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Linkage of G Protein-Coupled Receptors to the MAPK Signaling Pathway Through PI 3-Kinase γ

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Science  17 Jan 1997:
Vol. 275, Issue 5298, pp. 394-397
DOI: 10.1126/science.275.5298.394

Abstract

The tyrosine kinase class of receptors induces mitogen-activated protein kinase (MAPK) activation through the sequential interaction of the signaling proteins Grb2, Sos, Ras, Raf, and MEK. Receptors coupled to heterotrimeric guanine triphosphate-binding protein (G protein) stimulate MAPK through Gβγ subunits, but the subsequent intervening molecules are still poorly defined. Overexpression of phosphoinositide 3-kinase γ (PI3Kγ) in COS-7 cells activated MAPK in a Gβγ-dependent fashion, and expression of a catalytically inactive mutant of PI3Kγ abolished the stimulation of MAPK by Gβγ or in response to stimulation of muscarinic (m2) G protein-coupled receptors. Signaling from PI3Kγ to MAPK appears to require a tyrosine kinase, Shc, Grb2, Sos, Ras, and Raf. These findings indicate that PI3Kγ mediates Gβγ-dependent regulation of the MAPK signaling pathway.

The muscarinic receptor m2 was expressed in COS-7 cells together with an epitope-tagged MAPK (HA-ERK2) (1). Treatment of cells with the agonist carbachol induced activation of MAPK, and wortmannin, an inhibitor of phosphoinositide 3-kinases (PI3Ks), nearly abolished this effect (Fig. 1A). Furthermore, MAPK activation induced by transient expression of Gβγ or by coexpression of Gβγ and the guanine nucleotide exchange factor Sos was also inhibited by wortmannin (Fig. 1B). In contrast, no effect of wortmannin was observed when MAPK was stimulated by epidermal growth factor (EGF); by a mutationally activated form of MEK, MEK E; or by a membrane-targeted form of Sos, myrSos (Fig. 1, A and B). These results support an essential role for wortmannin-sensitive PI3K in signal transduction from G protein-coupled receptors to MAPK (2), separately from the EGF signaling pathway and upstream of Sos and MEK.

Fig. 1.

Inhibition of MAPK activation in COS-7 cells by wortmannin (Wort.). (A) Effect of wortmannin on the stimulation of MAPK induced by the m2 muscarinic receptor and carbachol (m2), EGF, or MEK E. C, control. Two days after transfection (18) with expression plasmids of HA-MAPK and, as indicated, the m2 receptor or MEK E, cells were treated for 5 min with 1 mM carbachol or EGF (100 ng/ml) and the indicated concentration of wortmannin, which was added 20 min before agonist addition. MAPK activity was assayed in cellular lysates (19). (B) Wortmannin blocked MAPK activation induced by Gβγ. COS-7 cells transfected with expression plasmids for the G-protein subunits (Gβ1 and Gγ2), alone or together with Sos, or cells transfected with myrSos alone were treated with wortmannin, and MAPK activity was assayed.

Several species of PI3K have been cloned and characterized. Heterodimeric PI3Kα and PI3Kβ, consisting of p110 catalytic subunits and different p85 adapter molecules, are regulated by receptors with intrinsic or associated tyrosine kinase activity (3). Another PI3K isotype, termed PI3Kγ, can be activated in vitro by both α and βγ subunits of heterotrimeric G proteins but does not interact with p85 (4). We expressed the α and γ forms of PI3K in COS-7 cells and investigated their ability to induce MAPK activity (Fig. 2, A and B). PI3Kγ induced a concentration-dependent stimulation of MAPK. In contrast, expression of PI3Kα or a mutant of PI3Kγ lacking lipid kinase activity, PI3Kγ K799R (5), did not affect MAPK activity (Fig. 2, B and C). Stimulation of MAPK by overexpression of PI3Kγ (5) was abolished by wortmannin (Fig. 2C). These observations indicate that PI3Kγ may mediate the wortmannin-sensitive activation of MAPK by receptors linked to heterotrimeric G proteins.

Fig. 2.

MAPK activation by PI3Kγ in COS-7 cells. (A) Stimulation of MAPK activity by overexpression of PI3Kγ. COS-7 cells were transfected with the indicated amounts of the expression plasmid pEF-BOS PI3Kγ. After 2 days, cells were lysed, MAPK activity was assayed (19), and protein immunoblot analysis with antiserum to PI3Kγ or with antibody to HA (for the HA-tagged MAPK) was performed (20). (B) Overexpression of PI3Kγ but not PI3Kα enhances MAPK activity. COS-7 cells were transfected with 1 μg of expression plasmids for PI3Kγ or PI3Kα. After 2 days, cells were lysed, MAPK activity was assayed (19), and protein immunoblot analysis with antiserum to PI3Kγ and PI3Kα was performed. Phospholipid analysis revealed a fivefold and twofold increase in total levels of PIP3 in PI3Kα- and PI3Kγ-transfected cells, respectively, as assayed by lipid extraction, deacylation, and separation by high-performance liquid chromatography (9). (C) Inhibition of PI3Kγ induced MAPK activation by wortmannin. COS-7 cells were transfected with 1 μg of expression plasmids for PI3Kγ, PI3Kγ K799R (DK) (6), or myrSos. After 48 hours, cells were treated with wortmannin for 30 min, and MAPK activity was determined. (D) Effects of a K799R catalytically inactive mutant of PI3Kγ on MAPK activation induced by carbachol through m2 receptors, Gβγ, or EGF. Empty vector or expression plasmids for PI3Kγ wild type (WT), the PI3Kγ K799R mutant (6), and the m2 receptor or Gβγ were used for transfection. MAPK activity was assayed after 48 hours. (E) Effects of the K799R catalytically inactive mutant of PI3Kγ on phosphatidylinositol hydrolysis induced by Gβγ. IP, inositol phosphates. Empty vector or expression plasmids for PI3Kγ WT, the PI3Kγ K799R mutant (DK), Gβγ, PLC-β2, CD8, or CD8-βARK were used for transfection. Forty-eight hours later, the accumulation of [3H]IP for 30 min after addition of LiCl was determined (21). (F) Requirement for interaction of PI3Kγ with Gβγ and its possible role in membrane localization. COS-7 cells were cotransfected with different constructs of PI3Kγ in the presence or absence of CD8 or the CD8-βARK expression plasmid, and the effect on MAPK activity was assayed.

We found that expression of the mutated PI3Kγ that lacks lipid kinase activity nearly abolished MAPK activation induced by expression of m2 receptors and by stimulation with carbachol, or by expression of Gβγ (Fig. 2D). However, no inhibitory effect by this mutant was observed when MAPK was stimulated with EGF or in Gβγ-induced activation of phospholipase C-β2 (PLC-β2) (Fig. 2E). Thus, the mutated PI3Kγ appears to specifically inhibit the MAPK response to Gβγ. We also expressed a chimeric molecule combining the extracellular and transmembrane domain of CD8 fused to the COOH-terminal domain of βARK, which includes the βγ-binding region (6). This chimeric molecule expressing the CD8 antigen at the cell surface and the βARK COOH-terminal domain at the inner face of the plasma membrane is expected to bind and sequester free βγ, thus blocking βγ-dependent pathways (6). As expected, CD8-βARK inhibited activation of PLC-β2 by Gβγ (Fig. 2E). Coexpression of CD8-βARK with PI3Kγ nearly abolished MAPK activation by PI3Kγ, whereas CD8 alone had no demonstrable effect (Fig. 2F). CD8-βARK was ineffective in inhibiting MAPK activation by PI3Kγ when this kinase was expressed as a myristoylated form, upon fusing its coding region to that of the NH2-terminal myrstoylation, membrane localization signal from c-Src (7). These results indicate that one function of Gβγ is to localize PI3Kγ to the plasma membrane, thereby allowing access to lipid substrates. We can conclude that PI3Kγ has a critical role linking G protein-coupled receptors and Gβγ to the MAPK signaling pathway.

We tested whether the small guanosine triphospate (GTP)-binding protein Ras participates in signal transduction from PI3Kγ to the MAPK cascade. The dominant negative mutant N17-Ras suppressed the increase of MAPK activity induced by PI3Kγ, but the negative mutants of the small guanosine triphosphatases (GTPases) RhoA, Rac, and Cdc42 did not (Fig. 3, A and B). Expression of a dominant negative mutant of Raf or a mutant Sos protein lacking the domain involved in Ras-specific guanine nucleotide exchange activity, SosΔcdc25, also inhibited MAPK stimulation by PI3Kγ without affecting MAPK elevation by the activated form of MEK, MEK E (Fig. 3, C and D). These results support a crucial role for Ras, Raf, and Sos in signaling from G protein-dependent receptors, Gβγ, and PI3Kγ to the MAPK pathway.

Fig. 3.

Ras and Sos mediate MAPK activation by PI3Kγ. (A) Inhibition of PI3Kγ induced MAPK activation by dominant negative N17ras. Expression plasmids of PI3Kγ WT, PI3Kγ K799R (DK), or MEK E were transfected together with N17ras (18) and MAPK was assayed (19). (B) Effects of dominant negative mutants of Rac (N17rac-1), Rho (N19rho-A), Cdc42 (N17cdc42), and Ras N17ras) on MAPK stimulation induced by PI3Kγ. Cells were cotransfected with expression plasmids for PI3Kγ and the indicated small GTPases. Data are from a representative experiment that was repeated three times with nearly identical results. (C) Inhibition of PI3Kγ induced MAPK activation by the dominant negative SosΔcdc25. COS-7 cells were cotransfected with PI3Kγ WT, PI3Kγ K799R (DK), or MEK E and an expression plasmid of SosΔcdc25. MAPK activity was assayed. (D) Inhibition of PI3Kγ induced MAPK activation by the dominant negative Raf. COS-7 cells were cotransfected with PI3Kγ WT, PI3Kγ K799R (DK), or MEK E and an expression plasmid for a dominant negative mutant of Raf (DN raf) (1). MAPK activity was assayed.

Finally, we investigated the possible involvement of Shc and Grb2 in this signaling route. These elements of the receptor tyrosine kinase-stimulated signaling cascade participate in Gβγ-dependent signal transduction (8). Wortmannin inhibited binding of Shc and Grb2 induced by carbachol (Fig. 4A). Association of Shc with Grb2 was induced by expression of PI3Kγ in COS-7 cells (9). Expression of PI3Kγ also stimulated tyrosine-phosphorylation of Shc (Fig. 4B). Furthermore, a mutant of Shc lacking the tyrosine-phosphorylation site, Y317F (10), suppressed the stimulation of MAPK induced by lysophosphatidic acid (LPA), expression of Gβγ, carbachol in m2-transfected cells, expression of PI3Kγ, or expression of the Src-related tyrosine kinase Fyn (11) (Fig. 4C). In contrast, the Shc mutant did not affect MAPK activation induced by coexpression of v-Ras. Thus, stimulation of MAPK by PI3Kγ apparently requires a tyrosine kinase that, in turn, phosphorylates Shc and induces its association with Grb2 and leads to a Ras-dependent activation of MAPK. Consistent with this conclusion, the nonspecific tyrosine kinase inhibitor genistein or the Src-like specific inhibitor PP1 (12) potently blocked MAPK activation by PI3Kγ (9).

Fig. 4.

Involvement of Shc and Grb2 in MAPK activation by PI3Kγ. (A) Inhibition by wortmannin of association of Shc and Grb2 induced by carbachol and m2 receptors. The m2 muscarinic receptors or PI3Kγ were expressed together with an epitope-tagged (HA) Shc in COS-7 cells. The cells were treated as indicated with wortmannin for 30 min and lysed. Proteins were immunoprecipitated with antiserum to Grb2, and association with Shc was assayed by protein immunoblotting with antiserum to Shc (20). (B) PI3Kγ induced increase of phosphotyrosine in Shc; inhibition by wortmannin. COS-7 cells transfected with PI3Kγ or m2 receptors were treated with the indicated effectors and lysed. Proteins immunoprecipitated with antibodies to Shc were probed with antibodies to phosphotyrosine (20). (C) Dominant negative Shc Y317F inhibits MAPK activation induced by LPA, m2 + carbachol, PI3Kγ, and activated Fyn (Fyn-c). COS-7 cells were transfected with the Shc Y317F mutant and constructs for PI3Kγ, the m2 receptor, v-Ras, and Fyn-c and treated with the indicated effectors; MAPK was then assayed (19).

The emerging picture is that agonist-activated G protein-coupled receptors first cause the exchange of guanosine diphosphate bound to Gα for GTP, thereby causing the dissociation of Gβγ from GTP-bound Gα. Free Gβγ would then recruit PI3Kγ to the plasma membrane, enhancing the activity of a Src-like kinase (13), which in turn leads to the activation of the Shc-Grb2-Sos-Ras pathway, resulting in increased MAPK activity.

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