Pleiotropic Coupling of G Protein-coupled Receptors to the Mitogen-activated Protein Kinase Cascade

G protein-coupled receptors (GPCRs) initiate Ras-dependent activation of the Erk 1/2 mitogen-activated protein kinase cascade by stimulating recruitment of Ras guanine nucleotide exchange factors to the plasma membrane. Both integrin-based focal adhesion complexes and receptor tyrosine kinases have been proposed as scaffolds upon which the GPCR-induced Ras activation complex may assemble. Using specific inhibitors of focal adhesion complex assembly and receptor tyrosine kinase activation, we have determined the relative contribution of each to activation of the Erk 1/2 cascade following stimulation of endogenous GPCRs in three different cell types. The tetrapeptide RGDS, which inhibits integrin dimerization, and cytochalasin D, which depolymerizes the actin cytoskeleton, disrupt the assembly of focal adhesions. In PC12 rat pheochromocytoma cells, both agents block lysophosphatidic acid (LPA)- and bradykinin-stimulated Erk 1/2 phosphorylation, suggesting that intact focal adhesion complexes are required for GPCR-induced mitogen-activated protein kinase activation in these cells. In Rat 1 fibroblasts, Erk 1/2 activation via LPA and thrombin receptors is completely insensitive to both agents. Conversely, the epidermal growth factor receptor-specific tyrphostin AG1478 inhibits GPCR-mediated Erk 1/2 activation in Rat 1 cells but has no effect in PC12 cells. In HEK-293 human embryonic kidney cells, LPA and thrombin receptor-mediated Erk 1/2 activation is partially sensitive to both the RGDS peptide and tyrphostin AG1478, suggesting that both focal adhesion and receptor tyrosine kinase scaffolds are employed in these cells. The dependence of GPCR-mediated Erk 1/2 activation on intact focal adhesions correlates with expression of the calcium-regulated focal adhesion kinase, Pyk2. In all three cell types, GPCR-stimulated Erk 1/2 activation is significantly inhibited by the Src kinase inhibitors, herbimycin A and 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-d-3,4-pyrimidine (PP1), suggesting that Src family nonreceptor tyrosine kinases represent a point of convergence for signals originating from either scaffold.

Many GPCRs 1 initiate Ras-dependent activation of the Erk 1/2 MAP kinase cascade by inducing the tyrosine phosphorylation of proteins that serve as scaffolds for the plasma membrane recruitment of Ras guanine nucleotide exchange factors.
Receptor stimulation results in a rapid increase in the tyrosine phosphorylation of docking proteins, such as Shc (1,2) and Gab1 (3), followed by the Grb2-mediated recruitment of the Ras guanine nucleotide exchange factor, mSos, to the plasma membrane (4). These tyrosine phosphorylation events are sensitive to the inhibition of Src family nonreceptor tyrosine kinases in many cell types (2,3,5).
Although the requirement for tyrosine kinases in GPCRmediated Erk 1/2 activation has been well documented, the proximal signaling events whereby these receptors initiate tyrosine phosphorylation remain poorly understood. Recent data have implicated FAK family kinases and RTKs, both of which regulate the activity of Src kinases, as proximal mediators of GPCR-induced tyrosine phosphorylation. FAKs are nonreceptor tyrosine kinases that compose part of the focal adhesion complex. These complexes assemble on ␣␤ integrin heterodimers following integrin engagement of extracellular matrix proteins. Following recruitment, FAKs autophosphorylate and provide docking sites for several signaling proteins, including c-Src and Grb2 (6). In many cell types, stimulation of G i -or G q -coupled receptors causes FAK activation (7)(8)(9). This activation is cell adhesion-dependent, because disruption of focal adhesions prevents the response (10). In neuronal cells, stimulation of either LPA or bradykinin receptors activates the calcium-regulated FAK family kinase, Pyk2 (11), and overexpression of Pyk2 mutants that are either catalytically inactive or unable to bind to c-Src prevents GPCR-induced Erk 1/2 activation (5,11). In other systems, however, GPCR-mediated Erk 1/2 activation is apparently dissociable from FAK phosphorylation (7)(8)(9).
Classical RTKs, such as the receptor for epidermal growth factor (EGF), are single transmembrane domain proteins that dimerize and transphosphorylate upon ligand binding. Tyrosine phosphorylation of RTKs promotes their association with SH2 or PTB domain-containing signaling proteins, which assemble on the receptor to form a Ras activation complex (12). "Transactivation" of RTKs following GPCR stimulation has been implicated in GCPR-mediated activation of Erk 1/2 (13)(14)(15). In Rat 1 fibroblasts and COS-7 cells, inhibition of EGF receptor function inhibits LPA-, endothelin-1-, and thrombin receptor-mediated tyrosine phosphorylation of Shc and Gab1 and activation of Erk 1/2 (3,15). In this model, the transactivated RTK forms the structural core of a GPCR-induced mitogenic signaling complex, as receptor phosphorylation creates docking sites for the components of the Ras activation complex.
We compared the role of focal adhesions and EGF receptors in mediating Erk 1/2 activation via endogenously expressed LPA, thrombin, and bradykinin receptors in three different cell types: PC12 rat pheochromocytoma cells, Rat 1 fibroblasts, and HEK-293 embryonic kidney cells. Surprisingly, we found that the preferred scaffold was independent of the specific G i /G qcoupled GPCR being stimulated. Rather, the utilization of scaffolds varied between cell types, with PC-12 cells and Rat 1 fibroblasts apparently representing opposite ends of a continuum. In PC-12 cells GPCR-mediated Erk 1/2 activation was almost exclusively focal adhesion-dependent, whereas in Rat 1 fibroblasts it was almost exclusively RTK-dependent. In HEK-293 cells, both scaffolds contributed to the GPCR signal. Utilization of the focal adhesion scaffold correlated with signaling via pertussis toxin-insensitive G proteins and with cellular expression of the calcium-regulated FAK family kinase, Pyk2. In each case, GPCR-stimulated Erk 1/2 activation was sensitive to Src kinase inhibitors, suggesting that a critical role of both scaffolds is to support the GPCR-induced activation of Src family nonreceptor tyrosine kinases.
Immunoprecipitation and Immunoblotting-For the determination of p125 FAK and Pyk2 tyrosine phosphorylation, cells grown to confluence in 100-mm dishes were incubated in serum-free medium (Dulbecco's modified Eagle's medium, 10 mM HEPES, pH 7.4, 0.1% bovine serum albumin, 50 g/ml gentamicin) for 24 h before assay. Agonist stimulation was performed at 37°C in serum-free medium following preincubation with inhibitors, as described in the figure legends. After stimulation, monolayers were washed once with ice-cold phosphatebuffered saline and lysed in ice-cold RIPA buffer (150 mM NaCl, 50 mM Tris-Cl, pH 8.0, 0.25% w/v sodium deoxycholate, 0.1% v/v Nonidet P-40, 1 mM NaF, 1 mM sodium pyrophosphate, 100 M NaVO 4 , 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, 10 /ml aprotinin). Cell lysates were clarified by centrifugation and diluted to a protein concentration of 1 mg/ml. Before immunoprecipitation, a 50-l aliquot of the whole cell lysate was added to 2ϫ Laemmli sample buffer for SDSpolyacrylamide gel electrophoresis (PAGE) and assay of Erk 1/2 phosphorylation (below). Immunoprecipitation of p125 FAK was performed using mouse monoclonal anti-p125 FAK IgG (clone 2A7, Upstate Biotechnology, Inc.) plus 50 l of a 50% slurry of protein G plus/protein A-agarose (Calbiochem) with overnight agitation at 4°C. Immune complexes were washed twice with ice-cold RIPA buffer and once with phosphate-buffered saline, denatured in 2ϫ Laemmli sample buffer, and resolved by SDS-PAGE.
Tyrosine phosphorylation or the presence of immunoprecipitated proteins was detected by protein immunoblotting. Phosphotyrosine was detected using a 1:1000 dilution of horseradish peroxidase-conjugated anti-phosphotyrosine monoclonal antibody PY20H (Transduction Lab-oratories). FAK was detected using a 1:1000 dilution of mouse monoclonal anti-p125 FAK IgG (Transduction Laboratories), and Pyk2 was detected using a 1:1000 dilution of mouse monoclonal anti-Pyk2 IgG (Transduction Laboratories), each with horseradish peroxidase-conjugated anti-mouse IgG (Jackson Laboratories) as secondary antibody. Immunoprecipitated proteins on nitrocellulose were visualized by enzyme-linked chemiluminescence (Amersham Pharmacia Biotech) and quantified by scanning laser densitometry.
Erk 1/2 Phosphorylation-For the determination of Erk 1/2 phosphorylation, 15 g of clarified whole cell lysate protein/lane were resolved by SDS-PAGE, and Erk 1/2 phosphorylation was detected by protein immunoblotting using a 1:1000 dilution of rabbit polyclonal phospho-specific MAP kinase IgG (New England Biolabs) with alkaline phosphatase-conjugated goat anti-rabbit IgG (Amersham Pharmacia Biotech) as secondary antibody. Quantitation of Erk 1/2 phosphorylation was performed after exposure of nitrocellulose membranes to Vistra ECF reagent (Amersham Pharmacia Biotech) and scanning on a Storm PhosphorImager (Molecular Dynamics). After quantitation of Erk 1/2 phosphorylation, nitrocellulose membranes were stripped of immunoglobulin and reprobed using rabbit polyclonal anti-Erk2 IgG (Santa Cruz Biotechnology) to confirm equal loading of Erk2 protein.

Erk 1/2 Phosphorylation following Stimulation of Endogenous GPCRs in PC12, Rat 1, and HEK-293
Cells-To select endogenous GPCRs capable of activating the Erk 1/2 cascade in a variety of cell types, we assayed Erk 1/2 phosphorylation following stimulation of PC12, Rat 1, and HEK-293 cells with agonists for LPA, thrombin, or bradykinin receptors. Each of these receptors has been shown to mediate both pertussis toxin-sensitive and -insensitive signals resulting from dual coupling to G i/o -family and G q/11 -family heterotrimeric G proteins (16 -18). Erk 1/2 activation via endogenous EGF receptors was also determined as a control for cellular responsiveness and inhibitor specificity. Fig. 1 compares GPCR-induced Erk 1/2 phosphorylation in each of the three cell lines. In PC12 cells, both LPA-and bradykinin-stimulated Erk 1/2 phosphorylation was pertussis toxin-insensitive (Fig. 1A). In these cells, the thrombin agonist peptide, SFLLRN, provoked a less than 2-fold stimulation of Erk 1/2 phosphorylation (data not shown). In contrast, LPAand SFLLRN-stimulated Erk 1/2 phosphorylation in Rat 1 fibroblasts was completely pertussis toxin-sensitive (Fig. 1B). In HEK-293 cells, pertussis toxin only partially blocked the LPA and thrombin receptor responses (Fig. 1C). Here, LPAstimulated Erk 1/2 phosphorylation was predominantly pertussis toxin-sensitive, whereas the response to SFLLRN was predominantly pertussis toxin-insensitive. This differential sensitivity to pertussis toxin indicates that dual G i /G q -coupled GPCRs activate the Erk 1/2 cascade via distinct G protein pools in different cell types. As expected, EGF-stimulated Erk 1/2 phosphorylation was insensitive to pertussis toxin in all three cell lines.
As shown in Fig. 2, the Src-selective tyrosine kinase inhibitors herbimycin A (left panels) and PP1 (right panels) significantly inhibited LPA-, bradykinin-and SFLLRN-stimulated Erk 1/2 phosphorylation in PC12, Rat 1, and HEK-293 cells. Although neither inhibitor affected EGF receptor autophosphorylation at the concentrations employed (data not shown), both herbimycin A and PP1 also impaired EGF-stimulated Erk 1/2 phosphorylation. These data suggest that Src kinase activation contributes to both the GPCR-and RTK-mediated Erk 1/2 cascades.
Because Src family kinases associate with both integrinbased focal adhesion complexes (19) and receptor tyrosine kinases (12), it is likely that signals originating from either locus would be sensitive to Src inhibitors. If G i /G q -coupled receptors in different cell types selectively employ focal adhesions or RTKs as signaling platforms, then the differential use of these scaffolds might account for some of the observed heterogeneity in GPCR-mediated Erk 1/2 activation. To test this hypothesis, we determined the relative dependence of GPCR signals on the presence of functional focal adhesions and receptor tyrosine kinases in PC12, Rat 1, and HEK-293 cells.
Intact focal adhesions are required for the activation of FAK family kinases and formation of FAK⅐c-Src complexes (20 -22). Proper assembly of focal adhesions requires both cytoskeletal rearrangement (23) and integrin-mediated attachment to the extracellular matrix (24). Peptides containing the motif RGD, which mimic the integrin ligand found in extracellular matrix proteins such as fibronectin, have been shown to block integrin heterodimerization (25,26) and thereby disrupt the formation of focal adhesions. Similar effects are produced by depolymerization of actin stress fibers following exposure to cytochalasin D. In HEK 293 cells, blocking integrin dimerization using the synthetic oligopeptide GRGDS inhibits m1 and m3 muscarinic receptor-stimulated tyrosine phosphorylation of p125 FAK and paxillin (10).
To determine the extent to which intact focal adhesions might be required for GPCR-stimulated Erk 1/2 activation, we determined the effects of RGD peptides and cytochalasin D on Erk 1/2 phosphorylation in PC12, Rat 1, and HEK-293 cells. In each experiment, agonist-stimulated tyrosine phosphorylation of p125 FAK was measured as a marker for the integrity of focal adhesion complexes. As shown in Fig. 4, stimulation of LPA, thrombin, bradykinin, or EGF receptors rapidly induced the tyrosine phosphorylation of p125 FAK , indicating that each of these receptors promoted focal adhesion complex assembly.
Preincubation of cells with the RGDS peptide, but not the control RGES peptide, inhibited agonist-stimulated p125 FAK phosphorylation in each of the three cell lines (Fig. 4, A-C, left panels). In PC12 cells, LPA-and bradykinin-stimulated Erk 1/2 phosphorylation, like p125 FAK phosphorylation, was markedly inhibited by the RGDS peptide (Fig. 4A, right panel). In contrast, LPA and SFLLRN-stimulated Erk 1/2 phosphorylation in Rat 1 fibroblasts was completely insensitive to the RGDS peptide despite the significant inhibition of agonist-induced p125 FAK phosphorylation (Fig. 4B, right panel). In HEK-293 cells, LPA and SFLLRN-stimulated Erk 1/2 phosphorylation was partially inhibited (Fig. 4C, right panel). EGF receptormediated Erk 1/2 activation was insensitive to the RGDS peptide in all three cell lines, indicating that intact focal adhesions are not required for acute stimulation of Erk 1/2 by RTKs.
RTKs as Scaffolds for GPCR-stimulated Erk 1/2 Activation-Like FAKs, RTKs including the EGF (3,15,27), plateletderived growth factor (13), and insulin-like growth factor-1 (14) receptors can be activated in response to GPCR stimulation. In Rat 1 and COS-7 cells, inhibition of EGF receptor transactivation blocks GPCR-mediated MAP kinase activation (3,15). Because both activated RTKs and focal adhesions represent potential docking sites for proteins involved in the regulation of mitogenesis, either or both might function as a scaffold for GPCR-mediated activation of Erk 1/2.
As shown in Fig. 6, the EGF receptor-specific tyrphostin AG1478 has markedly different effects on GPCR-stimulated Erk 1/2 phosphorylation in PC12, Rat 1, and HEK-293 cells. As expected, exposure to tyrphostin AG1478 had no effect on LPA-, bradykinin-or SFLLRN-stimulated FAK phosphoryla- tion in any of the three cell lines, whereas producing marked inhibition the EGF effect (Fig. 6, A-C, left panels). At concentrations sufficient to abolish EGF-stimulated Erk 1/2 activation, LPA-and bradykinin-stimulated Erk 1/2 phosphorylation in PC12 cells was insensitive to tyrphostin AG1478 (Fig. 6A,  right panel). In contrast, tyrphostin AG1478 inhibited both LPA-and SFLLRN-stimulated Erk 1/2 activation in Rat 1 fibroblasts (Fig. 6B, right panel). In HEK-293 cells, the tyrphostin also produced partial inhibition of LPA-and SFLLRNstimulated Erk 1/2 phosphorylation (Fig. 6C, right panel). Because the sensitivity of GPCR-stimulated Erk 1/2 activation to tyrphostin AG1478 was opposite the effects of inhibitors of focal adhesion assembly, these results suggest that focal adhesions and RTKs can function independently as scaffolds for GPCRmediated Erk 1/2 activation. DISCUSSION Our data indicate that both focal adhesions and RTKs can function independently to support activation of the Erk 1/2 MAP kinase cascade following activation of endogenous G i /G qcoupled receptors. Fig. 7 schematically depicts a model consistent with these data. In PC12 cells, G i /G q -coupled receptors such as those for LPA and bradykinin mediate Erk 1/2 activation predominantly via pertussis toxin-insensitive G proteins and a focal adhesion-based scaffold. The lack of pertussis toxin sensitivity is consistent with the recent report that the G icoupled ␣2A adrenergic receptor does not mediate Erk 1/2 activation in stably transfected PC12 cells (28). Despite the presence of functional receptors, EGF receptor transactivation does not contribute detectably to GPCR-stimulated Erk 1/2 activation in these cells.
Rat 1 fibroblasts apparently represent the opposite end of a continuum. In these cells, LPA and thrombin receptors mediate Erk 1/2 activation largely via pertussis toxin-sensitive G proteins and transactivation of the EGF receptor. Unlike PC12 cells, GPCR-stimulated Erk 1/2 activation in these cells is unaffected by the disruption of focal adhesion complexes. HEK-293 cells apparently employ both scaffolds, as these cells exhibit LPA-and thrombin-stimulated Erk 1/2 activation that is partially pertussis toxin-sensitive and partially sensitive to inhibitors of focal adhesion complex assembly and of EGF receptor transactivation. Signals arising from either scaffold apparently converge on Src family nonreceptor tyrosine kinases, as Src inhibitors impair Erk 1/2 activation in each cell type.
G i /G q -coupled receptors are known to activate the Erk pathway via both tyrosine kinase-dependent and -independent pathways (29). Our data indicate that about a third of the Erk 1/2 phosphorylation mediated by LPA receptors in Rat 1 fibroblasts and by LPA and thrombin receptors in HEK-293 cells is insensitive to both Src-and EGF receptor-selective kinase inhibitors. This residual, tyrosine kinase-independent signal may reflect protein kinase C-mediated Erk 1/2 activation, which we have shown is Ras-independent and herbimycin Ainsensitive in HEK-293 cells (29).
Although it is clear that GPCR-mediated Erk 1/2 activation arising from focal adhesion-and RTK-based scaffolds are dissociable, the factors that determine scaffold preference are poorly understood. Our data suggest that cell type-specific expression of calcium-regulated FAK kinases such as Pyk2 in neuronal (11) or hematopoeitic cells (30)  GPCRs employ the focal adhesion complex as a signaling scaffold. Pyk2 and p125 FAK share approximately 60% sequence identity within their catalytic domain and 40% within their Nand C-terminal domains (11) but appear to differ significantly in their regulation by extracellular stimuli. Unlike p125 FAK , activation of the Pyk2 homologue CADTK apparently occurs by a two-stage process dependent upon both cellular adhesion and a costimulatory calcium-or protein kinase C-dependent signal (11,22). Similarly, phosphorylation of both endogenous Pyk2 and p125 FAK occurs following cell adhesion in rat aortic smooth muscle cells, but Pyk2 phosphorylation is further increased by costimulation with calcium ionophore or angiotensin II (31). This differential regulation of Pyk2 and p125 FAK activity suggests a basis for their distinct roles in the regulation of MAP kinase pathways. Consistent with this, GPCR-induced p125 FAK phosphorylation is dissociated from Erk 1/2 activation in Rat 1 cells (8,9), which do not detectably express Pyk2.
Conversely, overexpression of Pyk2 in 293T cells is sufficient to confer robust LPA-stimulated Erk 1/2 activation that is calcium-and Src kinase-dependent (5).
Several distinct RTKs, including those for platelet-derived growth factor, EGF, and insulin-like growth factor-1, can undergo transactivation (13)(14)(15). In a given cell type, GPCRstimulated Erk 1/2 activation may involve transactivation of multiple RTKs. For example, in Chinese hamster ovary cells, which lack endogenous EGF receptors, LPA stimulation results in Erk 1/2 activation that is dependent upon transactivation of platelet-derived growth factor receptors. However, when EGF receptors are expressed in these cells, signaling proceeds in an EGF receptor-dependent manner (32). Although such data suggest that "generic" mechanisms for the pleiotropic transactivation of RTKs may exist, the molecular mechanisms behind RTK transactivation are poorly understood. In COS-7 cells, EGF receptor transactivation is pertussis toxin-sensitive and inhibited by sequestration of free G protein G␤␥ subunits (27,32).  , 1 M). Cells were stimulated for 5 min with vehicle (NS), LPA, bradykinin, SFLLRN, or EGF as indicated, and RIPA buffer lysates were prepared. Tyrosine phosphorylation of p125 FAK (left panels) was determined by anti-phosphotyrosine immunoblotting of p125 FAK immunoprecipitates. Erk 1/2 phosphorylation (right panels) was determined from an aliquot of each RIPA buffer lysate. Data are presented as fold increase of basal Erk 1/2 phosphorylation, where the basal amount of Erk 1/2 phosphorylation in untreated cells is assigned a value of 1.0. Data shown represent the mean ϮS.E. values of duplicate determinations from six separate experiments. *, less than control; p Ͻ 0.05, paired t test.
Considerable evidence supports the role of Src family kinases in GPCR stimulation of Erk 1/2. Activation of Src kinases by the ␣-thrombin (34), LPA (2), angiotensin II (35), N-formylmethionyl peptide chemoattractant (1), ␣2A adrenergic (2, 34), and m1 muscarinic (34) receptors has been reported. Recruitment of c-Src to Pyk2 is required for its action, because a point mutant of Pyk2 that cannot complex with c-Src behaves as a dominant negative inhibitor of GPCR-stimulated Erk 1/2 activation (5). Similarly, inhibition of Src kinase activity using either dominant inhibitory c-Src mutants (2) or pharmacologic agents (3) dramatically reduces LPA receptor-mediated tyrosine phosphorylation of Shc and Gab1 and Erk 1/2 activation in COS-7 cells. Although these data clearly support a role for Src kinases "downstream" of both FAK family kinases and trans-activated RTKs, additional evidence suggests that Src kinase activity may also play an "upstream" role in GPCR-induced RTK transactivation. Overexpression of Src inhibitor kinase Csk impairs LPA and ␣2A adrenergic receptor-mediated EGF receptor phosphorylation in COS-7 cells (27). In addition, angiotensin II stimulation has recently been shown to induce association of activated c-Src with the EGF receptor independent of EGF receptor catalytic activity, suggesting that c-Src activation may precede EGF receptor transactivation (36).
Previous work has revealed significant heterogeneity in the mechanisms whereby GPCRs mediate activation of the Erk 1/2 MAP kinase pathway. In most systems studied, GPCR-stimulated Erk 1/2 activation involves the assembly of a Ras activation complex on the plasma membrane, which is dependent upon regulated tyrosine phosphorylation of adapter proteins such as Shc and Gab1 and recruitment of Grb2-mSos. Our data strongly suggest that both focal adhesions and RTKs can function as independently regulated scaffolds for the assembly of this complex and indicate that the preferred scaffold is determined primarily by the cellular context in which the receptor is expressed. Considerable care is therefore warranted in using ectopic expression systems to characterize the signal transduction pathways employed by GPCRs which exhibit tissue-specific expression in vivo. Further examination of the functional significance of these different scaffolds will ultimately enhance our understanding of the diversity of proliferative and differentiative signals originating from GPCRs.  Rat 1 (B), or HEK-293 (C) cells were incubated in the presence or absence (black bars, control) of tyrphostin AG1478 (shaded bars, 250 nM). Cells were stimulated for 5 min with vehicle (NS), LPA, bradykinin, SFLLRN, or EGF as indicated, and RIPA buffer lysates were prepared. Tyrosine phosphorylation of p125 FAK (left panels) was determined by anti-phosphotyrosine immunoblotting of p125 FAK immunoprecipitates. Erk 1/2 phosphorylation (right panels) was determined from an aliquot of each RIPA buffer lysate. Data are presented as fold increase of basal Erk 1/2 phosphorylation, where the basal amount of Erk 1/2 phosphorylation in untreated cells is assigned a value of 1.0. Data shown represent the mean ϮS.E. values of duplicate determinations from six separate experiments. Exposure to tyrphostin AG1478 did not significantly inhibit Erk 1/2 phosphorylation in response to a 5-min exposure to 100 nM phorbol ester (data not shown). *, less than control; p Ͻ 0.05, paired t test.

FIG. 7. Focal adhesions and transactivated RTKs as independent scaffolds for GPCR-mediated Erk 1/2 activation.
Stimulation of endogenous G i /G q -coupled receptors results in both tyrosine kinaseindependent (e.g. protein kinase C-mediated) and tyrosine kinase-dependent activation of Erk 1/2. GPCR-mediated focal adhesion complex assembly or RTK transactivation creates scaffolds for tyrosine kinasedependent activation of the Erk 1/2 cascade. Depending on cell type, a GPCR may signal via either, or both, scaffolds. Signals originating at either scaffold converge on Src family kinases. MAPK, MAP kinase.