Signaling from G Protein-coupled Receptors to c-Jun Kinase Involves βγ Subunits of Heterotrimeric G Proteins Acting on a Ras and Rac1-dependent Pathway

Abstract

Stimulation of a variety of cell surface receptors enhances the enzymatic activity of mitogen-activated protein kinases (MAPKs). MAPKs have been classified in three subfamilies: extracellular signal-regulated kinases (ERKs), stress-activated protein kinases or c-Jun NH2-terminal kinases (SAPKs/JNKs), and p38 kinase. Whereas the pathway linking cell surface receptors to ERKs has been partially elucidated, the mechanism of activation of JNKs is still poorly understood. Recently, we have shown that stimulation of G protein-coupled receptors can effectively induce JNK in NIH 3T3 cells (Coso, O. A., Chiariello, M., Kalinec, G., Kyriakis, J. M., Woodgett, J., and Gutkind, J. S.(1995) J. Biol. Chem. 270, 5620-5624). In the present study, we have used the transient expression in COS-7 cells of m1 and m2 muscarinic receptors (mAChRs) as a model system to study the signaling pathway linking G protein-coupled receptors to JNK. We show that stimulation of either muscarinic receptor subtype leads to JNK activation; however, this effect was not mimicked by expression of activated forms of αs, αi2, αq, or α13 G protein α subunits. In contrast, overexpression of Gβγ subunits potently induced JNK activity. Furthermore, we show that signaling from m1 and m2 mAChRs to JNK involves βγ subunits of heterotrimeric G proteins, acting on a Ras and Rac1-dependent pathway. Stimulation of a variety of cell surface receptors enhances the enzymatic activity of mitogen-activated protein kinases (MAPKs). MAPKs have been classified in three subfamilies: extracellular signal-regulated kinases (ERKs), stress-activated protein kinases or c-Jun NH2-terminal kinases (SAPKs/JNKs), and p38 kinase. Whereas the pathway linking cell surface receptors to ERKs has been partially elucidated, the mechanism of activation of JNKs is still poorly understood. Recently, we have shown that stimulation of G protein-coupled receptors can effectively induce JNK in NIH 3T3 cells (Coso, O. A., Chiariello, M., Kalinec, G., Kyriakis, J. M., Woodgett, J., and Gutkind, J. S.(1995) J. Biol. Chem. 270, 5620-5624). In the present study, we have used the transient expression in COS-7 cells of m1 and m2 muscarinic receptors (mAChRs) as a model system to study the signaling pathway linking G protein-coupled receptors to JNK. We show that stimulation of either muscarinic receptor subtype leads to JNK activation; however, this effect was not mimicked by expression of activated forms of αs, αi2, αq, or α13 G protein α subunits. In contrast, overexpression of Gβγ subunits potently induced JNK activity. Furthermore, we show that signaling from m1 and m2 mAChRs to JNK involves βγ subunits of heterotrimeric G proteins, acting on a Ras and Rac1-dependent pathway. INTRODUCTIONStimulation of a variety of cell surface receptors causes a rapid elevation of the enzymatic activity of a family of closely related serine-threonine kinases, known as mitogen-activated protein kinases (MAPKs)( 1The abbreviations used are: MAPKmitogen-activated protein kinaseERKextracellular signal-regulated kinaseJNK (or SAPK)c-Jun NH2-terminal kinase (or stress-activated protein kinase).) (2Cano E. Mahadevan L.C. Trends Biochem. Sci. 1995; 20: 117-122Abstract Full Text PDF PubMed Scopus (996) Google Scholar). The function of MAPKs is to convert extracellular stimuli to intracellular signals which, in turn, control the expression of genes that are essential for many cellular processes, including cell growth and differentiation(3Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4223) Google Scholar). MAPKs have been classified in three subfamilies: extracellular signal regulated kinases (ERKs), including ERK1 and ERK2, also known as p44mapk and p42mapk, respectively; stress-activated protein kinases (SAPKs), also termed c-Jun NH2-terminal kinases (JNKs); and p38 kinase(2Cano E. Mahadevan L.C. Trends Biochem. Sci. 1995; 20: 117-122Abstract Full Text PDF PubMed Scopus (996) Google Scholar). ERKs phosphorylate and regulate the activity of certain enzymes, including phospholipase A2 and p90rsk, and nuclear proteins, such as the ternary complex factor p62TCF or Elk-1(4Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar). The latter represents a critical event in controlling the expression of several genes, including c-fos(5Hill C.S. Treisman R. Cell. 1995; 80: 199-211Abstract Full Text PDF PubMed Scopus (1195) Google Scholar). JNKs phosphorylate the amino-terminal transactivating domain of c-Jun and ATF2(6Gupta S. Campbell D. Derijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1336) Google Scholar), thereby increasing their transcriptional activity. p38 is the homologue of the Saccharomyces cerevisiae HOG1 gene, and its function is still unknown, although recently available information suggests that p38 might play a critical role in the inflammatory response(7Lee J.C. Laydon J.T. Mc Donnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3121) Google Scholar).Recent findings have helped to unveil the pathway linking cell surface receptors to ERKs(3Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4223) Google Scholar). In contrast, the mechanism of activation of JNKs is still poorly understood. JNKs were shown to be activated by a variety of stimuli distinct from those that elevate the enzymatic activity of ERKs, including protein synthesis inhibitors, heat shock, changes in osmolarity, and ultraviolet irradiation(6Gupta S. Campbell D. Derijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1336) Google Scholar, 8Kyriakis J. Banerjee P. Nikolakaki E. Dai T. Rubie E. Ahmad M. Avruch J. Woodgett J. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2409) Google Scholar). JNKs can be also activated by agents acting on cell surface receptors, such as tumor necrosis factor-α, interleukin-1, or epidermal growth factor (8Kyriakis J. Banerjee P. Nikolakaki E. Dai T. Rubie E. Ahmad M. Avruch J. Woodgett J. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2409) Google Scholar). Furthermore, available evidence suggest that whereas Ras controls the activation of ERKs, members of the Rho family of small GTP-binding proteins, Rac1 and Cdc42, regulate the activity of JNKs(9Coso O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1559) Google Scholar).Recently, we have shown that stimulation of certain G protein-coupled receptors expressed in NIH 3T3 cells can effectively induce JNK activity, however, following a time course clearly distinct from that of ERK activation(1Coso O.A. Chiariello M. Kalinec G. Kyriakis J.M. Woodgett J. Gutkind J.S. J. Biol. Chem. 1995; 270: 5620-5624Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). In the present study, we have used the transient expression in COS-7 cells of m1 and m2 muscarinic receptors (mAChRs) as a model system to study the biochemical route connecting G protein-coupled receptors to an epitope-tagged JNK. We present evidence that signaling from m1 and m2 mAChRs to JNK involves βγ subunits of heterotrimeric G proteins, acting on a Ras- and Rac1-dependent pathway.EXPERIMENTAL PROCEDURESCell Lines and TransfectionCOS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% of fetal bovine serum. Cells were transfected by the DEAE-dextran technique, adjusting the total amount of DNA to 5-10 μg/plate with vector DNA (pcDNA3; Invitrogen), when necessary(9Coso O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1559) Google Scholar).Expression PlasmidsExpression plasmids for an epitope-tagged JNK (pcDNA3-HA-JNK), for m1 and m2 mAChRs, for the dominant-negative mutants of the small GTP-binding proteins Ras, RhoA, Rac1, and Cdc42, as well as expression plasmids for β1 and γ2 subunits of heterotrimeric G proteins, or the isoprenylation defective mutant of γ2, γ2∗, have been described(9Coso O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1559) Google Scholar, 10Crespo P. Xu N. Simonds W.F. Gutkind J.S. Nature. 1994; 369: 418-420Crossref PubMed Scopus (758) Google Scholar). Plasmids expressing GTPase-deficient, constitutively activated forms of representative α subunits of heterotrimeric G proteins have been described previously. In each case, a critical glutamine residue in the G3 region of Gαs (codon 227), Gαi2 (codon 205), Gαq (codon 209), and Gα13 (codon 226) was replaced for leucine (QL mutants)(10Crespo P. Xu N. Simonds W.F. Gutkind J.S. Nature. 1994; 369: 418-420Crossref PubMed Scopus (758) Google Scholar, 11Crespo P. Cachero T.G. Xu N. Gutkind J.S. J. Biol. Chem. 1995; 270: 25259-25265Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). An expression plasmid for a chimeric molecule between the extracellular and transmembrane domains of CD8 fused to the COOH-terminal domain of βARK (pcDNA-CD8-βARK) has been described recently(11Crespo P. Cachero T.G. Xu N. Gutkind J.S. J. Biol. Chem. 1995; 270: 25259-25265Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar).JNK Assay and Western BlotsJNK activity in lysates from COS-7 cells transfected with an expression vector for an epitope-tagged JNK (pcDNA3-HA-JNK) was determined upon immunoprecipitation with the anti-HA-specific monoclonal antibody 12CA5 (Babco) using bacterially expressed GST-ATF2(96) fusion protein as a substrate, as described previously(9Coso O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1559) Google Scholar). The products of the kinase reactions were fractionated in SDS-12% polyacrylamide electrophoresis gels, and radioactivity incorporated into GST-ATF2(96) was determined with the use of a PhosphorImager system (Molecular Dynamics). Parallel immunoprecipitates were analyzed by Western blotting after SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with the anti-HA monoclonal antibody. Immunocomplexes were visualized by enhanced chemiluminescence detection (Amersham Corp.) using goat anti-mouse serum coupled to horseradish peroxidase as a secondary antibody (Cappel).RESULTS AND DISCUSSIONIn order to study the mechanism controlling the activation of JNK by m1 and m2 G protein-coupled receptors, we coexpressed these receptors together with an epitope-tagged JNK (HA-JNK) in COS-7 cells. We observed that when cotransfected with the HA-JNK cDNA, both m1 and m2 were expressed at similar high levels (data not shown), and the epitope-tagged JNK was efficiently expressed and readily detectable upon immunoprecipitation with the anti-HA monoclonal antibody(9Coso O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1559) Google Scholar). In cells expressing either muscarinic receptor, the cholinergic agonist carbachol induced an increase in JNK activity, as judged by its in vitro phosphorylating activity using GST-ATF2(96) as a substrate (Fig. 1). Whether mediated by m1 or m2 receptors, induction of GST-ATF2(96) phosphorylation showed a peak at approximately 15 min after stimulation, being of 2-4-fold higher than the control with a slow decrease at later times (Fig. 1). Thus, JNK is activated by either m1 or m2 G protein-coupled receptors when expressed in COS-7 cells. Whereas m1 receptors are typical of those coupled through G proteins of the Gq family to phospholipase C activation, m2 is known to couple through Gi to a number of effector pathways, including to the inhibition of adenylyl cyclases (12.Ashkenazi, A., Peralta, E. G., Winslow, J. W., Ramachandran, J., Capon, D. J. (1989) Trends. Pharmacol. Sci. (suppl.) 16-22Google Scholar). Interestingly, both m1 and m2 mAChRs appear to activate JNK irrespective of their G protein-coupling specificity.As an approach to investigate which G proteins mediate the activation of JNK, we took advantage of the observation that the expression of GTPase-deficient, mutationally activated forms of G protein α subunits can activate effector pathways by obviating the need for receptor stimulation(13Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4682) Google Scholar). Thus, we coexpressed the epitope-tagged JNK together with GTPase-deficient mutants for Gαs, Gαi2, Gαq, and Gα13, which are representative members for each of the four Gα subunit families (14Neer E. Cell. 1995; 80: 249-257Abstract Full Text PDF PubMed Scopus (1283) Google Scholar). Expression of each G protein α subunit, when transfected into COS-7 cells, could be demonstrated by immunoblotting with subtype-specific antibodies (data not shown; (11Crespo P. Cachero T.G. Xu N. Gutkind J.S. J. Biol. Chem. 1995; 270: 25259-25265Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar) and (12.Ashkenazi, A., Peralta, E. G., Winslow, J. W., Ramachandran, J., Capon, D. J. (1989) Trends. Pharmacol. Sci. (suppl.) 16-22Google Scholar)). However, these activated mutants enhanced JNK activity only to a very limited extent, either when each Gα subunit was expressed alone (Fig. 2) (15Prasad M.V. Dermott J.M. Heasley L.E. Johnson G.L. Dhanasekaran N. J. Biol. Chem. 1995; 270: 18655-18659Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar) or in all possible combinations (data not shown). Thus, α subunits of heterotrimeric G proteins might not mediate JNK activation by G protein-coupled receptors.Figure 2:Overexpression of βγ subunits of heterotrimeric G proteins results in stimulation of JNK activity. COS-7 cells were transfected with pcDNA3-HA-JNK (1 μg/plate) together with pcDNA3 vector (control) or with an expression vector carrying cDNAs for the activated (QL) forms of the α subunits of Gs, Gi2, Gq, and G13, or expressing β1, γ2, or γ2∗ G protein subunits, alone or in combination, as indicated (2 μg/plate in each case). Kinase reactions and Western blot (WB) analysis were performed in anti-HA immunoprecipitates from the corresponding lysates, as described under “Experimental Procedures.” Autoradiograms correspond to representative experiments.32P-Labeled products as well as specific bands detected by the anti-HA antibody are indicated with an arrow. Data represent the mean of four to expressed as increase with to cells receptors to G proteins the of by to the α subunit and induce the of from βγ the α subunits were to be for receptors to has a critical role for βγ in signal P. Xu N. Simonds W.F. Gutkind J.S. Nature. 1994; 369: 418-420Crossref PubMed Scopus (758) Google Scholar, E. Cell. 1995; 80: 249-257Abstract Full Text PDF PubMed Scopus (1283) Google Scholar). Thus, the of mutationally activated G protein α subunits to elevate JNK activity to βγ in signaling to JNK. We observed when subunits induce a increase in the phosphorylating activity of the epitope-tagged JNK, although expression of the HA-JNK was similar for each transfected cell (Fig. results were when subunits were expressed (data not shown). In contrast, JNK was poorly activated when coexpressed with β1 or γ2 alone or when cotransfected with β1 and an of the γ2 subunit, γ2∗, that an isoprenylation signal and to to the P. Xu N. Simonds W.F. Gutkind J.S. Nature. 1994; 369: 418-420Crossref PubMed Scopus (758) Google Scholar) (Fig. We that βγ subunits of heterotrimeric G proteins, not Gα subunits, can effectively JNK activity in COS-7 a role for βγ in JNK stimulation by approach this we a chimeric molecule the extracellular and transmembrane domain of CD8 fused to the domain of which the P. Cachero T.G. Xu N. Gutkind J.S. J. Biol. Chem. 1995; 270: 25259-25265Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). chimeric molecule the CD8 at the cell the βARK domain to the of the The is to and βγ when from Gα subunits upon receptor stimulation, P. Cachero T.G. Xu N. Gutkind J.S. J. Biol. Chem. 1995; 270: 25259-25265Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). shown in of with the m1 or m2 mAChRs the activation of JNK in to CD8 alone In contrast, JNK activation by agents such as O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1559) Google Scholar) was by the of this approach (Fig. these findings suggest that signaling from m1 and m2 mAChRs to JNK is mediated by βγ subunits of heterotrimeric G of a βγ protein in JNK pcDNA3-HA-JNK was cotransfected with expression plasmids for m1 or m2 mAChRs (1 and β1 and γ2 subunits (2 together with vector alone or plasmids expressing the CD8 receptor or a chimeric molecule as Cells were with carbachol or for 15 min or of JNK activity present in anti-HA immunoprecipitates was performed as in Data represent the mean of three expressed as increase with to have recently shown that the small GTP-binding proteins Rac1 and are of signaling pathways linking certain cell surface receptors to O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1559) Google Scholar). Thus, we to investigate these small mediate JNK activation by G protein-coupled receptors, using as a the expression of dominant-negative mutants for Ras, RhoA, Rac1, and of these small GTP-binding proteins the JNK activation in to (Fig. In contrast, as shown in of and JNK activation by either mAChRs, or when induced by βγ JNK stimulation by m1 mAChRs, and the dominant-negative of not we have recently shown that JNK activation by in COS-7 cells is also by and O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1559) Google Scholar). Thus, these findings a role for Ras and Rac1 or in linking both kinase and G protein-coupled receptors to the JNK of dominant-negative mutants for Ras, RhoA, Rac1, and on JNK COS-7 cells were transfected with pcDNA3-HA-JNK and expression plasmids for m1 and m2 receptors (1 μg/plate) or for β1 and γ2 G protein subunits (2 together with either pcDNA3 or carrying cDNAs for or (1 and cells were or with carbachol or for 15 as lysates were as in Data represent the mean of four to expressed as of activation with to the corresponding vector cotransfected control Ras controls the activity of ERKs, available evidence suggests that Rac1 regulate JNK O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1559) Google Scholar). Thus, distinct small GTP-binding proteins appear to cell surface receptors with signaling pathways to the activation of each of the In this study, we present evidence a role for βγ subunits of heterotrimeric G proteins in G protein-coupled receptors with the JNK acting on a Ras- and Rac1-dependent biochemical Furthermore, we and have shown that βγ this of cell surface receptors to ERKs, in this acting through P. Xu N. Simonds W.F. Gutkind J.S. Nature. 1994; 369: 418-420Crossref PubMed Scopus (758) Google Scholar, T. E. M. R.J. Nature. 1995; PubMed Scopus Google Scholar). Thus, these findings together we can that βγ a between heterotrimeric G proteins and small GTP-binding for this is still poorly T. E. M. R.J. Nature. 1995; PubMed Scopus Google Scholar). However, is similar to that of the pathway linking the G protein-coupled receptors to in the S. In this case, extracellular or a activate receptors which, in turn, induce the of a heterotrimeric G protein into α and βγ subunits C.J. J. Biol. 1994; PubMed Scopus Google Scholar) for βγ activate a serine-threonine kinase activity from a of kinases, including and the and for linking βγ to has to the that in B. J.R. S. Nature. 1995; PubMed Scopus Google Scholar), and that might and activate a factor for T. E. Cell. Biol. 1995; PubMed Scopus Google Scholar). suggest that proteins might also be including a protein which a role as a or T. E. Science. 1995; PubMed Scopus Google Scholar), and small GTP-binding the Ras homologue J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). The latter also to play an role by by this factor the its with and its J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). represent an of between and biochemical and might also a the pathway connecting βγ to JNK. upon these in and present we can that upon receptor stimulation βγ might a to be factor for Rac1 and that Ras be for the activation of these GTP-binding proteins and the stimulation of JNK. We that present study might represent a of a signal pathway from to and might an model to the of those linking heterotrimeric G proteins to small GTP-binding INTRODUCTIONStimulation of a variety of cell surface receptors causes a rapid elevation of the enzymatic activity of a family of closely related serine-threonine kinases, known as mitogen-activated protein kinases (MAPKs)( 1The abbreviations used are: MAPKmitogen-activated protein kinaseERKextracellular signal-regulated kinaseJNK (or SAPK)c-Jun NH2-terminal kinase (or stress-activated protein kinase).) (2Cano E. Mahadevan L.C. Trends Biochem. Sci. 1995; 20: 117-122Abstract Full Text PDF PubMed Scopus (996) Google Scholar). The function of MAPKs is to convert extracellular stimuli to intracellular signals which, in turn, control the expression of genes that are essential for many cellular processes, including cell growth and differentiation(3Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4223) Google Scholar). MAPKs have been classified in three subfamilies: extracellular signal regulated kinases (ERKs), including ERK1 and ERK2, also known as p44mapk and p42mapk, respectively; stress-activated protein kinases (SAPKs), also termed c-Jun NH2-terminal kinases (JNKs); and p38 kinase(2Cano E. Mahadevan L.C. Trends Biochem. Sci. 1995; 20: 117-122Abstract Full Text PDF PubMed Scopus (996) Google Scholar). ERKs phosphorylate and regulate the activity of certain enzymes, including phospholipase A2 and p90rsk, and nuclear proteins, such as the ternary complex factor p62TCF or Elk-1(4Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar). The latter represents a critical event in controlling the expression of several genes, including c-fos(5Hill C.S. Treisman R. Cell. 1995; 80: 199-211Abstract Full Text PDF PubMed Scopus (1195) Google Scholar). JNKs phosphorylate the amino-terminal transactivating domain of c-Jun and ATF2(6Gupta S. Campbell D. Derijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1336) Google Scholar), thereby increasing their transcriptional activity. p38 is the homologue of the Saccharomyces cerevisiae HOG1 gene, and its function is still unknown, although recently available information suggests that p38 might play a critical role in the inflammatory response(7Lee J.C. Laydon J.T. Mc Donnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3121) Google Scholar).Recent findings have helped to unveil the pathway linking cell surface receptors to ERKs(3Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4223) Google Scholar). In contrast, the mechanism of activation of JNKs is still poorly understood. JNKs were shown to be activated by a variety of stimuli distinct from those that elevate the enzymatic activity of ERKs, including protein synthesis inhibitors, heat shock, changes in osmolarity, and ultraviolet irradiation(6Gupta S. Campbell D. Derijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1336) Google Scholar, 8Kyriakis J. Banerjee P. Nikolakaki E. Dai T. Rubie E. Ahmad M. Avruch J. Woodgett J. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2409) Google Scholar). JNKs can be also activated by agents acting on cell surface receptors, such as tumor necrosis factor-α, interleukin-1, or epidermal growth factor (8Kyriakis J. Banerjee P. Nikolakaki E. Dai T. Rubie E. Ahmad M. Avruch J. Woodgett J. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2409) Google Scholar). Furthermore, available evidence suggest that whereas Ras controls the activation of ERKs, members of the Rho family of small GTP-binding proteins, Rac1 and Cdc42, regulate the activity of JNKs(9Coso O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1559) Google Scholar).Recently, we have shown that stimulation of certain G protein-coupled receptors expressed in NIH 3T3 cells can effectively induce JNK activity, however, following a time course clearly distinct from that of ERK activation(1Coso O.A. Chiariello M. Kalinec G. Kyriakis J.M. Woodgett J. Gutkind J.S. J. Biol. Chem. 1995; 270: 5620-5624Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). In the present study, we have used the transient expression in COS-7 cells of m1 and m2 muscarinic receptors (mAChRs) as a model system to study the biochemical route connecting G protein-coupled receptors to an epitope-tagged JNK. We present evidence that signaling from m1 and m2 mAChRs to JNK involves βγ subunits of heterotrimeric G proteins, acting on a Ras- and Rac1-dependent pathway.