Activation of Mitogen-activated Protein Kinase by the A2A-adenosine Receptor via a rap1-dependent and via a p21 -dependent Pathway

Abstract

The A2A-adenosine receptor, a prototypical Gs-coupled receptor, activates mitogen-activated protein (MAP) kinase in a manner independent of cAMP in primary human endothelial cells. In order to delineate signaling pathways that link the receptor to the regulation of MAP kinase, the human A2A receptor was heterologously expressed in Chinese hamster ovary (CHO) and HEK293 cells. In both cell lines, A2A agonist-mediated cAMP accumulation was accompanied by activation of the small G protein rap1. However, rap1 mediates A2A receptor-dependent activation of MAP kinase only in CHO cells, the signaling cascade being composed of Gs, adenylyl cyclase, rap1, and the p68 isoform of B-raf. This isoform was absent in HEK293 cells. Contrary to CHO cells, in HEK293 cells activation of MAP kinase by A2A agonists was not mimicked by 8-bromo-cAMP, was independent of Gαs, and was associated with activation of p21ras. Accordingly, overexpression of the inactive S17N mutant of p21ras and of a dominant negative version of mSos (the exchange factor of p21ras) blocked MAP kinase stimulation by the A2A receptor in HEK 293 but not in CHO cells. In spite of the close homology between p21ras and rap1, the S17N mutant of rap1 was not dominant negative because (i) overexpression of rap1(S17N) failed to inhibit A2A receptor-dependent MAP kinase activation, (ii) rap1(S17N) was recovered in the active form with a GST fusion protein comprising the rap1-binding domain of ralGDS after A2A receptor activation, and (iii) A2A agonists promoted the association of rap1(S17N) with the 68-kDa isoform of B-raf in CHO cells. We conclude that the A2A receptor has the capacity two activate MAP kinase via at least two signaling pathways, which depend on two distinct small G proteins, namely p21ras and rap1. Our observations also show that the S17N version of rap1 cannot be assumed a priori to act as a dominant negative interfering mutant. The A2A-adenosine receptor, a prototypical Gs-coupled receptor, activates mitogen-activated protein (MAP) kinase in a manner independent of cAMP in primary human endothelial cells. In order to delineate signaling pathways that link the receptor to the regulation of MAP kinase, the human A2A receptor was heterologously expressed in Chinese hamster ovary (CHO) and HEK293 cells. In both cell lines, A2A agonist-mediated cAMP accumulation was accompanied by activation of the small G protein rap1. However, rap1 mediates A2A receptor-dependent activation of MAP kinase only in CHO cells, the signaling cascade being composed of Gs, adenylyl cyclase, rap1, and the p68 isoform of B-raf. This isoform was absent in HEK293 cells. Contrary to CHO cells, in HEK293 cells activation of MAP kinase by A2A agonists was not mimicked by 8-bromo-cAMP, was independent of Gαs, and was associated with activation of p21ras. Accordingly, overexpression of the inactive S17N mutant of p21ras and of a dominant negative version of mSos (the exchange factor of p21ras) blocked MAP kinase stimulation by the A2A receptor in HEK 293 but not in CHO cells. In spite of the close homology between p21ras and rap1, the S17N mutant of rap1 was not dominant negative because (i) overexpression of rap1(S17N) failed to inhibit A2A receptor-dependent MAP kinase activation, (ii) rap1(S17N) was recovered in the active form with a GST fusion protein comprising the rap1-binding domain of ralGDS after A2A receptor activation, and (iii) A2A agonists promoted the association of rap1(S17N) with the 68-kDa isoform of B-raf in CHO cells. We conclude that the A2A receptor has the capacity two activate MAP kinase via at least two signaling pathways, which depend on two distinct small G proteins, namely p21ras and rap1. Our observations also show that the S17N version of rap1 cannot be assumed a priori to act as a dominant negative interfering mutant. 8-bromo-cAMP mitogen-activated protein kinase (two isoforms of which exist: erk1 and erk2) influenza hemagglutinin epitope MAP kinase kinase protein kinase A (cAMP-dependent protein kinase) protein kinase C glutathione S-transferase 5′-N-ethylcarboxamidoadenosine N-ethylcarboxamido-2-[4-(2-carboxyethyl)phenylethyl]adenosine N 6-cyclopentyladenosine xanthine amine congener 2′-amino-3′-methoxyflavone bisindoylmaleimide I basic fibroblast growth factor phorbol 12,13-dibutyrate N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide Chinese hamster ovary Adenosine is ubiquitously released from metabolically active cells and is rapidly generated in the extracellular space by dephosphorylation of ATP that has been released from nerve terminals as a cotransmitter. Hypoxia leads to accumulation of excessive amounts of the nucleoside. In the extracellular space, adenosine serves as an autacoid that regulates the function of virtually every organ and tissue via four different classes of G protein-coupled receptor subtypes, designated A1-, A2A-, A2B, and A3-adenosine receptor (1Collis M.G. Hourani S.M.O. Trends Pharmacol. Sci. 1993; 14: 360-366Abstract Full Text PDF PubMed Scopus (388) Google Scholar). These differ by their affinity for the endogenous agonist as well as by their pharmacological specificity. In addition, they operate through distinct signaling mechanisms. The A1- and A3-adenosine receptors control most, if not all, their cellular effectors via pertussis toxin-sensitive G proteins of the Gi and Go family; in contrast, both A2A- and A2B-adenosine receptors couple to Gs and thereby stimulate cAMP formation (2Fredholm B.B. Abbracchio M.P. Burnstock G. Daly J.W. Harden T.K. Jacobson K.A. Leff P. Williams M. Pharmacol. Rev. 1994; 46: 143-156PubMed Google Scholar). In primary cultures of endothelial cells from various species and vascular beds, adenosine stimulates proliferation (3Meininger C.J. Granger H.J. Am. J. Physiol. 1990; 258: H198-H206PubMed Google Scholar, 4Ethier M.F. Chander V. Dobson J.G. Am. J. Physiol. 1993; 265: H131-H138PubMed Google Scholar), an observation that is consistent with a possible role of adenosine in angiogenesis (5Folkman J. Shing Y. J. Biol. Chem. 1992; 267: 10931-10934Abstract Full Text PDF PubMed Google Scholar). In human endothelial cells derived from the umbilical vein, the mitogenic effect is mediated by the A2A-adenosine receptor (6Sexl V. Mancusi G. Baumgartner-Parzer S. Schütz W. Freissmuth M. Br. J Pharmacol. 1995; 114: 1577-1586Crossref PubMed Scopus (68) Google Scholar); however, the available evidence indicates that growth stimulation is not mediated by cAMP, because forskolin, an activator of adenylyl cyclase, and membrane-permeable cAMP analogs such as 8-Br-cAMP1 inhibit endothelial cell growth (6Sexl V. Mancusi G. Baumgartner-Parzer S. Schütz W. Freissmuth M. Br. J Pharmacol. 1995; 114: 1577-1586Crossref PubMed Scopus (68) Google Scholar, 7Leitman D.C. Fiscus R.R. Murad F. J. Cell. Physiol. 1986; 127: 237-243Crossref PubMed Scopus (45) Google Scholar). In addition, the endothelial A2A-adenosine receptor stimulates phosphorylation of the MAP kinase isoforms erk1 and erk2 (8Sexl V. Mancusi G. Höller C. Gloria-Maercker E. Schütz W. Freissmuth M. J. Biol. Chem. 1997; 272: 5792-5799Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) and of p70S6 kinase (9Höller C. Milligan G. Anderson N. Freissmuth M. Drug Dev. Res. 1998; 45: 145-150Crossref Google Scholar), effects that cannot be mimicked by 8-Br-cAMP. It has long been appreciated that elevated levels of cAMP inhibit the proliferation of many cells (10Pastan I.H. Johnson G.S. Anderson W.B. Annu. Rev. Biochem. 1975; 44: 491-522Crossref PubMed Scopus (546) Google Scholar). This effect is thought to arise, at least in part, from inhibition of the interaction of p21ras with the downstream kinase raf-1, an effect that leads to cAMP-mediated suppression of the MAP kinase pathway (11Wu J. Dent P. Jelinek T. Wolfmann A. Weber M.J Sturgill T.W. Science. 1993; 262: 1066-1069Crossref Scopus (824) Google Scholar, 12Cook S.J. McCormick F. Science. 1993; 262: 1069-1072Crossref PubMed Scopus (865) Google Scholar, 13Burgering B.M. Pronk G.J. van Weeren P.C. Chardin P. Bos J.L. EMBO J. 1993; 12: 4211-4220Crossref PubMed Scopus (316) Google Scholar, 14Graves L.M. Bornfeldt K.E. Raines E.W. Potts B.C. Macdonald S.G. Ross R. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10300-10304Crossref PubMed Scopus (404) Google Scholar). However, in some cells activation of PKA does to activation of MAP kinase, the being in cell the stimulation of MAP kinase by analogs of cAMP has been to activation of B-raf via rap1, of the of small proteins M.G. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). These that adenylyl activation to activate MAP kinase in a manner on rap1 but independent of p21ras. This has been in cells the receptor Gs and rap1, but not to to MAP kinase Y. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). In contrast, in HEK293 cells, the receptor has been to activate MAP kinase via a pathway that phosphorylation of the the G protein of the receptor to from Gs to the activation of in G protein to the stimulation of MAP kinase via activation of p21ras Y. L.M. 1997; PubMed Scopus Google Scholar). It is not if of is to Gs-coupled In the the by which the A2A-adenosine receptor, a Gs-coupled receptor, regulates MAP kinase after in two cell lines, HEK293 and In both cell lines, cAMP was generated and MAP kinase was in to agonist However, MAP kinase activation was via distinct signaling pathways in the two cell In cells, MAP kinase activation Gαs, cAMP, rap1, and in contrast, in cells, MAP kinase activation was independent of Gs and cAMP and p21ras. adenosine basic fibroblast growth factor the and from was from was from and from The for from was from and from was from cAMP, protein forskolin, pertussis and from The and the of erk1 and erk2 of was from The of protein kinase A was from The protein was from and from was from and protein was from and tissue from and from and from The for the human A2A-adenosine receptor in the and the HEK293 as well as the CHO cell the human A2A-adenosine receptor J. J. C. Pharmacol. 1998; PubMed Scopus Google Scholar), by M. J. of The the for of a MAP kinase from U. and J. of the for of dominant negative mSos from Y. and R. J. the of the domain of to GST as well as for and from and A. the for of a GST fusion protein of the rap1 domain of ralGDS and for of a version of from and J. Bos of The and derived was from was from and and from The on by from HEK293 cells in CHO cells in at and with for the of cells with in order to the at a of and with of the the cells at a of the of a a of protein from the as a control to by in with the to the of The was to after and with adenosine was on after to the MAP kinase at cells also cells from on a four The for and for and MAP kinase stimulation was as cultures of human endothelial cells from umbilical and as D.C. Fiscus R.R. Murad F. J. Cell. Physiol. 1986; 127: 237-243Crossref PubMed Scopus (45) Google Scholar). to on and by for to MAP kinase in order to The was with adenosine to of cells with was also for in adenosine not cells by of agonists at for in order to that the of which in of the levels of MAP kinase phosphorylation the to The to agonists was by rapidly with the was in cells by of of to with The cellular was by at for and the protein was to cells of in and to MAP kinase activation was by with an that only the of and MAP in order to that the to the of amounts of control also with an both the and The by of the proteins was in an the GST fusion protein of the domain of J. Bos J.L. 1997; 14: PubMed Scopus Google Scholar) as well as of the rap1-binding domain of ralGDS Bos J.L. EMBO J. 1997; PubMed Scopus Google Scholar) expressed in by as GST fusion proteins by for at with in in order to GST fusion for the in a manner to that for MAP kinase and with adenosine for if not with agonists was for by rapidly with and of to cell by for The with the of a of GST fusion for to for the association of p21ras rap1 with the fusion in in and to p21ras rap1 in a of to in was to stimulation of the cells and was the at the and adenosine of the agonists for the was for the in the in The formation of was to Y. PubMed Scopus Google Scholar). was at least of and cells with the agonist in a accumulation of cAMP effect was not in control cells for CHO in The of in formation of was in both cell levels of accumulation in in In and cells and MAP kinase a that was to that for adenylyl activation and that was to that for MAP kinase activation in primary human endothelial cells of MAP kinase was mediated by the A2A-adenosine receptor, because (i) the agonist was for in and and (ii) the adenosine receptor the activation of MAP kinase a of for in (iii) MAP kinase stimulation by was not in control cells not In the of an of MAP kinase kinase A. P. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), the stimulation of MAP kinase by was for in is an of MAP kinase in the signaling cascade that the A2A-adenosine receptor to MAP of the of the protein to This leads to activation of Gαs, in a stimulation of downstream in cells, the effect is to of after long G. Biochem. J. 262: PubMed Scopus Google Scholar). We and cells to for from to and the of a different was in the two cell In cells accumulation of cAMP the was after of to and was by a of cAMP accumulation such that at after of cAMP to the with a that the levels of not In contrast, in cells, the of cAMP at was cells, cAMP levels and a that from to was A of was also not in cells by if cells for to to not Accordingly, of cAMP after of with in in the of but was virtually in We to the of in cells to by the of and of the cAMP in was to if MAP kinase stimulation by the A2A receptor be in the of the of MAP kinase to the A2A agonist was in cells that been for with This that in HEK cells the A2A-adenosine signaling in to in the of Gs that the MAP kinase In contrast, after with the of MAP kinase phosphorylation was in cells and failed to stimulation of MAP kinase of by the phorbol MAP kinase phosphorylation in cells that the of an A2A agonist effect a of the cells. It is that the of MAP kinase phosphorylation is by the of cAMP which stimulation of MAP kinase by on MAP kinase In HEK293 cells, the receptor G protein from Gs to for pertussis toxin-sensitive activation of MAP kinase in to agonists Y. L.M. 1997; PubMed Scopus Google Scholar). We the A2A-adenosine MAP kinase activation was on pertussis toxin-sensitive G However, of by pertussis of and of cells effect on MAP kinase phosphorylation agonist In in the of cells with pertussis signaling by prototypical receptors such as the and receptors and of the to be not also M. E. Milligan G. Freissmuth M. C. Pharmacol. 1998; Google Scholar). also with the of and pertussis This many cells to from the in the cells MAP kinase stimulation was stimulation not isoforms be by receptors via of which to activation and isoforms Annu. Rev. Pharmacol. 1997; PubMed Scopus Google Scholar) as to stimulation of by also cells with an of the phosphorylation of MAP kinase was by the stimulation by We that pathway is in the MAP kinase stimulation of the A2A-adenosine receptor in HEK293 cells. of cells with the phosphorylation of MAP kinase in the of effect to activation of PKA to a of released from a membrane-permeable cAMP to the effect of This was the of to in MAP kinase activation the stimulation of MAP kinase phosphorylation by both and was blocked by the protein kinase A In contrast, of to not stimulate MAP kinase to of A and activation by a of was The of effect is not to the of to because the the effect of stimulation of adenylyl by in MAP kinase phosphorylation only in but not in cells not in cells, MAP kinase activation by the be for by the of in contrast, in cells, signaling pathway is The small p21ras a role as an of MAP In MAP kinase cAMP an effect by the activation of (11Wu J. Dent P. Jelinek T. Wolfmann A. Weber M.J Sturgill T.W. Science. 1993; 262: 1066-1069Crossref Scopus (824) Google Scholar, 12Cook S.J. McCormick F. Science. 1993; 262: 1069-1072Crossref PubMed Scopus (865) Google Scholar, 13Burgering B.M. Pronk G.J. van Weeren P.C. Chardin P. Bos J.L. EMBO J. 1993; 12: 4211-4220Crossref PubMed Scopus (316) Google Scholar, 14Graves L.M. Bornfeldt K.E. Raines E.W. Potts B.C. Macdonald S.G. Ross R. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10300-10304Crossref PubMed Scopus (404) Google Scholar). However, in some cell M.G. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), cAMP stimulates MAP kinase and effect is thought to be mediated via rap1, a small of the Y. T. Y. M. Cell. Full Text PDF PubMed Scopus Google Scholar). We the of the A2A-adenosine receptor to activate rap1 in and The for of rap1 is on the to the domain of ralGDS J. Bos J.L. 1997; 14: PubMed Scopus Google Scholar); ralGDS to rap1 with an affinity of affinity for is by of C. G. M. A. J. Biol. Chem. Full Text PDF PubMed Scopus Google Scholar). A GST fusion protein comprising the as to rap1 from control cells cells in the of adenosine and from cells with of rap1 was after stimulation with and the of stimulation was in both cell This observation is consistent with a role of rap1 in the A2A-adenosine receptor to MAP kinase in cells. In contrast, failed to activate of MAP kinase in cells that rap1 in A2A MAP kinase activation, the protein is expressed and is in cells. p21ras a role in the activation of MAP kinase by both kinase receptors and G protein-coupled for in p21ras activation of the receptor in both and The domain of to GST was as a to J. Bos J.L. 1997; 14: PubMed Scopus Google Scholar, C. G. F. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). In to rap1, activation of p21ras was only in not cells stimulation with that p21ras is in the activation of MAP kinase in cells. that a dominant negative mutant of p21ras the A2A-adenosine activation of MAP kinase in but not cells. We and cells with the dominant negative mutant and the MAP kinase In cells, of dominant negative p21ras only the activation of MAP kinase by the stimulation by A2A agonist and In contrast, of the MAP kinase by the A2A agonist and in cells via p21ras is for cell after of in negative However, in that was in both cell not the with the levels of accumulation of is from the in that was the in the levels in cannot for the in MAP kinase recovered in the in A. of the cells with a dominant negative version of mSos Y. L.M. 1997; PubMed Scopus Google Scholar), the exchange factor for the effect of of in cells MAP kinase activation by but not by and In contrast, in cells with the to both and was A dominant negative version of rap1 to regulation of MAP kinase in cells. We a however, expressed rap1(S17N) not the activation of MAP kinase in cells of the activator The dominant negative of rap1(S17N) has been N. G A. 1997; PubMed Scopus Google Scholar). Our observations also with a dominant negative effect of for the if cells a version of rap1(S17N) with was with from of cells. The of is that of endogenous rap1. be from endogenous rap1 by the the epitope for The of activation of and of endogenous rap1 was We also that rap1(S17N) is not inactive by to with an endogenous namely with B-raf of B-raf is thought to be in activation of PKA to stimulation of MAP kinase M.G. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, T. S. Y. J. Biol. Chem. 1995; Full Text Full Text PDF Scopus Google Scholar). that the of B-raf is distinct in and cells, for the in signaling pathways by the A2A-adenosine receptor in the two cell of cell with a the of B-raf that the 68-kDa isoform of B-raf was expressed in but not in cells in The role of B-raf isoform was as rap1(S17N) was expressed in and cells by the A2A agonist with as a and the of B-raf was by with an the epitope by for B-raf and In from association of B-raf to was by In cells, an association of the p68 isoform of B-raf with both activate rap1 in isoforms to be generated from the B-raf by C. A. G. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). However, not isoform p68 in the with rap1. We after stimulation of with the levels of p68 B-raf to not in a version of rap1 was levels of p68 B-raf recovered in the after stimulation with and not It is also that the levels of active after stimulation with endogenous rap1 A and observations that the rap1(S17N) a dominant negative effect on the generated by the receptor but not on that of the A2A G protein-coupled receptors control the of the MAP kinase cascade via a activation of T. E. M. L.M. 1995; PubMed Scopus Google Scholar), M. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar) and activation of isoforms M. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar), signaling via M.P. P. S. R. Science. 1997; PubMed Scopus Google Scholar), and activation of PKA M.G. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). In the show that the A2A-adenosine receptor at least two distinct signaling pathways that to MAP kinase is from observations that the cellular of signaling which pathway is In CHO cells, the A2A-adenosine receptor regulates MAP kinase phosphorylation via a cascade composed of Gαs, adenylyl cyclase, rap1, p68 and In contrast, in HEK293 cells, Gαs, cAMP, and rap1 not in the MAP kinase because the p68 isoform of B-raf is not the A2A-adenosine receptor on activation of p21ras. The receptor, which is expressed in HEK 293 cells, activates p21ras via from a phosphorylation of the receptor, which G from Gs to Gi Y. L.M. 1997; PubMed Scopus Google Scholar) and of the receptor Y. L.M. S. G. M.G. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). However, in the that on the A2A of cells with pertussis with the MAP kinase which in addition, to the PKA This is also consistent with the that the A2A-adenosine receptor phosphorylation for PKA T. T.W. Jacobson K.A. Pharmacol. 1994; 45: Google Scholar). in and isoforms of protein kinase C not in MAP kinase activation by the A2A This is also for endothelial cells (9Höller C. Milligan G. Anderson N. Freissmuth M. Drug Dev. Res. 1998; 45: 145-150Crossref Google Scholar), the mitogenic effect of adenosine cannot be for by activation of protein kinase C because leads to inhibition of cell growth J. V. C. A. Schütz W. Freissmuth M. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The small protein rap1 was as of the because was of the by Y. T. Y. M. Cell. Full Text PDF PubMed Scopus Google Scholar). This the of rap1 to p21ras from J. E. S.J. J. 1993; PubMed Scopus Google Scholar) and effectors M. J. Chardin P. A. R. McCormick F. A. Science. 1990; PubMed Scopus Google Scholar, N. G. C. A. McCormick F. A. 1995; PubMed Scopus Google Scholar). a form of rap1 stimulation of MAP kinase via p21ras S.J. McCormick F. EMBO J. 1993; 12: PubMed Scopus Google Scholar). However, rap1 also MAP kinase activation M.G. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). Accordingly, in cells, rap1 as an Y. M. Y. T. T. A. Y. Cell. Biol. 1992; 12: PubMed Scopus Google Scholar, F. Proc. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). to activation of mediated by an exchange factor N. G A. 1997; PubMed Scopus Google Scholar) and phosphorylation by J. Bos J.L. 1997; 14: PubMed Scopus Google Scholar); the between the two of regulation is not at J.L. 1997; PubMed Scopus Google Scholar). The S17N mutant of rap1 is thought to act in a dominant negative is on the with the in p21ras Cell. Biol. PubMed Scopus Google Scholar) and the of homology that the two proteins In the of by with the of of and thereby of the protein from the exchange such as and N. G A. 1997; PubMed Scopus Google Scholar, C.J. S. 1994; Google Scholar, J. 1998; PubMed Scopus Google Scholar). However, in the that of mutant the effect as a active version of rap1 C.J. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). show in rap1(S17N) is by and released from the exchange factor the mutant also to activation of rap1 N. G A. 1997; PubMed Scopus Google Scholar). These rap1(S17N) does not cellular Our in with because failed to a dominant negative of stimulation of cells by the A2A active rap1(S17N) was generated that is of with an domain from and an endogenous the p68 isoform of In contrast, rap1(S17N) has been to activation of MAP kinase M.G. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The of is exchange for rap1 N. S. M. M. Science. 1998; PubMed Scopus Google Scholar, J. A. Bos J.L. 1998; PubMed Scopus Google Scholar); rap1(S17N) to from some of The of to the association of rap1(S17N) with p68 B-raf In activation of MAP kinase by the A2A-adenosine receptor of the in endothelial cells (8Sexl V. Mancusi G. Höller C. Gloria-Maercker E. Schütz W. Freissmuth M. J. Biol. Chem. 1997; 272: 5792-5799Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), because signaling is independent of and cAMP but p21ras. The of that link activation of p21ras to the is not The only G which the A2A-adenosine receptor has been to couple is Gs C. S. M. Schütz W. Freissmuth M. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). However, has been appreciated that G protein signaling by receptors via a interaction with the of the receptors D.C. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, J. A. S. S. 1998; PubMed Scopus Google Scholar). The A2A-adenosine receptor has an which an role in signaling C. Jacobson K.A. Pharmacol. Google Scholar, C. J. Res. 1993; PubMed Scopus Google Scholar). role in MAP kinase activation is being We J. Y. R. J. M. J. U. J. and A. for and A. for in the