suggested in smooth muscle contractile responses elicited by angiotensin II and growth factors (31) .

Angiotensin II increases PDGF A chain, transforming growth factor-1 and basic fibroblast growth factor expression in vascular smooth muscle cells (32) . In view of this, we evaluated whether autocrine release of PDGF accounted for PDGF receptor involvement in angiotensin II signal transduction. Since maximal stimulation of ShcGRB2 complex formation in response to angiotensin II corresponded with PDGF at 10 ng/ml, we anticipated that release of PDGF in response to angiotensin II would approach a similar value. Several assays capable of detecting PDGF at concentrations much lower that 10 ng/ml ruled out secretion of PDGF as the mechanism for activation of the signaling pathway by angiotensin II. First, media from cells exposed to angiotensin II contained undetectable levels of PDGF as determined by a sensitive radioimmunoassay (data not shown). This agrees with previous reports that adult rat aortic smooth muscle cells synthesize and release little PDGF (33) . Second, media from cultures of RASMC exposed to various concentrations of angiotensin II contained no activity capable of inducing PDGF receptor phosphorylation in the angiotensin II-insensitive rat A10 vascular smooth muscle cell line. PDGF concentrations as low as 100 pg/ml stimulated detectable phosphorylation on PDGF receptors in A10 cell cultures and verified this system as a sensitive bioassay for PDGF. Lack of activity in the angiotensin II-conditioned media, therefore, indicated a PDGF concentration below 100 pg/ml. Finally, a PDGF neutralizing antibody failed to block activation of the signaling pathway when added to angiotensin II-conditioned media. Taken together, these data eliminate PDGF release as the mechanism for PDGF -receptor phosphorylation in response to angiotensin II.

Media transfer experiments did, however, suggest liberation of a factor distinct from PDGF. The angiotensin II type I receptor antagonist, losartan, fully blocked activation of the pathway when angiotensin II was added directly to cells. In contrast, losartan only partially blocked the activity present in angiotensin II-conditioned media. The identity of this factor requires further investigation, but it may fall in the lipid category given the profile of rapid production and the inability of smooth muscle cells to rapidly secrete proteins. In this regard, angiotensin II stimulates phospholipases A, C, and D activity in vascular smooth muscle cultures with a time course similar to activation of the Shc/GRB2 pathway (34, 35, 36) .

We have demonstrated a novel mechanism wherein angiotensin II receptor signal transduction links with PDGF -receptor signaling cascades and results in activation of proteins upstream of p21. The direct involvement of the PDGF receptor in this process presents a novel paradigm for cross-talk between G-protein-linked and growth factor receptor signaling pathways. The activation of this pathway by either PDGF or angiotensin II is paradoxical in that angiotensin II does not elicit a mitogenic response typical of PDGF. This suggests that additional, PDGF-specific pathways are required for full mitogenic potential or that divergence of the pathways, downstream of Shc/GRB2, facilitates the specific biological responses characteristic of these two growth factors.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: The Upjohn Company, 7243-209-317, 301 Henrietta St., Kalamazoo, MI 49001. Tel.: 616-385-7433; Fax: 616-385-5262.

The abbreviations used are: GRB2, growth factor receptor binding protein-2; PDGF, platelet-derived growth factor; SH2, src homology-2; SH3, src homology-3; RASMC, rat aortic smooth muscle cells; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline.


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