Gβγ Subunits Mediate Src-dependent Phosphorylation of the Epidermal Growth Factor Receptor

In many cells, stimulation of mitogen-activated protein kinases by both receptor tyrosine kinases and receptors that couple to pertussis toxin-sensitive heterotrimeric G proteins proceed via convergent signaling pathways. Both signals are sensitive to inhibitors of tyrosine protein kinases and require Ras activation via phosphotyrosine-dependent recruitment of Ras guanine nucleotide exchange factors. Receptor tyrosine kinase stimulation mediates ligand-induced receptor autophosphorylation, which creates the initial binding sites for SH2 domain-containing docking proteins. However, the mechanism whereby G protein-coupled receptors mediate the phosphotyrosine-dependent assembly of a mitogenic signaling complex is poorly understood. We have studied the role of Src family nonreceptor tyrosine kinases in G protein-coupled receptor-mediated tyrosine phosphorylation in a transiently transfected COS-7 cell system. Stimulation of Gi-coupled lysophosphatidic acid and α2A adrenergic receptors or overexpression of Gβ1γ2 subunits leads to tyrosine phosphorylation of the Shc adapter protein, which then associates with tyrosine phosphoproteins of approximately 130 and 180 kDa, as well as Grb2. The 180-kDa Shc-associated tyrosine phosphoprotein band contains both epidermal growth factor (EGF) receptor and p185neu. 3-5-fold increases in EGF receptor but not p185neu tyrosine phosphorylation occur following Gi-coupled receptor stimulation. Inhibition of endogenous Src family kinase activity by cellular expression of a dominant negative kinase-inactive mutant of c-Src inhibits Gβ1γ2 subunit-mediated and Gi-coupled receptor-mediated phosphorylation of both EGF receptor and Shc. Expression of Csk, which inactivates Src family kinases by phosphorylating the regulatory carboxyl-terminal tyrosine residue, has the same effect. The Gi-coupled receptor-mediated increase in EGF receptor phosphorylation does not reflect increased EGF receptor autophosphorylation, assayed using an autophosphorylation-specific EGF receptor monoclonal antibody. Lysophosphatidic acid stimulates binding of EGF receptor to a GST fusion protein containing the c-Src SH2 domain, and this too is blocked by Csk expression. These data suggest that Gβγ subunit-mediated activation of Src family nonreceptor tyrosine kinases can account for the Gi-coupled receptor-mediated tyrosine phosphorylation events that direct recruitment of the Shc and Grb2 adapter proteins to the membrane.

monoclonal antibody. Lysophosphatidic acid stimulates binding of EGF receptor to a GST fusion protein containing the c-Src SH2 domain, and this too is blocked by Csk expression. These data suggest that G␤␥ subunitmediated activation of Src family nonreceptor tyrosine kinases can account for the G i -coupled receptor-mediated tyrosine phosphorylation events that direct recruitment of the Shc and Grb2 adapter proteins to the membrane.
The low molecular weight G protein Ras functions as a signaling intermediate in many pathways involved in the regulation of cellular mitogenesis and differentiation. Ras activation by growth factor receptors that possess intrinsic tyrosine kinase activity follows ligand-induced phosphorylation of specific docking sites on the receptor itself or adapter proteins, such as Shc and insulin receptor substrate-1, which serve to recruit Ras guanine nucleotide exchange factors to the plasma membrane (1,2). Recently, several receptors that couple to heterotrimeric G proteins, including the lysophosphatidic acid (LPA) 1 (3,4), ␣-thrombin (5), angiotensin II (6,7), ␣2A adrenergic (AR) (8,9), M2 muscarinic acetylcholine, D2 dopamine, and A1 adenosine receptors (10), have been shown to mediate Ras-dependent mitogenic signals. In COS-7 cells, Ras-dependent activation of mitogen-activated protein kinases via the ␣2A AR, M2 muscarinic acetylcholine, D2 dopamine, and A1 adenosine receptors is mediated largely by G␤␥ subunits released from pertussis toxin-sensitive G proteins (8,9). These G␤␥ subunitmediated signals are sensitive to inhibitors of tyrosine protein kinases (8), associated with increased tyrosine protein phosphorylation, and dependent upon recruitment of Ras guanine nucleotide exchange factors to the membrane (9), indicating that the pathway converges with the receptor tyrosine kinase pathway at an early point.
The mechanism whereby G protein-coupled receptors stimulate tyrosine protein phosphorylation is poorly understood. The observation that the receptors for PDGF (16) and EGF (17) undergo tyrosine phosphorylation following G protein-coupled receptor activation has led to the hypothesis that the intrinsic tyrosine kinase activity of these receptors becomes activated by an unknown mechanism. G protein-coupled receptor-mediated activation of nonreceptor tyrosine kinases has also been reported. Recently, activation of Src family kinases by the ␣-thrombin (18), LPA (19), angiotensin II (20), N-formyl methionyl peptide chemoattractant (21), ␣2A AR (18,19), and M1 muscarinic acetylcholine (18) receptors has been reported. Furthermore, inhibition of Src family kinases has been shown to inhibit angiotensin II-stimulated Ras (22) and phospholipase C-␥1 (23) activation in rat aortic smooth muscle cells, LPA and ␣2A AR-stimulated MAP kinase activation in COS-7 cells (19), M1 and M2 muscarinic acetylcholine-stimulated MAP kinase activation in avian B cells (24), and endothelin-1-stimulated transcriptional activation in rat glomerular mesangial cells (25).
We have previously shown in transiently transfected COS-7 cells that pertussis toxin-sensitive G protein-coupled receptors mediate G␤␥ subunit-dependent activation of c-Src and that inhibition of Src family kinases by cellular expression of Csk antagonizes G protein-coupled receptor-mediated MAP kinase activation (19). Here, we examine the role of Src family nonreceptor tyrosine kinases in mediating G␤␥ subunit-dependent tyrosine phosphorylation of receptor tyrosine kinases and Shc. Our data suggest that activation of Src family kinases by G protein-coupled receptors can account for the G i -coupled receptor-mediated tyrosine phosphorylation events that direct recruitment of the Shc and Grb2 adapter proteins to the membrane using the EGF receptor as a scaffold.

EXPERIMENTAL PROCEDURES
DNA Constructs-The cDNA encoding the ␣2A AR was cloned in our laboratory. The cDNAs encoding G␤1 and G␥2 were provided by M. Simon (California Institute of Technology, Pasadena, CA). The cDNA encoding human p60 c-src was provided by D. Fujita (University of Calgary, Alberta, Canada), and the cDNA encoding p50 csk was provided by H. Hanafusa (Rockefeller University, New York, NY). The constitutively activated Y530F p60 c-src (TAC(Y) 3 TTC(F)), in which the regulatory carboxyl-terminal tyrosine residue has been mutated, and catalytically inactive K298M p60 c-src (AAA(K) 3 ATG(M)) mutants were prepared as described (19). All cDNAs were subcloned into pRK5 or pcDNA eukaryotic expression vectors for transient transfection.
Cell Culture and Transfection-COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 g/ml gentamicin at 37°C in a humidified 5% CO 2 atmosphere. Transfections were performed on 80 -90% confluent monolayers in 100-mm dishes. For transient transfection, cells were incubated at 37°C in 4 ml serum-free Dulbecco's modified Eagle's medium containing 6 -10 g of DNA/100-mm dish plus 6 l of LipofectAMINE reagent (Life Technologies, Inc.)/g of DNA. Empty pRK5 vector was added to transfections as needed to keep the total mass of DNA added per dish constant within an experiment. After 3-5 h of exposure to the transfection medium, monolayers were refed with growth medium and incubated overnight. Transfected monolayers were serum starved in Dulbecco's modified Eagle's medium supplemented with 0.1% bovine serum albumin and 10 mM Hepes, pH 7.4, for 16 -20 h prior to stimulation. Assays were performed 48 h after transfection. LipofectAMINE transfection of COS-7 cells consistently resulted in transfection efficiencies of greater than 80% (data not shown). Transient expression of G␤1 and G␥2 subunits, Csk, and mutant c-Src proteins was confirmed by immunoblotting of transfected whole cell lysates using commercially available antisera.
Immunoprecipitation and Immunoblotting-Stimulations were carried out at 37°C in serum-free medium as described in the figure legends. After stimulation, monolayers were washed once with ice-cold phosphate-buffered saline and lysed in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.25% sodium deoxycholate, 0.1% Nonidet P-40, 100 M NaVO 4 , 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotonin, 10 g/ml leupeptin) for immunoprecipitation under nondenaturing conditions or RIPA/SDS buffer (RIPA buffer containing 0.1% SDS) for immunoprecipitation under denaturing conditions. Cell lysates were sonicated briefly, clarified by centrifugation, and diluted to a protein concentration of 2 mg/ml. Immunoprecipitations from 1 ml of lysate were performed using the appropriate primary antibody plus 50 l of a 50% slurry of protein G plus/protein A agarose (Oncogene Science) agitated for 1 h at 4°C. Immune complexes were washed twice with ice-cold RIPA buffer and once with phosphate-buffered saline, denatured in Laemmli sample buffer, and resolved by SDS-polyacrylamide gel electrophoresis (PAGE). Shc immunoprecipitations were performed using rabbit polyclonal anti-Shc antibody (Transduction Laboratories). EGF receptor was immunoprecipitated using monoclonal anti-EGF receptor antibody (Transduction Laboratories), and p185 neu was immunoprecipitated using rabbit polyclonal anti-HER2 (Santa Cruz Biotechnology).
Tyrosine phosphorylation or the presence of coprecipitated proteins was detected by protein immunoblotting. Phosphotyrosine was detected using a 1:1000 dilution of horseradish peroxidase-conjugated anti-phosphotyrosine monoclonal antibody (Transduction Laboratories). Shc protein was detected using a 1:1000 dilution of rabbit polyclonal anti-Shc IgG (Transduction Laboratories), p185 neu was detected using a 1:1000 dilution of rabbit polyclonal anti-HER2 IgG (Santa Cruz Biotechnology), and Grb2 was detected using a 1:1000 dilution of rabbit polyclonal anti-Grb2 IgG (Santa Cruz Biotechnology), each with horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham Corp.) as secondary antibody. C-Src was detected using 1:500 dilution of mouse monoclonal anti-c-Src antibody 327 with horseradish peroxidase-conjugated donkey anti-mouse IgG (Jackson Laboratories) as secondary antibody. EGF receptor was detected using 1:1000 dilution of sheep antihuman EGF receptor IgG with horseradish peroxidase-conjugated donkey anti-sheep IgG (Jackson Laboratories) as secondary antibody. Immunoblots for autophosphorylated EGF receptor were performed using mouse monoclonal anti-activated EGF receptor IgG (26) with horseradish peroxidase-conjugated donkey anti-mouse IgG (Jackson Laboratories) as secondary antibody. Immune complexes on nitrocellulose were visualized by enzyme-linked chemiluminescence (Amersham Corp.) and quantified by scanning laser densitometry.
GST Fusion Proteins Containing the c-Src SH2 and SH3 Domains-GST fusion proteins containing the human c-Src SH2 (amino acids 144 -249), SH3 (amino acids 87-143), or SH2 and SH3 (amino acids 87-249) domains were prepared as GST Sepharose conjugates as described previously (27). For the detection of c-Src SH2 or SH3 domainbinding proteins, appropriately transfected and stimulated COS-7 cells were lysed in RIPA/SDS buffer containing 5 mM dithiothreitol, sonicated, clarified by centrifugation, precleared with 6 g/ml GST Sepharose for 1 h and incubated with 6 g/ml of the GST fusion protein Sepharose for 3 h at 4°C. After incubation, fusion protein complexes were washed twice with ice-cold RIPA buffer and once with phosphatebuffered saline, denatured in Laemmli sample buffer, and resolved by SDS-PAGE. Coprecipitated tyrosine phosphoproteins and EGF receptor were detected by protein immunoblotting as described. Fig. 1A, stimulation of endogenous LPA receptors in COS-7 cells leads to a rapid 3-5-fold increase in tyrosine phosphorylation of each of the three Shc isoforms. The phosphorylation is maximal within 2 min of stimulation and declines slowly thereafter (9,19). Under nondenaturing conditions, Shc coprecipitates with two major tyrosine phosphoprotein bands of approximately 130 and 180 kDa and with the adapter protein Grb2. The association of Shc with the p130 and p180 phosphoproteins is modulated with a time course that parallels the time course of Shc phosphorylation and Shc-Grb2 complex formation, suggesting that LPA stimu-lation induces association of these proteins. As shown in Fig.  1B, similar increases in Shc phosphorylation and Shc-p180 association result from transient expression of G␤1␥2 subunits or stimulation of endogenous LPA or transiently expressed ␣2A AR receptors. Stimulation of endogenous EGF receptors or transient overexpression of a constitutively activated mutant human c-Src (Y530F p60 c-src ; Refs. 28 -30) has similar, although more robust effects, indicating that activation of either the receptor tyrosine kinase or nonreceptor Src kinase can mimic the G protein-mediated effects. As shown in Fig. 1 (C and D), G i -coupled receptor-mediated but not EGF receptormediated Shc phosphorylation and Shc-p180 association are pertussis toxin-sensitive in these cells.

G i -coupled Receptors and G␤␥ Subunits Mediate Formation of a Mitogenic Signaling Complex Containing EGF Receptor, Shc, and Grb2-As shown in
Because G protein-coupled receptor-mediated tyrosine phosphorylation of PDGF receptor (16), EGF receptor, and p185 neu (17) has been reported, we performed immunoblots for EGF receptor and p185 neu on Shc immunoprecipitates from nondenatured cell lysates following stimulation of LPA, ␣2A AR, or EGF receptors to determine whether these receptor tyrosine kinases are present in the Shc-associated p180 phosphotyrosine band. COS-7 cells lack detectable expression of PDGF receptor (31). As shown in Fig. 2A, EGF receptor is not detectable in Shc immunoprecipitates from nonstimulated cells, but stimulation of either G i -coupled receptor results in Shc-EGF receptor coprecipitation. In contrast, Shc-p185 neu complexes are present in nonstimulated cells and do not increase detectably following LPA or ␣2A AR receptor stimulation. As expected, EGF stimulation results in both Shc-EGF receptor and Shc-p185 neu association, which may reflect heterodimerization and transphosphorylation of the two related receptor tyrosine kinases (32). As shown in Fig. 2B, the tyrosine phosphorylation states of Shc, EGF receptor, and p185 neu , determined following direct immunoprecipitation of each protein, reflect the changes in Shc-receptor tyrosine kinase association. Shc and EGF receptor phosphorylation is increased following LPA, ␣2A AR, or EGF receptor stimulation. P185 neu exhibits significant basal tyrosine phosphorylation, consistent with the detection of Shc-p185 neu complexes in nonstimulated cells, which detectably increases only following EGF receptor stimulation.
To confirm that Shc, Grb2, and EGF receptor directly associate following G i -coupled receptor stimulation, EGF receptor immunoprecipitates were assayed for the presence of Shc and Grb2 following LPA stimulation. As shown in Fig. 2C, stimulation with either LPA or EGF resulted in the association of Shc and Grb2 with EGF receptor. G i -coupled receptor-induced association of Src family nonreceptor tyrosine kinases with Shc has been reported (19,21). As shown, c-Src can also be detected in EGF receptor immunoprecipitates from LPA-or EGF-stim-

FIG. 1. G i -coupled receptor-and G␤␥ subunit-stimulated association of Shc with p130 and p180 tyrosine phosphoproteins and Grb2 in COS-7 cells.
A, time course of LPA-stimulated Shc tyrosine phosphorylation and association with p130 and p180 tyrosine phosphoproteins and Grb2. Serum-starved cells were stimulated for the indicated times with LPA (10 M). Immunoprecipitates of Shc from nondenatured RIPA buffer lysates were resolved by SDS-PAGE and immunoblotted with anti-phosphotyrosine (upper panel) or anti-Grb2 (lower panel) as described. The position of tyrosine phosphorylated Shc isoforms, Shc-associated p130 and p180 phosphoproteins, and Grb2 are as indicated. B, effect of ␣2A AR, LPA, or EGF receptor stimulation and G␤␥ subunit or Y530F p60 c-src expression on Shc tyrosine phosphorylation and association with p130 and p180 tyrosine phosphoproteins and Grb2. Cells were transiently transfected with empty pRK5 vector, G␤1 and G␥2, ␣2A AR, or Y530F p60 c-src as described. Duplicate plates of serum-starved cells were stimulated for 2 min with the ␣2A AR agonist UK14304 (UK, 10 M), LPA (10 M), or EGF (10 ng/ml) as indicated. Immunoprecipitates of Shc from nondenatured RIPA buffer lysates were resolved by SDS-PAGE and immunoblotted with anti-phosphotyrosine (upper panel) or anti-Grb2 (lower panel) as described. C, pertussis toxin-sensitivity of ␣2A AR-and LPA-stimulated Shc, p130, and p180 tyrosine phosphorylation. Cells were transiently transfected with empty pRK5 vector or ␣2A AR and serum-starved overnight in the presence or the absence of pertussis toxin (Ptx, 100 ng/ml) prior to stimulation with LPA, UK14304, or EGF as indicated. Immunoprecipitates of Shc from nondenatured RIPA buffer lysates were resolved by SDS-PAGE and immunoblotted with antiphosphotyrosine. D, quantitation of the effects of pertussis toxin treatment on ␣2A AR-and LPA-stimulated tyrosine phosphorylation of p52 shc . UK14304-, LPA-, and EGF-stimulated p52shc phosphorylation was determined as described. Autoradiographs were quantified by scanning laser densitometry, and the data were presented as fold increase over nonstimulated or empty pRK5 vector transfected controls. The data shown represent the means Ϯ S.E. for three separate experiments. NS, nonstimulated. ulated cell lysates, suggesting that activation of the G i -coupled receptor results in association of EGF receptor, c-Src, Shc, and Grb2 in a multiprotein complex.
Src Family Kinase Activity Is Required for Both G i -coupled Receptor and G␤␥ Subunit-mediated Tyrosine Phosphorylation of EGF Receptor and Shc-G i -coupled receptor-mediated increases in EGF receptor phosphotyrosine might result from ligand-independent activation of the receptor tyrosine kinase, phosphorylation by an activated nonreceptor tyrosine kinase, or inhibition of a phosphotyrosine phosphatase. To distinguish between these alternative mechanisms, we employed a monoclonal anti-EGF receptor antibody specific for autophosphorylated EGF receptor. This antibody selectively recognizes activated EGF receptor via an epitope distal to amino acid 1052 (26), which is distinct from the major in vitro c-Src phosphorylation sites (33). As shown in Fig. 3 (A and B), antiphosphotyrosine immunoblots of EGF receptor immunoprecipitated from EGF-stimulated cells, from cells transiently expressing Y530F p60 c-src , and from cells in which phosphotyrosine phosphatase activity is inhibited by incubation with sodium orthovanadate, each exhibit increased total receptor phosphorylation. Identical immunoblots probed with the anti-activated EGF receptor antibody give increased signals from EGF-stimulated and sodium orthovanadate-treated cells but not from Y530F p60 c-src -transfected cells. Thus, the anti-activated EGF receptor antibody is able to discriminate between increased autophosphorylation resulting from activation of the intrinsic tyrosine kinase activity of the EGF receptor or inhibition of a phosphotyrosine phosphatase versus phosphorylation of the EGF receptor mediated by the c-Src nonreceptor tyrosine kinase. As shown, this antibody does not detect EGF receptor autophosphorylation following stimulation of LPA or ␣2A AR receptors, despite a 3-5-fold increase in total EGF receptor phosphotyrosine, suggesting that the increase in receptor tyrosine phosphorylation does not reflect activation of the intrinsic tyrosine kinase.
Because expression of activated mutant c-Src is sufficient to mediate EGF receptor phosphorylation in the absence of ligand, we tested the hypothesis that G i -coupled receptor-mediated activation of Src family kinases can account for the observed tyrosine phosphorylation of Shc and EGF receptor. To inhibit endogenous Src family kinases, cells were transiently transfected with cDNA encoding either Csk or a kinase-inactive dominant negative mutant c-Src (K298M p60 c-src ; Ref. 34). Csk is a cytoplasmic tyrosine protein kinase (35) that inactivates Src family kinases by phosphorylating the regulatory carboxyl-terminal tyrosine residue. Csk overexpression has been shown to impair G protein-coupled receptor-mediated MAP kinase activation in COS-7 cells (19) and c-fos transcription in rat glomerular mesangial cells (25). As shown in Fig. 4  (A and B), coexpression of either Csk or K298M p60 c-src markedly inhibits G␤1␥2 subunit-, ␣2A AR-, and LPA receptor-

FIG. 2. Correlation between G i -coupled receptor-stimulated EGF receptor and p185 neu tyrosine phosphorylation with Shc complex formation.
A, coprecipitation of endogenous EGF receptor and p185 neu with Shc following G i -coupled receptor or EGF receptor stimulation. Serum-starved cells, transiently transfected with empty pRK5 vector or ␣2A AR, were stimulated for 2 min with UK14304 (UK), LPA, or EGF as indicated. Immunoprecipitates of Shc from nondenatured RIPA buffer lysates were resolved by SDS-PAGE and immunoblotted with anti-EGF receptor (top panel), anti-p185 neu (HER2, center panel), or anti-Shc (bottom panel) as described. The position of EGF receptor, p185 neu , and Shc isoforms are as indicated. B, tyrosine phosphorylation of endogenous EGF receptor and p185 neu following G i -coupled receptor or EGF receptor stimulation. Serum-starved cells, transiently transfected with empty pRK5 vector or ␣2A AR, were stimulated with UK14304, LPA, or EGF as indicated. Immunoprecipitates of EGF receptor (top panel), p185 neu (center panel), or Shc (bottom panel) from RIPA/SDS buffer lysates were resolved by SDS-PAGE and immunoblotted with anti-phosphotyrosine as described. C, coprecipitation of Grb2, Shc, and c-Src with endogenous EGF receptor following LPA receptor or EGF receptor stimulation. Serum-starved cells were stimulated for 2 min with LPA or EGF as indicated. Immunoprecipitates of EGF receptor from nondenatured RIPA buffer lysates were resolved by SDS-PAGE and immunoblotted with anti-EGF receptor, anti-Grb2, anti-Shc, or anti-c-Src as described. The position of EGF receptor, Grb2, Shc isoforms, and c-Src are as indicated. NS, nonstimulated. mediated tyrosine phosphorylation of both Shc and EGF receptor. EGF-stimulated Shc and EGF receptor phosphorylation were less dramatically effected. Shc and EGF receptor phosphorylation mediated by Y530F, which is not a substrate for Csk, is not significantly attenuated by Csk overexpression.
c-Src SH2 Domain GST Fusion Proteins Bind EGF Receptor Following G i -coupled Receptor Activation-To determine whether G i -coupled receptor-stimulated EGF receptor phosphorylation can induce binding of Src kinases directly to the EGF receptor, GST-fusion proteins containing either the c-Src SH2, SH3, or SH2-SH3 domains (27) were assayed for the ability to precipitate phosphorylated EGF receptor from lysates of LPA-stimulated cells. As shown in Fig. 5A, the c-Src SH2 and SH2-SH3 domain GST fusion proteins but not the c-Src SH3 domain GST fusion protein precipitate a 180-kDa tyrosine phosphoprotein band that increases in intensity following LPA or EGF receptor stimulation. Immunoblots of EGF receptor in c-Src GST SH2 domain precipitates, shown in Fig. 5B, reveal increased association of EGF receptor with the c-Src SH2 domain following LPA or EGF stimulation, suggesting that LPAstimulated tyrosine phosphorylation of the EGF receptor is responsible for recognition of EGF receptor by the c-Src SH2 domain.
In vitro mapping of phosphorylation sites on the EGF receptor has suggested that phosphorylation of the putative c-Src SH2 domain recognition site (Tyr 891 ) is mediated by the c-Src kinase rather than the intrinsic receptor tyrosine kinase (33).
To determine if phosphorylation of this site is mediated by endogenous Src kinases in the intact cell following G i -coupled receptor stimulation, we tested the effect of Csk overexpression on LPA-stimulated phosphorylation of the c-Src SH2 domain binding site. As shown in Fig. 5C, the ability of the c-Src SH2 domain GST fusion protein to precipitate EGF receptor following LPA stimulation is markedly attenuated in Csk-expressing cells. Because Src kinases also mediate phosphorylation of this site following receptor tyrosine kinase activation, EGF receptor precipitation by the c-Src SH2 domain GST fusion protein following stimulation with EGF is also significantly attenuated. These data suggest that G i -coupled receptor stimulation results in both c-Src mediated phosphorylation of the EGF receptor and SH2 domain-dependent c-Src-EGF receptor complex formation. DISCUSSION Several lines of evidence suggest that Src family kinases play a key role in the transduction of mitogenic signals by G proteincoupled receptors. Pertussis toxin-sensitive activation of the Src family kinases Src, Fyn, Yes, and Lyn (18 -21) in various cell types has been reported, and inhibition of Src family kinases has been shown to block G protein-coupled receptormediated Ras and phospholipase C-␥1 activation, MAP kinase activation, and c-fos transcription (19,(22)(23)(24). Our data demonstrate that in COS-7 cells, G i -coupled receptor-stimulated tyrosine phosphorylation of the EGF receptor results in formation of a complex between the membrane-associated EGF receptor and the cytosolic adapter proteins Shc and Grb2, thus providing a scaffold for the assembly of a mitogenic signaling complex. The G i -coupled receptor effects can be mimicked by cellular overexpression of G␤␥ subunits, suggesting that the process is G␤␥ subunit-mediated. Because inhibition of endogenous Src kinases blocks both G protein-coupled receptor-mediated EGF receptor phosphorylation and binding of the EGF receptor to the c-Src SH2 domain, the data also suggest that Src family kinases directly associate with and phosphorylate the EGF receptor following G i -coupled receptor stimulation. Fig. 6 depicts a model of G␤␥ subunit-mediated, Ras-dependent MAP kinase activation that is consistent with these data. G␤␥ subunit-dependent activation of endogenous Src family nonreceptor tyrosine kinases is an early event following G icoupled receptor stimulation (19). Once activated, the Src kinases mediate phosphorylation of several intracellular targets, including receptor tyrosine kinases, adapter proteins such as Shc and insulin receptor substrate-1, and possibly cytoskeletally associated Src substrates such as focal adhesion kinase and paxillin. Once phosphorylated, membrane-associated proteins such as the receptor tyrosine kinases and focal adhesion kinase would provide docking sites for the SH2 domains of the Shc and Grb2 adapter molecules, resulting in the recruitment of Ras guanine nucleotide exchange factors, and potentially of other components of the mitogenic signaling complex, to the plasma membrane. The ensuing activation of Ras would recruit the Raf kinase to the membrane and initiate the phosphoryla- FIG. 3. Discrimination between Src kinase-mediated EGF receptor phosphorylation and EGF receptor autophosphorylation using anti-activated EGF receptor antibody. A, comparison of anti-phosphotyrosine immunoblots of endogenous EGF receptor with anti-activated EGF receptor immunoblots following G i -coupled receptor and EGF receptor stimulation, expression of Y530F, and inhibition of phosphotyrosine phosphatase activity. Serum-starved cells, transiently transfected with empty pRK5 vector, ␣2A AR, or Y530F p60 c-src were stimulated for 2 min with UK14304 (UK), LPA, or EGF or incubated for 20 min in the presence of sodium orthovanadate (VO 4 Ϫ3 , 10 M) as indicated. Duplicate immunoprecipitates of EGF receptor from RIPA/ SDS buffer lysates were resolved by SDS-PAGE and immunoblotted with either anti-phosphotyrosine (upper panel) or anti-activated EGF receptor antibody (lower panel) as described. B, quantitation of antiphosphotyrosine immunoblots of EGF receptor and anti-activated EGF receptor immunoblots. UK14304-, LPA-, EGF-, sodium orthovanadate-, and Y530F p60 c-src -stimulated EGF receptor total tyrosine phosphorylation and autophosphorylation were determined as described. Autoradiographs were quantified by scanning laser densitometry, and the data were presented as fold increase over nonstimulated or empty pRK5 vector transfected controls. The data shown represent the means Ϯ S.E. for three separate experiments. NS, nonstimulated. tion cascade leading to MAP kinase activation.
Depending upon cell type, the G protein-coupled receptors for angiotensin II, LPA, and ␣-thrombin have been shown to stimulate ligand-independent tyrosine phosphorylation of PDGF receptor (16), insulin-like growth factor-1 receptor ␤

FIG. 4. Effect of Csk and K298M p60 c-src expression on G i -coupled receptor-mediated Shc and EGF receptor tyrosine phosphorylation.
A, Immunoblots of Shc phosphotyrosine following ␣2A AR, LPA or EGF receptor stimulation and G␤␥ subunit or Y530F p60 c-src expression. Cells were transiently cotransfected with empty vector (Control) or expression plasmid encoding Csk or K298M p60 c-src , plus empty pRK5 vector, G␤1 and G␥2, ␣2A AR, or Y530F p60 c-src . Serum-starved cells were stimulated for 2 min with UK14304 (UK), LPA, or EGF as indicated, and immunoprecipitates of Shc from RIPA/ SDS buffer lysates were immunoblotted with anti-phosphotyrosine as described. The position of tyrosine phosphorylated Shc isoforms are as indicated. B, immunoblots of EGF receptor phosphotyrosine following ␣2A AR, LPA, or EGF receptor stimulation and G␤␥ subunit or Y530F p60 c-src expression. Serum-starved, transiently cotransfected cells were stimulated as described and immunoprecipitates of EGF receptor from RIPA/SDS buffer lysates were immunoblotted with anti-phosphotyrosine as described. The position of tyrosine phosphorylated EGF receptor is as indicated. C, quantitation of the effects of Csk and K298M p60 c-src coexpression on G␤␥ subunit-, ␣2A AR-, LPA-, EGF-, and Y530F p60 c-src -stimulated Shc and EGF receptor tyrosine phosphorylation. Shc and EGF receptor phosphotyrosine were determined as described following ␣2A AR, LPA, or EGF receptor stimulation and G␤␥ subunit or Y530F p60 c-src expression. Autoradiographs were quantified by scanning laser densitometry, and the data were presented as fold increase over nonstimulated or empty pRK5 vector transfected controls. The data shown represent the means Ϯ S.E. for three to five separate experiments. NS, nonstimulated. subunit (14), EGF receptor, and p185 neu (17). The finding that several receptor tyrosine kinases undergo G protein-coupled receptor-mediated phosphorylation suggests the existence of a common mechanism that is not receptor tyrosine kinase-specific, such as activation of a nonreceptor tyrosine kinase or inhibition of a phosphotyrosine phosphatase. Our data, demonstrating inhibition of G i -coupled receptor-mediated tyrosine phosphorylation of the EGF receptor by specific inhibitors of Src family kinases, support the hypothesis that activation of Src kinases can account for the observed receptor tyrosine kinase phosphorylation.
The role of the intrinsic tyrosine kinase activity of receptor tyrosine kinases in G i -coupled receptor-mediated tyrosine phosphorylation is unclear. Daub et al. (17) have reported that inhibition of EGF receptor function in Rat1 cells, by either an EGF receptor-selective tyrphostin, AG1478, or expression of a dominant negative mutant EGF receptor, blocks endothelin-1, LPA, and ␣-thrombin receptor-mediated EGF receptor/HER2 phosphorylation and MAP kinase activation. They conclude that a ligand-independent transactivation of the EGF receptor/ HER2 tyrosine kinase is responsible for G protein-coupled receptor-mediated tyrosine phosphorylation. Our data suggest that activation of Src family nonreceptor tyrosine kinases by G i -coupled receptors can account for tyrosine phosphorylation of both the EGF receptor and the Shc adapter protein in COS-7 cells. The finding that inhibition of endogenous Src kinase activity blocks G i -coupled receptor-mediated EGF receptor phosphorylation suggests that Src kinase activation precedes receptor tyrosine kinase phosphorylation but does not preclude the possibility that Src-mediated phosphorylation modulates the activity of the receptor tyrosine kinase. However, using a monoclonal antibody that can discriminate between c-Src-me-diated phosphorylation and EGF receptor autophosphorylation, we have been unable to detect increased EGF receptor autophosphorylation following either overexpression of Y530F p60 c-src or G i -coupled receptor stimulation.
The mechanism whereby effectors of activated G proteincoupled receptors stimulate Src family kinases is unknown. Stimulation of phosphatidylinositol 3-kinase activity may play a role in Ras-dependent MAP kinase activation in some cells. G␤␥ subunit-mediated PI3K activity has been described in neutrophils and platelets (36,37), a G␤␥ subunit-regulated isoform of p110 PI3K has been cloned, and G␤␥ subunits may contribute to the regulation of the conventional p85/p110 PI3K (38). We have previously reported that G i -coupled receptor-and G␤␥ subunit-mediated MAP kinase activation in COS-7 and CHO cells is sensitive to the PI3K inhibitors wortmannin and LY294002 and to expression of a dominant negative form of the p85 regulatory subunit of PI3K (39). Interestingly, MAP kinase activation by transiently expressed Y530F p60 c-src , 2 , mSos, and constitutively activated mutants of Ras and MAP kinase/erk kinase (39) is wortmannin-insensitive, suggesting that the PI3Kdependent step in the pathway may lie upstream of Src kinase activation. The recent report that the c-Src SH2 domain can bind with high affinity to phosphatidylinositol 3,4,5-trisphosphate, the product of PI3K (40), may provide an explanation for this phenomenon.
Interaction between Src kinases and novel G␤␥ subunitregulated nonreceptor tyrosine kinases might also contribute to the regulation of Src kinase activity. In neuronal cells, G qcoupled receptors have been shown to stimulate the Ca 2ϩ and protein kinase C dependent tyrosine protein kinase, PYK2 (41). PYK2 is a member of the focal adhesion kinase family of integrin receptor-associated tyrosine kinases and like p125 FAK (42) can complex with activated c-Src upon stimulation (43). However, phospholipase C activation and Ca 2ϩ mobilization are apparently unable to account for G protein-coupled receptormediated tyrosine phosphorylation in many noneuronal cells (4,44,45). Bruton's tyrosine kinase (Btk) and Tsk, two members of a family of pleckstrin homology domain-containing tyrosine protein kinases that includes Btk, Itk, Tsk and Tec A, are reportedly regulated by G␤␥ subunits (46). In hematopoietic cells, Btk interacts with the Src family kinases Fyn, Lyn, and Hck (47), and Src-Btk interaction is associated with Btk autoactivation (48). This is unlikely to be a general mechanism for G protein-coupled receptor regulation of Src kinases, however, because the pleckstrin homology domain-containing tyrosine kinases appear to have limited tissue distribution and are not known to be involved in the regulation of Ras.
The data presented in this report suggest that both Src family kinases and receptor tyrosine kinases play central roles in directing the assembly of membrane-associated mitogenic signaling complexes in response to G i -coupled receptor activation in some cells. An understanding of the mechanisms whereby G protein-coupled receptors regulate tyrosine protein phosphorylation and of the basis for cross-talk between G protein-coupled receptor and receptor tyrosine kinase signaling pathways may ultimately provide strategies for selective activation or inhibition of cellular proliferation.
FIG. 6. Model of G i -coupled receptor-mediated, Ras-dependent MAP kinase activation in COS-7 cells. Activation of Src family kinases following release of G␤␥ subunits from pertussis toxin-sensitive heterotrimeric G proteins precedes tyrosine phosphorylation of several putative scaffolding molecules, such as receptor tyrosine kinases (RTK) and focal adhesion kinase, leading to the phosphotyroine-dependent, SH2 domain-mediated recruitment of Ras guanine nucleotide exchange factor (mSos) to the plasma membrane. The subsequent activation of Ras initiates the Raf, MAP kinase/erk kinase, MAP kinase phosphorylation cascade that leads to MAP kinase (ERK 1/2) activation.