Analysis of the Gs/Mitogen-activated Protein Kinase Pathway in Mutant S49 Cells

Heterotrimeric G protein-coupled receptors can activate the mitogen-activated protein kinase (MAPK) cascade. Recent studies using pharmacological inhibitors or dominant-negative mutants of signaling molecules have advanced our understanding of the pathways from G protein-coupled receptors to MAPK. However, molecular genetic analysis of these pathways is inadequate in mammalian cells. Here, using the well characterized Gsα- and protein kinase A-deficient S49 mouse lymphoma cells, we provide the molecular genetic evidence that Gsα is responsible for transducing the β-adrenergic receptor signal to MAPK in a protein kinase A-dependent pathway involving Rap1 and Raf (but not Ras) molecules. Heterotrimeric G protein-coupled receptors can activate the mitogen-activated protein kinase (MAPK) cascade. Recent studies using pharmacological inhibitors or dominant-negative mutants of signaling molecules have advanced our understanding of the pathways from G protein-coupled receptors to MAPK. However, molecular genetic analysis of these pathways is inadequate in mammalian cells. Here, using the well characterized Gsα- and protein kinase A-deficient S49 mouse lymphoma cells, we provide the molecular genetic evidence that Gsα is responsible for transducing the β-adrenergic receptor signal to MAPK in a protein kinase A-dependent pathway involving Rap1 and Raf (but not Ras) molecules. G proteins serve their physiological roles by transducing signals from a broad class of cell-surface receptors to specific effector proteins (1Gilman A. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4682) Google Scholar, 2Bourne H.R. Sanders D.A. McCormick F. Nature. 1991; 349: 117-127Crossref PubMed Scopus (2660) Google Scholar, 3Simon M.I. Strathmann M.P. Gautam N. Science. 1991; 252: 802-808Crossref PubMed Scopus (1574) Google Scholar, 4Clapham D.E. Neer E.J. Nature. 1993; 365: 403-406Crossref PubMed Scopus (587) Google Scholar). A variety of intracellular signal transduction pathways are regulated by G proteins, including the mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated protein kinase kinase; PKA, protein kinase A. 1The abbreviations used are: MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated protein kinase kinase; PKA, protein kinase A.pathway (5Post G.R. Brown J.H. FASEB J. 1996; 10: 741-749Crossref PubMed Scopus (198) Google Scholar). Although the activation mechanism of the MAPK cascade by receptors with intrinsic tyrosine kinase activity has been well studied, the route from G proteins to the MAPK cascade in mammalian cells is less understood (6Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3187) Google Scholar, 7Herskowitz I. Cell. 1995; 80: 187-197Abstract Full Text PDF PubMed Scopus (863) Google Scholar).Recent studies in cultured cell lines with pharmacological inhibitors and dominant-negative mutants of certain signaling molecules have revealed the participation of some molecular components in the regulation of MAPK by G protein-coupled receptors (for review, see Ref.5Post G.R. Brown J.H. FASEB J. 1996; 10: 741-749Crossref PubMed Scopus (198) Google Scholar). Although the detailed biochemical steps are far from clear, these studies have shown that G protein-coupled receptors use pathways very similar to those utilized by receptor tyrosine kinases to activate the prototype Raf/MEK/MAPK cascade. Gq- and Gi-coupled receptors transmit the signals to MAPK through a pathway involving tyrosine kinase, adapter proteins Shc and Grb2, guanine nucleotide exchange factor Sos, Ras, and Raf in most cases (for review, see Ref. 5Post G.R. Brown J.H. FASEB J. 1996; 10: 741-749Crossref PubMed Scopus (198) Google Scholar). Phosphatidylinositol 3-kinase has been implicated to act upstream of tyrosine kinases in the Gi/MAPK pathway in some cells (8Ferby I.M. Waga I. Sakanaka C. Kume K. Shimizu T. J. Biol. Chem. 1994; 269: 30485-30488Abstract Full Text PDF PubMed Google Scholar, 9Pace A.M. Faure M. Bourne H.R. Mol. Biol. Cell. 1995; 6: 1685-1695Crossref PubMed Scopus (65) Google Scholar, 10Hawes B.E. Luttrell L.M. van Biesen T. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 12133-12136Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 11Cowen D.S. Sowers R.S. Manning D.R. J. Biol. Chem. 1996; 271: 22297-22300Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 12Lopez-Ilasaca M. Crespo P. Pellici P.G. Gutkind J.S. Wetzker R. Science. 1997; 275: 394-397Crossref PubMed Scopus (627) Google Scholar).For receptors coupled to Gs, overexpressing Gβγ or Gsα subunits in COS-7 cells has shown that whereas Gβγ subunits have the capacity to stimulate MAPK, the ability of Gsα to stimulate MAPK is controversial (13Faure M. Voyno-Yasenetskaya T.A. Bourne H.R. J. Biol. Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google Scholar, 14Crespo 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). It is also unclear if cAMP and protein kinase A (PKA) participate in the Gs-coupled receptor/MAPK pathway. Whereas one group reported that cAMP, forskolin, and Gs-coupled receptors can stimulate MAPK in COS-7 cells (13Faure M. Voyno-Yasenetskaya T.A. Bourne H.R. J. Biol. Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google Scholar), another reported that cAMP and PKA do not mediate activation of MAPK by Gs-coupled receptors in COS-7 cells (14Crespo 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). It was proposed that the Gs-coupled β-adrenergic receptor used the Gβγ subunit to activate the MAPK pathway through Ras and used the Gsα subunit to inhibit MAPK activation through cAMP and PKA (14Crespo 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). These contradictory results regarding whether the α-subunit or the βγ-subunits of Gs protein mediate the receptor stimulation of MAPK and whether PKA is involved in the Gs/MAPK pathway in mammalian cells prompted us to address this question genetically. For the most part, signaling by heterotrimeric G proteins has not been studied genetically in mammalian cells.S49 mouse lymphoma cells have played an important historical role in G protein research (15Gilman A.G. Biosci. Rep. 1995; 15: 65-97Crossref PubMed Scopus (175) Google Scholar). A variant of S49 cells lacking Gsα was instrumental in defining the function of and characterizing Gsα (15Gilman A.G. Biosci. Rep. 1995; 15: 65-97Crossref PubMed Scopus (175) Google Scholar). Elevation of intracellular cAMP levels results in growth arrest in the G1 phase of the cell cycle and later (after several days) in cell death (16Coffino P. Bourne H.R. Tomkins G.M. J. Cell. Physiol. 1975; 85: 603-610Crossref PubMed Scopus (69) Google Scholar, 17Bourne H.R. Coffino P. Tomkins G.M. J. Cell. Physiol. 1975; 85: 611-620Crossref PubMed Scopus (51) Google Scholar). Mutants have been selected that are resistant to cytolysis. These mutants includecyc − (which lacks Gsα) (18Bourne H.R. Coffino P. Tomkins G.M. Science. 1975; 187: 750-752Crossref PubMed Scopus (144) Google Scholar),UNC(which has a mutation of arginine at position 372 of Gsα and thus uncouples the interaction of Gsα with the receptors) (19Haga T. Ross E.M. Anderson H.J. Gilma A.G. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 2016-2020Crossref PubMed Scopus (94) Google Scholar), andkin − (which lacks protein kinase A activity) (20Insel P.A. Bourne H.R. Coffino P. Tomkins G.M. Science. 1975; 190: 896-897Crossref PubMed Scopus (117) Google Scholar).These Gsα and PKA mutant S49 cells should be very useful in a molecular genetic study to understand the role of Gsα and PKA in the β-adrenergic receptor/MAPK signaling system. In this study, using these mutant S49 cells, we demonstrate that Gsα transduces the β-adrenergic receptor signal to MAPK in a protein kinase A-dependent pathway involving Rap1 and Raf (but not Ras) molecules.DISCUSSIONIn summary, using various mutant S49 mouse lymphoma cells, we have provided the first genetic evidence for Gsα and protein kinase A transducing the β-adrenergic receptor signal to MAPK. Incyc − mutant S49 cells that lack Gsα proteins, stimulation of the β-adrenergic receptor failed to activate MAPK. In UNC mutant S49 cells that Gsα is unable to couple to the β-adrenergic receptor, MAPK could not be stimulated by the β-receptor. Two Gsα mutants that can complex with the Gβγ subunit and β-receptor, but are unable to stimulate adenylyl cyclase, failed to rescue thecyc − mutant response to β-receptor stimulation of MAPK. Wild-type Gsα rescued thecyc − cell response. Furthermore, inkin − mutant S49 cells that lack protein kinase A stimulation of MAPK by the β-receptor was dominant-negative mutants of Rap1 or but not Ras, the MAPK These demonstrate that the Gs-coupled receptor transducing the signal to a and but pathway to MAPK activation in S49 mouse lymphoma Faure (13Faure M. Voyno-Yasenetskaya T.A. Bourne H.R. J. Biol. Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google have shown that overexpressing Gβγ or Gsα could to activation of MAPK and that cAMP, forskolin, and Gs-coupled receptors could stimulate MAPK in COS-7 cells. the Crespo (14Crespo 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 reported that of Gβγ but not the Gsα could MAPK activity in COS-7 cells. It was proposed that in COS-7 cells, whereas Gβγ transduces a signal to MAPK through protein kinase the MAPK stimulation (14Crespo 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). for this is not that Gβγ the β-adrenergic receptor signal to MAPK is of (14Crespo 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). is of Gβγ could to MAPK is that of a from the β-adrenergic receptor kinase protein or could the stimulation of MAPK by β-adrenergic unable to a genetic analysis of the role of Gβγ subunits to the lack of mutants of or subunits in S49 cells. Gβγ is in a role for the of G protein but is to a signaling role for the − mutant cells, the activity of MAPK is similar to that in cells. Gβγ is the signal in in − cells, MAPK could be in mutant cells in S. C. J. Cell. 1987; Full Text PDF PubMed Scopus Google I. M. N. C. A. K. K. K. Cell. 1987; Full Text PDF PubMed Scopus Google Scholar). mutant Gsα and subunits not rescue thecyc − cell response to Gβγ receptor in S49 cells, the of adenylyl can be stimulated by but not by Gβγ or A.G. Science. 1991; PubMed Scopus Google Scholar). These that if Gβγ is is for not for in S49 mouse lymphoma cells, not transduces the receptor signal to the the α-subunit of G protein the signal to the MAPK pathway T. M. M. Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar). In the S. the of G protein the receptor to the MAPK cascade C. J. Cell. 1987; Full Text PDF PubMed Scopus Google Scholar, I. M. N. C. A. K. K. K. Cell. 1987; Full Text PDF PubMed Scopus Google Scholar). that the α-subunit of Gs transduces Gs-coupled receptor signal to MAPK in S49 cells and that the of the from Gi-coupled receptors to MAPK (13Faure M. Voyno-Yasenetskaya T.A. Bourne H.R. J. Biol. Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google Scholar, P. Xu N. Gutkind J.S. Nature. 1994; PubMed Scopus Google Scholar, B.E. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar), these of the and subunits be of S. S. MAPK signaling mammalian cells have pathways that are utilized by of G cAMP and protein kinase A the MAPK pathway cell in some cells, are to the MAPK whereas in cells, are M. Bourne H.R. Mol. Biol. Cell. 1995; 6: PubMed Scopus Google Scholar, Biol. Sci. 1995; Full Text PDF PubMed Scopus Google Scholar). A study has that these or are by the of Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). kinase A the G protein in and to the activation of MAPK. that is in S49 cells and that could stimulate activity in S49 cells. we have the activation of MEK, an upstream of MAPK, and results similar to those for MAPK we the activation β-adrenergic pathway the first biochemical pathway for G G proteins serve their physiological roles by transducing signals from a broad class of cell-surface receptors to specific effector proteins (1Gilman A. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4682) Google Scholar, 2Bourne H.R. Sanders D.A. McCormick F. Nature. 1991; 349: 117-127Crossref PubMed Scopus (2660) Google Scholar, 3Simon M.I. Strathmann M.P. Gautam N. Science. 1991; 252: 802-808Crossref PubMed Scopus (1574) Google Scholar, 4Clapham D.E. Neer E.J. Nature. 1993; 365: 403-406Crossref PubMed Scopus (587) Google Scholar). A variety of intracellular signal transduction pathways are regulated by G proteins, including the mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated protein kinase kinase; PKA, protein kinase A. 1The abbreviations used are: MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated protein kinase kinase; PKA, protein kinase A.pathway (5Post G.R. Brown J.H. FASEB J. 1996; 10: 741-749Crossref PubMed Scopus (198) Google Scholar). Although the activation mechanism of the MAPK cascade by receptors with intrinsic tyrosine kinase activity has been well studied, the route from G proteins to the MAPK cascade in mammalian cells is less understood (6Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3187) Google Scholar, 7Herskowitz I. Cell. 1995; 80: 187-197Abstract Full Text PDF PubMed Scopus (863) Google Scholar). Recent studies in cultured cell lines with pharmacological inhibitors and dominant-negative mutants of certain signaling molecules have revealed the participation of some molecular components in the regulation of MAPK by G protein-coupled receptors (for review, see Ref.5Post G.R. Brown J.H. FASEB J. 1996; 10: 741-749Crossref PubMed Scopus (198) Google Scholar). Although the detailed biochemical steps are far from clear, these studies have shown that G protein-coupled receptors use pathways very similar to those utilized by receptor tyrosine kinases to activate the prototype Raf/MEK/MAPK cascade. Gq- and Gi-coupled receptors transmit the signals to MAPK through a pathway involving tyrosine kinase, adapter proteins Shc and Grb2, guanine nucleotide exchange factor Sos, Ras, and Raf in most cases (for review, see Ref. 5Post G.R. Brown J.H. FASEB J. 1996; 10: 741-749Crossref PubMed Scopus (198) Google Scholar). Phosphatidylinositol 3-kinase has been implicated to act upstream of tyrosine kinases in the Gi/MAPK pathway in some cells (8Ferby I.M. Waga I. Sakanaka C. Kume K. Shimizu T. J. Biol. Chem. 1994; 269: 30485-30488Abstract Full Text PDF PubMed Google Scholar, 9Pace A.M. Faure M. Bourne H.R. Mol. Biol. Cell. 1995; 6: 1685-1695Crossref PubMed Scopus (65) Google Scholar, 10Hawes B.E. Luttrell L.M. van Biesen T. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 12133-12136Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 11Cowen D.S. Sowers R.S. Manning D.R. J. Biol. Chem. 1996; 271: 22297-22300Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 12Lopez-Ilasaca M. Crespo P. Pellici P.G. Gutkind J.S. Wetzker R. Science. 1997; 275: 394-397Crossref PubMed Scopus (627) Google Scholar). For receptors coupled to Gs, overexpressing Gβγ or Gsα subunits in COS-7 cells has shown that whereas Gβγ subunits have the capacity to stimulate MAPK, the ability of Gsα to stimulate MAPK is controversial (13Faure M. Voyno-Yasenetskaya T.A. Bourne H.R. J. Biol. Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google Scholar, 14Crespo 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). It is also unclear if cAMP and protein kinase A (PKA) participate in the Gs-coupled receptor/MAPK pathway. Whereas one group reported that cAMP, forskolin, and Gs-coupled receptors can stimulate MAPK in COS-7 cells (13Faure M. Voyno-Yasenetskaya T.A. Bourne H.R. J. Biol. Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google Scholar), another reported that cAMP and PKA do not mediate activation of MAPK by Gs-coupled receptors in COS-7 cells (14Crespo 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). It was proposed that the Gs-coupled β-adrenergic receptor used the Gβγ subunit to activate the MAPK pathway through Ras and used the Gsα subunit to inhibit MAPK activation through cAMP and PKA (14Crespo 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). These contradictory results regarding whether the α-subunit or the βγ-subunits of Gs protein mediate the receptor stimulation of MAPK and whether PKA is involved in the Gs/MAPK pathway in mammalian cells prompted us to address this question genetically. For the most part, signaling by heterotrimeric G proteins has not been studied genetically in mammalian cells. S49 mouse lymphoma cells have played an important historical role in G protein research (15Gilman A.G. Biosci. Rep. 1995; 15: 65-97Crossref PubMed Scopus (175) Google Scholar). A variant of S49 cells lacking Gsα was instrumental in defining the function of and characterizing Gsα (15Gilman A.G. Biosci. Rep. 1995; 15: 65-97Crossref PubMed Scopus (175) Google Scholar). Elevation of intracellular cAMP levels results in growth arrest in the G1 phase of the cell cycle and later (after several days) in cell death (16Coffino P. Bourne H.R. Tomkins G.M. J. Cell. Physiol. 1975; 85: 603-610Crossref PubMed Scopus (69) Google Scholar, 17Bourne H.R. Coffino P. Tomkins G.M. J. Cell. Physiol. 1975; 85: 611-620Crossref PubMed Scopus (51) Google Scholar). Mutants have been selected that are resistant to cytolysis. These mutants includecyc − (which lacks Gsα) (18Bourne H.R. Coffino P. Tomkins G.M. Science. 1975; 187: 750-752Crossref PubMed Scopus (144) Google Scholar),UNC(which has a mutation of arginine at position 372 of Gsα and thus uncouples the interaction of Gsα with the receptors) (19Haga T. Ross E.M. Anderson H.J. Gilma A.G. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 2016-2020Crossref PubMed Scopus (94) Google Scholar), andkin − (which lacks protein kinase A activity) (20Insel P.A. Bourne H.R. Coffino P. Tomkins G.M. Science. 1975; 190: 896-897Crossref PubMed Scopus (117) Google Scholar). These Gsα and PKA mutant S49 cells should be very useful in a molecular genetic study to understand the role of Gsα and PKA in the β-adrenergic receptor/MAPK signaling system. In this study, using these mutant S49 cells, we demonstrate that Gsα transduces the β-adrenergic receptor signal to MAPK in a protein kinase A-dependent pathway involving Rap1 and Raf (but not Ras) molecules. summary, using various mutant S49 mouse lymphoma cells, we have provided the first genetic evidence for Gsα and protein kinase A transducing the β-adrenergic receptor signal to MAPK. Incyc − mutant S49 cells that lack Gsα proteins, stimulation of the β-adrenergic receptor failed to activate MAPK. In UNC mutant S49 cells that Gsα is unable to couple to the β-adrenergic receptor, MAPK could not be stimulated by the β-receptor. Two Gsα mutants that can complex with the Gβγ subunit and β-receptor, but are unable to stimulate adenylyl cyclase, failed to rescue thecyc − mutant response to β-receptor stimulation of MAPK. Wild-type Gsα rescued thecyc − cell response. Furthermore, inkin − mutant S49 cells that lack protein kinase A stimulation of MAPK by the β-receptor was dominant-negative mutants of Rap1 or but not Ras, the MAPK These demonstrate that the Gs-coupled receptor transducing the signal to a and but pathway to MAPK activation in S49 mouse lymphoma Faure (13Faure M. Voyno-Yasenetskaya T.A. Bourne H.R. J. Biol. Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google have shown that overexpressing Gβγ or Gsα could to activation of MAPK and that cAMP, forskolin, and Gs-coupled receptors could stimulate MAPK in COS-7 cells. the Crespo (14Crespo 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 reported that of Gβγ but not the Gsα could MAPK activity in COS-7 cells. It was proposed that in COS-7 cells, whereas Gβγ transduces a signal to MAPK through protein kinase the MAPK stimulation (14Crespo 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). for this is not that Gβγ the β-adrenergic receptor signal to MAPK is of (14Crespo 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). is of Gβγ could to MAPK is that of a from the β-adrenergic receptor kinase protein or could the stimulation of MAPK by β-adrenergic unable to a genetic analysis of the role of Gβγ subunits to the lack of mutants of or subunits in S49 cells. Gβγ is in a role for the of G protein but is to a signaling role for the − mutant cells, the activity of MAPK is similar to that in cells. Gβγ is the signal in in − cells, MAPK could be in mutant cells in S. C. J. Cell. 1987; Full Text PDF PubMed Scopus Google I. M. N. C. A. K. K. K. Cell. 1987; Full Text PDF PubMed Scopus Google Scholar). mutant Gsα and subunits not rescue thecyc − cell response to Gβγ receptor in S49 cells, the of adenylyl can be stimulated by but not by Gβγ or A.G. Science. 1991; PubMed Scopus Google Scholar). These that if Gβγ is is for not for in S49 mouse lymphoma cells, not transduces the receptor signal to the the α-subunit of G protein the signal to the MAPK pathway T. M. M. Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar). In the S. the of G protein the receptor to the MAPK cascade C. J. Cell. 1987; Full Text PDF PubMed Scopus Google Scholar, I. M. N. C. A. K. K. K. Cell. 1987; Full Text PDF PubMed Scopus Google Scholar). that the α-subunit of Gs transduces Gs-coupled receptor signal to MAPK in S49 cells and that the of the from Gi-coupled receptors to MAPK (13Faure M. Voyno-Yasenetskaya T.A. Bourne H.R. J. Biol. Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google Scholar, P. Xu N. Gutkind J.S. Nature. 1994; PubMed Scopus Google Scholar, B.E. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar), these of the and subunits be of S. S. MAPK signaling mammalian cells have pathways that are utilized by of G cAMP and protein kinase A the MAPK pathway cell in some cells, are to the MAPK whereas in cells, are M. Bourne H.R. Mol. Biol. Cell. 1995; 6: PubMed Scopus Google Scholar, Biol. Sci. 1995; Full Text PDF PubMed Scopus Google Scholar). A study has that these or are by the of Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). kinase A the G protein in and to the activation of MAPK. that is in S49 cells and that could stimulate activity in S49 cells. we have the activation of MEK, an upstream of MAPK, and results similar to those for MAPK we the activation β-adrenergic pathway the first biochemical pathway for G In summary, using various mutant S49 mouse lymphoma cells, we have provided the first genetic evidence for Gsα and protein kinase A transducing the β-adrenergic receptor signal to MAPK. Incyc − mutant S49 cells that lack Gsα proteins, stimulation of the β-adrenergic receptor failed to activate MAPK. In UNC mutant S49 cells that Gsα is unable to couple to the β-adrenergic receptor, MAPK could not be stimulated by the β-receptor. Two Gsα mutants that can complex with the Gβγ subunit and β-receptor, but are unable to stimulate adenylyl cyclase, failed to rescue thecyc − mutant response to β-receptor stimulation of MAPK. Wild-type Gsα rescued thecyc − cell response. Furthermore, inkin − mutant S49 cells that lack protein kinase A stimulation of MAPK by the β-receptor was dominant-negative mutants of Rap1 or but not Ras, the MAPK These demonstrate that the Gs-coupled receptor transducing the signal to a and but pathway to MAPK activation in S49 mouse lymphoma cells. Faure (13Faure M. Voyno-Yasenetskaya T.A. Bourne H.R. J. Biol. Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google have shown that overexpressing Gβγ or Gsα could to activation of MAPK and that cAMP, forskolin, and Gs-coupled receptors could stimulate MAPK in COS-7 cells. the Crespo (14Crespo 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 reported that of Gβγ but not the Gsα could MAPK activity in COS-7 cells. It was proposed that in COS-7 cells, whereas Gβγ transduces a signal to MAPK through protein kinase the MAPK stimulation (14Crespo 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). for this is not that Gβγ the β-adrenergic receptor signal to MAPK is of (14Crespo 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). is of Gβγ could to MAPK is that of a from the β-adrenergic receptor kinase protein or could the stimulation of MAPK by β-adrenergic unable to a genetic analysis of the role of Gβγ subunits to the lack of mutants of or subunits in S49 cells. Gβγ is in a role for the of G protein but is to a signaling role for the − mutant cells, the activity of MAPK is similar to that in cells. Gβγ is the signal in in − cells, MAPK could be in mutant cells in S. C. J. Cell. 1987; Full Text PDF PubMed Scopus Google I. M. N. C. A. K. K. K. Cell. 1987; Full Text PDF PubMed Scopus Google Scholar). mutant Gsα and subunits not rescue thecyc − cell response to Gβγ receptor in S49 cells, the of adenylyl can be stimulated by but not by Gβγ or A.G. Science. 1991; PubMed Scopus Google Scholar). These that if Gβγ is is for not for in S49 mouse lymphoma cells, not transduces the receptor signal to MAPK. In the the α-subunit of G protein the signal to the MAPK pathway T. M. M. Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar). In the S. the of G protein the receptor to the MAPK cascade C. J. Cell. 1987; Full Text PDF PubMed Scopus Google Scholar, I. M. N. C. A. K. K. K. Cell. 1987; Full Text PDF PubMed Scopus Google Scholar). that the α-subunit of Gs transduces Gs-coupled receptor signal to MAPK in S49 cells and that the of the from Gi-coupled receptors to MAPK (13Faure M. Voyno-Yasenetskaya T.A. Bourne H.R. J. Biol. Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google Scholar, P. Xu N. Gutkind J.S. Nature. 1994; PubMed Scopus Google Scholar, B.E. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar), these of the and subunits be of S. S. MAPK signaling mammalian cells have pathways that are utilized by of G cAMP and protein kinase A the MAPK pathway cell in some cells, are to the MAPK whereas in cells, are M. Bourne H.R. Mol. Biol. Cell. 1995; 6: PubMed Scopus Google Scholar, Biol. Sci. 1995; Full Text PDF PubMed Scopus Google Scholar). A study has that these or are by the of Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). kinase A the G protein in and to the activation of MAPK. that is in S49 cells and that could stimulate activity in S49 cells. we have the activation of MEK, an upstream of MAPK, and results similar to those for MAPK we the activation β-adrenergic pathway the first biochemical pathway for G T. A. and P. for are to R. T. and the of our for the