Key points are not available for this paper at this time.
The seven-membrane-spanning angiotensin II type 1A receptor activates the mitogen-activated protein kinases extracellular signal-regulated kinases 1 and 2 (ERK1/2) by distinct pathways dependent on either G protein (likely Gq/G11) or β-arrestin2. Here we sought to distinguish the kinetic and spatial patterns that characterize ERK1/2 activated by these two mechanisms. We utilized β-arrestin RNA interference, the protein kinase C inhibitor Ro-31-8425, a mutant angiotensin II receptor (DRY/AAY), and a mutant angiotensin II peptide (SII-angiotensin), which are incapable of activating G proteins, to isolate the two pathways in HEK-293 cells. G protein-dependent activation was rapid (peak <2 min), quite transient (t½ ∼2 min), and led to nuclear translocation of the activated ERK1/2 as assessed by confocal microscopy. In contrast, β-arrestin2-dependent activation was slower (peak 5–10 min), quite persistent with little decrement noted out to 90 min, and entirely confined to the cytoplasm. Moreover, ERK1/2 activated via β-arrestin2 accumulated in a pool of cytoplasmic endosomal vesicles that also contained the internalized receptors and β-arrestin. Such differential regulation of the temporal and spatial patterns of ERK1/2 activation via these two pathways strongly implies the existence of distinct physiological endpoints. The seven-membrane-spanning angiotensin II type 1A receptor activates the mitogen-activated protein kinases extracellular signal-regulated kinases 1 and 2 (ERK1/2) by distinct pathways dependent on either G protein (likely Gq/G11) or β-arrestin2. Here we sought to distinguish the kinetic and spatial patterns that characterize ERK1/2 activated by these two mechanisms. We utilized β-arrestin RNA interference, the protein kinase C inhibitor Ro-31-8425, a mutant angiotensin II receptor (DRY/AAY), and a mutant angiotensin II peptide (SII-angiotensin), which are incapable of activating G proteins, to isolate the two pathways in HEK-293 cells. G protein-dependent activation was rapid (peak <2 min), quite transient (t½ ∼2 min), and led to nuclear translocation of the activated ERK1/2 as assessed by confocal microscopy. In contrast, β-arrestin2-dependent activation was slower (peak 5–10 min), quite persistent with little decrement noted out to 90 min, and entirely confined to the cytoplasm. Moreover, ERK1/2 activated via β-arrestin2 accumulated in a pool of cytoplasmic endosomal vesicles that also contained the internalized receptors and β-arrestin. Such differential regulation of the temporal and spatial patterns of ERK1/2 activation via these two pathways strongly implies the existence of distinct physiological endpoints. Upon agonist binding, signaling via seven-membrane-spanning (7MS) 1The abbreviations used are: 7MS, seven-membrane-spanning; siRNA, small interfering RNA; AT1A, angiotensin II type 1A; AngII, angiotensin II; SII-AngII, (Sar1, Ile4, Ile8) angiotensin II; Sar, sarcosine; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; PKC, protein kinase C; CXCR4, CXC chemokine receptor 4; PMA, phorbol 12-myristate 13-acetate; HA, hemagglutinin; HEK, human embryonic kidney; RFP, red fluorescent protein. receptors is classically mediated by receptor coupling to G proteins, leading to dissociation of their α and βγ subunits, which in turn activate a variety of effectors, propagating the signal (1Hall R.A. Premont R.T. Lefkowitz R.J. J. Cell Biol. 1999; 145: 927-932Crossref PubMed Scopus (282) Google Scholar). Termination of this signaling is initiated by phosphorylation of the agonist-occupied receptor by G protein-coupled receptor kinases (GRKs), promoting high affinity binding of cytoplasmic β-arrestins to the receptor (2Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (906) Google Scholar). Binding of β-arrestins sterically inhibits coupling of the receptor to G protein (desensitization) (2Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (906) Google Scholar, 3Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1068) Google Scholar), as well as leading to removal of the receptor from the cell surface (internalization) by interaction with elements of the clathrin-mediated endocytic pathway (4Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1172) Google Scholar, 5Laporte S.A. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Crossref PubMed Scopus (524) Google Scholar, 6McDonald P.H. Cote N.L. Lin F.T. Premont R.T. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1999; 274: 10677-10680Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). In addition to these classical functions of β-arrestins as signal terminators for G protein-dependent 7MS receptor signaling, accumulating evidence over the last several years has drawn attention to a novel function of β-arrestins, as signal transducers that scaffold various signaling molecules upon activation of 7MS receptors (7Luttrell L.M. Lefkowitz R.J. J. Cell Sci. 2002; 115: 455-465Crossref PubMed Google Scholar). One such example is that of β-arrestin scaffolding components of mitogen-activated protein kinase (MAPK) cascades, leading to their activation (8McDonald P.H. Chow C.W. Miller W.E. Laporte S.A. Field M.E. Lin F.T. Davis R.J. Lefkowitz R.J. Science. 2000; 290: 1574-1577Crossref PubMed Google Scholar, 9Luttrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2449-2454Crossref PubMed Scopus (703) Google Scholar, 10DeFea K.A. Vaughn Z.D. O'Bryan E.M. Nishijima D. Dery O. Bunnett N.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11086-11091Crossref PubMed Scopus (353) Google Scholar, 11DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (689) Google Scholar). In the case of the extracellular signal-regulated kinase (ERK) cascade, it has been demonstrated that upon activation of angiotensin II type 1A (AT1A) (9Luttrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2449-2454Crossref PubMed Scopus (703) Google Scholar), neurokinin 1 (10DeFea K.A. Vaughn Z.D. O'Bryan E.M. Nishijima D. Dery O. Bunnett N.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11086-11091Crossref PubMed Scopus (353) Google Scholar), and protease-activated (11DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (689) Google Scholar) receptors, β-arrestin scaffolds the components of the ERK cascade, Raf-1, MEK1, and ERK1/2, into the receptor complex, leading to activation of ERK1/2. Furthermore, confocal microscopic studies have revealed that stimulation of the AT1A receptor causes colocalization of activated ERK1/2 with β-arrestin2 in a cytoplasmic vesicular compartment (9Luttrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2449-2454Crossref PubMed Scopus (703) Google Scholar, 12Tohgo A. Pierce K.L. Choy E.W. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 2002; 277: 9429-9436Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). Overexpression of β-arrestin also enhances cytoplasmic or β-arrestin-bound ERK1/2 activation following stimulation of AT1A or vasopressin V2 receptors (12Tohgo A. Pierce K.L. Choy E.W. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 2002; 277: 9429-9436Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 13Tohgo A. Choy E.W. Gesty-Palmer D. Pierce K.L. Laporte S. Oakley R.H. Caron M.G. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 2003; 278: 6258-6267Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). More recently, studies using the RNA interference technique have demonstrated that β-arrestin2 is involved in AT1A receptor and CXC chemokine receptor 4 (CXCR4)-mediated ERK1/2 activation (14Ahn S. Nelson C.D. Garrison T.R. Miller W.E. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1740-1744Crossref PubMed Scopus (190) Google Scholar, 15Wei H. Ahn S. Shenoy S.K. Karnik S.S. Hunyady L. Luttrell L.M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10782-10787Crossref PubMed Scopus (550) Google Scholar, 16Ahn S. Wei H. Garrison T.R. Lefkowitz R.J. J. Biol. Chem. 2004; 279: 7807-7811Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 17Sun Y. Cheng Z. Ma L. Pei G. J. Biol. Chem. 2002; 277: 49212-49219Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). Furthermore, some of these studies have revealed that in the case of ERK1/2 activation by AT1A receptor stimulation, β-arrestin2, but not β-arrestin1, mediates G protein-independent ERK1/2 activation (15Wei H. Ahn S. Shenoy S.K. Karnik S.S. Hunyady L. Luttrell L.M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10782-10787Crossref PubMed Scopus (550) Google Scholar, 16Ahn S. Wei H. Garrison T.R. Lefkowitz R.J. J. Biol. Chem. 2004; 279: 7807-7811Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). A wide variety of extracellular signals transduced via numerous cell surface receptors or integrins activate MAPKs including ERK1/2, which in turn play a major role in the integration of multiple biological responses such as cell proliferation, differentiation, and survival (18Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar, 19Schaeffer H.J. Weber M.J. Mol. Cell. Biol. 1999; 19: 2435-2444Crossref PubMed Scopus (1404) Google Scholar). Thus, regulation of activation is for the physiological from a such are the and of activated which in to H.J. Weber M.J. Mol. Cell. Biol. 1999; 19: 2435-2444Crossref PubMed Scopus (1404) Google Scholar, J. Biochem. 2002; PubMed Scopus Google Scholar). it has been that activation is in to that by chemokine receptors L. F. J. 2000; PubMed Scopus Google Scholar). protease-activated receptors have also been to have kinetic and patterns for activated ERK1/2 L. Y. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, it has been that ERK1/2 activation is confined to the (9Luttrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2449-2454Crossref PubMed Scopus (703) Google Scholar, 11DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (689) Google Scholar, 12Tohgo A. Pierce K.L. Choy E.W. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 2002; 277: 9429-9436Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, L. Y. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). to it has not been to the regulation of 7MS ERK activation by G protein signaling 7MS receptor stimulation of ERK the activation of Here we for the in these two by which the angiotensin II receptor activates ERK1/2 and in the spatial and temporal patterns of The the of the two and II was from (Sar1, Ile4, Ile8) was in the 12-myristate and from and from and from the type and mutant AT1A receptors, the in the is to have been (15Wei H. Ahn S. Shenoy S.K. Karnik S.S. Hunyady L. Luttrell L.M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10782-10787Crossref PubMed Scopus (550) Google Scholar). The β-arrestin2 was into the to of with RNA and from in and The β-arrestin2 is to the to the (14Ahn S. Nelson C.D. Garrison T.R. Miller W.E. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1740-1744Crossref PubMed Scopus (190) Google Scholar). A RNA as the was used as a Cell and RNA embryonic as R.H. 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Biochem. 2002; PubMed Scopus Google Scholar). the temporal patterns of ERK1/2 activation mediated via of these two we the of RNA of β-arrestin2 on the of ERK1/2 activation following stimulation of AT1A receptors in HEK-293 cells. A and that of β-arrestin2 of β-arrestin2 with on In ERK1/2 activation 2 of agonist for to min, and C and the of β-arrestin2, which the G protein-dependent pathway (15Wei H. Ahn S. Shenoy S.K. Karnik S.S. Hunyady L. Luttrell L.M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10782-10787Crossref PubMed Scopus (550) Google Scholar, 16Ahn S. Wei H. Garrison T.R. Lefkowitz R.J. J. Biol. Chem. 2004; 279: 7807-7811Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), to rapid and transient ERK1/2 which 2 of agonist and to stimulation C and this G protein-dependent from the of the β-arrestin2-dependent In the this kinetic of ERK1/2 a slower but persistent ERK1/2 activation that in cells. that ERK1/2 activation a from that to G protein-dependent ERK We demonstrated that activation of ERK1/2 by the mutant or by the mutant AT1A receptor that has in the (DRY/AAY), of which are to receptor coupling to G (15Wei H. Ahn S. Shenoy S.K. Karnik S.S. Hunyady L. Luttrell L.M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10782-10787Crossref PubMed Scopus (550) Google Scholar, H. L. J. S. Karnik S. M.J. Mol. 2002; PubMed Scopus Google Scholar, Z. G. Zhang A. Hunyady L. 2003; PubMed Scopus Google Scholar), is entirely β-arrestin2-dependent (15Wei H. Ahn S. Shenoy S.K. Karnik S.S. Hunyady L. Luttrell L.M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10782-10787Crossref PubMed Scopus (550) Google Scholar, 16Ahn S. Wei H. Garrison T.R. Lefkowitz R.J. J. Biol. Chem. 2004; 279: 7807-7811Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). to the kinetic for ERK1/2 activation by in by the temporal of ERK1/2 activation by this mutant and receptor in the HEK-293 cells. in A and stimulation of the type AT1A receptor to but persistent activation of ERK1/2, which by and which is for stimulation of the mutant receptor causes a kinetic of ERK1/2 activation C and In as is ERK1/2 activation agonist β-arrestin2 is The kinetic in 2 the in strongly the that ERK1/2 activation by is and as with G protein-dependent We have that stimulation of the pathway to ERK1/2 activation in HEK-293 is by the inhibitor (15Wei H. Ahn S. Shenoy S.K. Karnik S.S. Hunyady L. Luttrell L.M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10782-10787Crossref PubMed Scopus (550) Google Scholar, 16Ahn S. Wei H. Garrison T.R. Lefkowitz R.J. J. Biol. Chem. 2004; 279: 7807-7811Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Thus, to isolate the ERK1/2 activation pathway by is to this with the inhibitor in a in ERK1/2 activation stimulation, but little is A and of stimulation, is of ERK1/2 activation by In the is to the kinetic of ERK1/2 activation which is as in the rapid and transient G protein-dependent ERK1/2 activation by in is by with the inhibitor A and In and β-arrestin2 of ERK1/2 which are by not with the in 1 and these that the temporal patterns of β-arrestin2 and G ERK1/2 activation following stimulation of the AT1A receptor are quite distinct from is and to G protein-dependent which is rapid and Thus, for AT1A ERK1/2 activation in HEK-293 the of is via the G protein-dependent is mediated by the β-arrestin2 A of evidence has that ERK1/2 activated by signaling in the (9Luttrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2449-2454Crossref PubMed Scopus (703) Google Scholar, 11DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (689) Google Scholar, 12Tohgo A. Pierce K.L. Choy E.W. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 2002; 277: 9429-9436Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, L. Y. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). we the of activated ERK1/2 by confocal and in HEK-293 cells. demonstrated in these G protein and ERK1/2 in 2 stimulation, is in the and the but not of is the β-arrestin2 by RNA interference to on the 2 as with but cytoplasmic of the Such in the cytoplasmic of in is for the activation of the AT1A receptor is in the and of by for with the or of β-arrestin2 In and β-arrestin2 is also that upon stimulation of the AT1A a pool of ERK1/2 is activated via the G protein-dependent pathway into the it is In contrast, ERK1/2 activated via the β-arrestin2 pathway is entirely in the is not and is confocal microscopic studies using fluorescent protein have revealed that activation of some 7MS receptors in of endocytic vesicular receptors and β-arrestin R.H. Laporte S.A. Holt J.A. Caron M.G. Barak L.S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, it has been that a pool of activated ERK1/2 is with β-arrestin2 in this compartment (9Luttrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2449-2454Crossref PubMed Scopus (703) Google Scholar, 12Tohgo A. Pierce K.L. Choy E.W. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 2002; 277: 9429-9436Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). the of activated ERK1/2 in to stimulation of the G protein and β-arrestin2-dependent we the of in HEK-293 2 stimulation, of the AT1A receptors are on the cell surface but as into which is with in β-arrestin2 is in the as well as in the this that the β-arrestin2 not with translocation of activated ERK1/2 into the stimulation, is into cytoplasmic endosomal that also the internalized receptors and The colocalization of the to this In nuclear of is min, to the in In contrast, for in nuclear and cytoplasmic of but not to of in endosomal that ERK1/2 activated via signaling to cytoplasmic vesicular that the internalized receptors and β-arrestin2. the of ERK1/2 activation mediated via the β-arrestin2 we the of by the mutant AT1A receptor in In we that this mutant receptor G protein-dependent ERK1/2 in with (15Wei H. Ahn S. Shenoy S.K. Karnik S.S. Hunyady L. Luttrell L.M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10782-10787Crossref PubMed Scopus (550) Google Scholar). the receptor 4 and the receptor to nuclear of 2 the of stimulation is to that with the receptor activation by the the that activation of the receptor to colocalization of with in the distinct of ERK1/2 activation as with ERK1/2 activated via G as in 4 and we the of the inhibitor Ro-31-8425, which stimulation of G protein-dependent ERK1/2 activation in HEK-293 (15Wei H. Ahn S. Shenoy S.K. Karnik S.S. Hunyady L. Luttrell L.M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10782-10787Crossref PubMed Scopus (550) Google Scholar), on the of stimulation of the AT1A receptor in with the not is nuclear of but cytoplasmic to 2 colocalization of with the cell 2 min, which is not in the of the to cytoplasmic is also stimulation, with the inhibitor has on the colocalization of with in the cytoplasmic endosomal the is the as that in with inhibitor of of that following stimulation of the AT1A receptor in HEK-293 transient nuclear of is mediated by G protein-dependent signaling, cytoplasmic and endosomal of is entirely signaling is for the and of multiple such as proliferation, differentiation, and (18Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar, 19Schaeffer H.J. Weber M.J. Mol. Cell. Biol. 1999; 19: 2435-2444Crossref PubMed Scopus (1404) Google Scholar, J. Biochem. 2002; PubMed Scopus Google Scholar). a activation of ERK in on the that activation to the responses to a variety of the and of the to a the temporal and spatial of ERK1/2 activation following stimulation by have been well H.J. Weber M.J. Mol. Cell. Biol. 1999; 19: 2435-2444Crossref PubMed Scopus (1404) Google Scholar, J. Biochem. 2002; PubMed Scopus Google Scholar), these of 7MS ERK1/2 activation have been The is by the multiple signaling pathways from a which to ERK Moreover, the of such multiple pathways in cell S. Y. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar), as well as a cell type with of stimulation A. Choy E.W. Gesty-Palmer D. Pierce K.L. Laporte S. Oakley R.H. Caron M.G. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 2003; 278: 6258-6267Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, L. Y. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). that upon stimulation of the AT1A ERK1/2 are but activated via the G protein-dependent ERK1/2 activation is but ERK stimulation <2 is G that is The functions of β-arrestin are to turn G protein signaling the coupling of the activated 7MS receptor to G protein (2Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (906) Google Scholar, 3Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1068) Google Scholar) and of the receptor from the cell surface by receptor (4Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1172) Google Scholar, 5Laporte S.A. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Crossref PubMed Scopus (524) Google Scholar, 6McDonald P.H. Cote N.L. Lin F.T. Premont R.T. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1999; 274: 10677-10680Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Thus, it that of the that G protein-dependent ERK1/2 activation is mediated by binding of β-arrestin to the activated evidence (9Luttrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2449-2454Crossref PubMed Scopus (703) Google Scholar, 12Tohgo A. Pierce K.L. Choy E.W. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 2002; 277: 9429-9436Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 13Tohgo A. Choy E.W. Gesty-Palmer D. Pierce K.L. Laporte S. Oakley R.H. Caron M.G. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 2003; 278: 6258-6267Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 15Wei H. Ahn S. Shenoy S.K. Karnik S.S. Hunyady L. 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