* This work was supported in part by National Institutes of Health Grant HL16037 (to R. J. L.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ Recipient of a National Institutes of Health Clinical Investigator Development Award. § Supported by National Institutes of Health Medical Scientist Training Program Grant T32GM-07171. ¶ Present address: Neuroscience Research, Abbott Laboratories, D-4PM, AP10, 100 Abbott Park Rd., Abbott Park, IL 60064. 1 The abbreviations used are: LPAlysophosphatidic acidARadrenergic receptorPDGFplatelet-derived growth factorEGFepidermal growth factorPAGEpolyacrylamide gel electrophoresisGSTglutathione S-transferaseMAPmitogen-activated proteinPI3Kphosphatidylinositol 3-kinase.
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.
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 (
), have been shown to mediate Ras-dependent mitogenic signals. In COS-7 cells, Ras-dependent activation of mitogen-activated protein kinases via the α2A AR, M2 muscarinic acetylcholine, D2 dopamine, and A1 adenosine receptors is mediated largely by Gβγ subunits released from pertussis toxin-sensitive G proteins (
) 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 α-thrombin (
We have previously shown in transiently transfected COS-7 cells that pertussis toxin-sensitive G protein-coupled receptors mediate Gβγ 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 (
). Here, we examine the role of Src family nonreceptor tyrosine kinases in mediating Gβγ 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 Gi-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.
The cDNA encoding the α2A AR was cloned in our laboratory. The cDNAs encoding Gβ1 and Gγ2 were provided by M. Simon (California Institute of Technology, Pasadena, CA). The cDNA encoding human p60c-src was provided by D. Fujita (University of Calgary, Alberta, Canada), and the cDNA encoding p50csk was provided by H. Hanafusa (Rockefeller University, New York, NY). The constitutively activated Y530F p60c-src (TAC(Y) TTC(F)), in which the regulatory carboxyl-terminal tyrosine residue has been mutated, and catalytically inactive K298M p60c-src (AAA(K) ATG(M)) mutants were prepared as described (
). 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 μg/ml gentamicin at 37°C in a humidified 5% CO2 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 μg of DNA/100-mm dish plus 6 μl of LipofectAMINE reagent (Life Technologies, Inc.)/μg 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 Gβ1 and Gγ2 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 μM NaVO4, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotonin, 10 μg/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 μl 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 p185neu 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), p185neu 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 anti-human 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 (
) 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 (
). For the detection of c-Src SH2 or SH3 domain-binding 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 μg/ml GST Sepharose for 1 h and incubated with 6 μg/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 phosphate-buffered saline, denatured in Laemmli sample buffer, and resolved by SDS-PAGE. Coprecipitated tyrosine phosphoproteins and EGF receptor were detected by protein immunoblotting as described.
Gi-coupled Receptors and Gβγ Subunits Mediate Formation of a Mitogenic Signaling Complex Containing EGF Receptor, Shc, and Grb2
As shown in 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 (
). 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 stimulation induces association of these proteins. As shown in Fig. 1B, similar increases in Shc phosphorylation and Shc-p180 association result from transient expression of Gβ1γ2 subunits or stimulation of endogenous LPA or transiently expressed α2A AR receptors. Stimulation of endogenous EGF receptors or transient overexpression of a constitutively activated mutant human c-Src (Y530F p60c-src; Refs.
) 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), Gi-coupled receptor-mediated but not EGF receptor-mediated Shc phosphorylation and Shc-p180 association are pertussis toxin-sensitive in these cells.
Because G protein-coupled receptor-mediated tyrosine phosphorylation of PDGF receptor (
) has been reported, we performed immunoblots for EGF receptor and p185neu on Shc immunoprecipitates from nondenatured cell lysates following stimulation of LPA, α2A 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 (
). As shown in Fig. 2A, EGF receptor is not detectable in Shc immunoprecipitates from nonstimulated cells, but stimulation of either Gi-coupled receptor results in Shc-EGF receptor coprecipitation. In contrast, Shc-p185neu complexes are present in nonstimulated cells and do not increase detectably following LPA or α2A AR receptor stimulation. As expected, EGF stimulation results in both Shc-EGF receptor and Shc-p185neu association, which may reflect heterodimerization and transphosphorylation of the two related receptor tyrosine kinases (
). As shown in Fig. 2B, the tyrosine phosphorylation states of Shc, EGF receptor, and p185neu, 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, α2A AR, or EGF receptor stimulation. P185neu exhibits significant basal tyrosine phosphorylation, consistent with the detection of Shc-p185neu complexes in nonstimulated cells, which detectably increases only following EGF receptor stimulation.
To confirm that Shc, Grb2, and EGF receptor directly associate following Gi-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. Gi-coupled receptor-induced association of Src family nonreceptor tyrosine kinases with Shc has been reported (
). As shown, c-Src can also be detected in EGF receptor immunoprecipitates from LPA- or EGF-stimulated cell lysates, suggesting that activation of the Gi-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 Gi-coupled Receptor and Gβγ Subunit-mediated Tyrosine Phosphorylation of EGF Receptor and Shc
Gi-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 (
). As shown in Fig. 3 (A and B), antiphosphotyrosine immunoblots of EGF receptor immunoprecipitated from EGF-stimulated cells, from cells transiently expressing Y530F p60c-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 p60c-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 α2A 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 Gi-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 p60c-src; Ref.
) 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 (
). As shown in Fig. 4 (A and B), coexpression of either Csk or K298M p60c-src markedly inhibits Gβ1γ2 subunit-, α2A AR-, and LPA receptor-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.
To determine whether Gi-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 (
) 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 LPA-stimulated 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 (Tyr891) is mediated by the c-Src kinase rather than the intrinsic receptor tyrosine kinase (
). To determine if phosphorylation of this site is mediated by endogenous Src kinases in the intact cell following Gi-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 Gi-coupled receptor stimulation results in both c-Src mediated phosphorylation of the EGF receptor and SH2 domain-dependent c-Src-EGF receptor complex formation.
Several lines of evidence suggest that Src family kinases play a key role in the transduction of mitogenic signals by G protein-coupled receptors. Pertussis toxin-sensitive activation of the Src family kinases Src, Fyn, Yes, and Lyn (
) in various cell types has been reported, and inhibition of Src family kinases has been shown to block G protein-coupled receptor-mediated Ras and phospholipase C-γ1 activation, MAP kinase activation, and c-fos transcription (
). Our data demonstrate that in COS-7 cells, Gi-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 Gi-coupled receptor effects can be mimicked by cellular overexpression of Gβγ subunits, suggesting that the process is Gβγ 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 Gi-coupled receptor stimulation.
Fig. 6 depicts a model of Gβγ subunit-mediated, Ras-dependent MAP kinase activation that is consistent with these data. Gβγ subunit-dependent activation of endogenous Src family nonreceptor tyrosine kinases is an early event following Gi-coupled receptor stimulation (
). 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 phosphorylation cascade leading to MAP kinase activation.
Depending upon cell type, the G protein-coupled receptors for angiotensin II, LPA, and α-thrombin have been shown to stimulate ligand-independent tyrosine phosphorylation of PDGF receptor (
). 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 Gi-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 Gi-coupled receptor-mediated tyrosine phosphorylation is unclear. Daub et al. (
) 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 α-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 Gi-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 Gi-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-mediated phosphorylation and EGF receptor autophosphorylation, we have been unable to detect increased EGF receptor autophosphorylation following either overexpression of Y530F p60c-src or Gi-coupled receptor stimulation.
The mechanism whereby effectors of activated G protein-coupled 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. Gβγ subunit-mediated PI3K activity has been described in neutrophils and platelets (
). We have previously reported that Gi-coupled receptor- and Gβγ 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 (
) is wortmannin-insensitive, suggesting that the PI3K-dependent 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 (
), may provide an explanation for this phenomenon.
Interaction between Src kinases and novel Gβγ subunit-regulated nonreceptor tyrosine kinases might also contribute to the regulation of Src kinase activity. In neuronal cells, Gq-coupled receptors have been shown to stimulate the Ca2+ and protein kinase C dependent tyrosine protein kinase, PYK2 (
). 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 Gβγ subunits (
). 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 Gi-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.
We thank D. Addison and M. Holben for excellent secretarial assistance.