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• W2020589005 abstract "G protein-coupled receptors form the largest family of membrane receptors and transmit diverse ligand signals to modulate various cellular responses. After activation by their ligands, some of these G protein-coupled receptors are desensitized, internalized (endocytosed), and down-regulated (degraded). In HEK 293 cells, the Gs-coupled β2-adrenergic receptor was postulated to initiate a second wave of signaling, such as the activation of the mitogen-activated protein kinase (MAPK) pathway after the receptor is internalized. The tyrosine kinase c-Src plays a critical role in these events. Here we used mouse embryonic fibroblast (MEF) cells deficient in Src family tyrosine kinases to examine the role of Src in β2-adrenergic receptor signaling to the MAPK pathway and in receptor internalization. We found that in Src-deficient cells the β2-adrenergic receptor could activate the MAPK pathway. However, the internalization of β2-adrenergic receptors was blocked in Src-deficient MEF cells. Furthermore, we observed that in MEF cells deficient in β-arrestin 2 the internalization of the β2-adrenergic receptor was impaired, whereas the activation of the MAPK pathway by the β2-adrenergic receptor was normal. Our data demonstrate that although Src and β-arrestin 2 play essential roles in β2-adrenergic receptor internalization, they are not required for the activation of the MAPK pathway by the β2-adrenergic receptor. In other words, our finding suggests that receptor internalization is not required for β2-adrenergic receptor signaling to the MAPK pathway in MEF cells. G protein-coupled receptors form the largest family of membrane receptors and transmit diverse ligand signals to modulate various cellular responses. After activation by their ligands, some of these G protein-coupled receptors are desensitized, internalized (endocytosed), and down-regulated (degraded). In HEK 293 cells, the Gs-coupled β2-adrenergic receptor was postulated to initiate a second wave of signaling, such as the activation of the mitogen-activated protein kinase (MAPK) pathway after the receptor is internalized. The tyrosine kinase c-Src plays a critical role in these events. Here we used mouse embryonic fibroblast (MEF) cells deficient in Src family tyrosine kinases to examine the role of Src in β2-adrenergic receptor signaling to the MAPK pathway and in receptor internalization. We found that in Src-deficient cells the β2-adrenergic receptor could activate the MAPK pathway. However, the internalization of β2-adrenergic receptors was blocked in Src-deficient MEF cells. Furthermore, we observed that in MEF cells deficient in β-arrestin 2 the internalization of the β2-adrenergic receptor was impaired, whereas the activation of the MAPK pathway by the β2-adrenergic receptor was normal. Our data demonstrate that although Src and β-arrestin 2 play essential roles in β2-adrenergic receptor internalization, they are not required for the activation of the MAPK pathway by the β2-adrenergic receptor. In other words, our finding suggests that receptor internalization is not required for β2-adrenergic receptor signaling to the MAPK pathway in MEF cells. G protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G protein-coupled receptor; β2-AR, β2-adrenergic receptor; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEF, mouse embryonic fibroblast; GFP, green fluorescent protein. mediate transmembrane signaling for a large number of ligands including hormones, neurotransmitters, photons, odorants, pheromones, chemokines, and other stimuli (1Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4728) Google Scholar, 2Dohlman H.G. Thorner J. Caron M.G. Lefkowitz R.J. Annu. Rev. Biochem. 1991; 60: 653-688Crossref PubMed Scopus (1137) Google Scholar, 3Morris A.J. Malbon C.C. Physiol. Rev. 1999; 79: 1373-1430Crossref PubMed Scopus (398) Google Scholar). These receptors relay the signals to heterotrimeric G proteins, which directly modulate the activity of enzymes or other effector molecules. One of the prototypes of GPCRs is the β2-adrenergic receptor (β2-AR) that transduces signals from catecholamines, norepinephrine, and epinephrine to the Gs protein, which in turn activates downstream effectors (2Dohlman H.G. Thorner J. Caron M.G. Lefkowitz R.J. Annu. Rev. Biochem. 1991; 60: 653-688Crossref PubMed Scopus (1137) Google Scholar). After agonist activation, β2-AR is phosphorylated by GPCR kinases with the subsequent binding of β-arrestin 2 to the phosphorylated β2-AR leading to the internalization (endocytosis) through clathrin-coated pits (4Marchese A. Chen C. Kim Y.M. Benovic J.L. Trends Biochem. Sci. 2003; 28: 369-376Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 5Kohout T.A. Lefkowitz R.J. Mol. Pharmacol. 2003; 63: 9-18Crossref PubMed Scopus (364) Google Scholar). GPCRs can relay ligand signals to various cellular signaling pathways. One of these cellular pathways is the mitogen-activated protein kinase (MAPK) pathway (6Gutkind J.S. J. Biol. Chem. 1998; 273: 1839-1842Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar). The MAPK cascade, an evolutionarily conserved signaling module, stimulates numerous physiological responses including cell growth and differentiation (7Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3218) Google Scholar). The pathway consists of a MAP kinase kinase kinase that phosphorylates and activates a MAP kinase kinase, which in turn phosphorylates the TXY activation loop of MAP kinase (8Chang L. Karin M. Nature. 2001; 410: 37-40Crossref PubMed Scopus (4420) Google Scholar). The first characterized subfamily of MAP kinases, termed extracellular signal-regulated kinases (ERKs), and is activated by growth factor receptors, G protein-coupled receptors, and other types of receptors. The most common MAPK pathway involves Ras, Raf kinase (a MAP kinase kinase kinase), and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (a MAP kinase kinase) (9Chen Z. Gibson T.B. Robinson F. Silvestro L. Pearson G. Xu B. Wright A. Vanderbilt C. Cobb M.H. Chem. Rev. 2001; 101: 2449-2476Crossref PubMed Scopus (794) Google Scholar). GPCRs seem to activate the ERK MAPK pathway by diverse signaling pathways depending on the receptors and the cell types. Some of these activation pathways from GPCRs to ERK MAPK involve tyrosine kinases (10Chen J. Iyengar R. Science. 1994; 263: 1278-1281Crossref PubMed Scopus (107) Google Scholar, 11Wan Y. Kurosaki T. Huang X.Y. Nature. 1996; 380: 541-544Crossref PubMed Scopus (259) Google Scholar, 12Wan Y. Bence K. Hata A. Kurosaki T. Veillette A. Huang X.Y. J. Biol. Chem. 1997; 272: 17209-17215Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 13Luttrell L.M. Hawes B.E. van Biesen T. Luttrell D.K. Lansing T.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 19443-19450Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar, 14Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (880) Google Scholar, 15Schieffer B. Paxton W.G. Chai Q. Marrero M.B. Bernstein K.E. J. Biol. Chem. 1996; 271: 10329-10333Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 16Simonson M.S. Wang Y. Herman W.H. J. Biol. Chem. 1996; 271: 77-82Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). For example, previously we showed that Src family tyrosine kinases are essential for Gq signaling to ERK MAPK in DT40 chicken B lymphoma cells (11Wan Y. Kurosaki T. Huang X.Y. Nature. 1996; 380: 541-544Crossref PubMed Scopus (259) Google Scholar). In other GPCR signaling to ERK MAPK or in other cell types, tyrosine kinases might not be essential. We have shown that in S49 mouse T lymphoma cells, Gs signaling to the ERK MAPK pathway is independent of Src family tyrosine kinases. Instead, a protein kinase A-dependent pathway was used (17Wan Y. Huang X.Y. J. Biol. Chem. 1998; 273: 14533-14537Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 18Gu C. Ma Y.C. Benjamin J. Littman D. Chao M.V. Huang X.Y. J. Biol. Chem. 2000; 275: 20726-20733Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). In HEK 293 cells, it was reported that isoproterenol stimulation of β2-AR leading to the activation of ERK MAPK requires Src and receptor internalization (19Luttrell L.M. Ferguson S.S. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Science. 1999; 283: 655-661Crossref PubMed Scopus (1264) Google Scholar). Src family tyrosine kinases are a major group of cellular signal transducers (20Thomas S.M. Brugge J.S. Annu. Rev. Cell Dev. Biol. 1997; 13: 513-609Crossref PubMed Scopus (2175) Google Scholar). c-Src was the first identified protein tyrosine kinase (21Bishop J.M. Annu. Rev. Biochem. 1983; 52: 301-354Crossref PubMed Scopus (1052) Google Scholar, 22Hunter T. Cooper J.A. Annu. Rev. Biochem. 1985; 54: 897-930Crossref PubMed Google Scholar). These tyrosine kinases can be activated by various extracellular signals and modulate a variety of cellular functions including proliferation, survival, adhesion, and migration (20Thomas S.M. Brugge J.S. Annu. Rev. Cell Dev. Biol. 1997; 13: 513-609Crossref PubMed Scopus (2175) Google Scholar). GPCRs have been shown to stimulate tyrosine phosphorylation of cellular proteins (20Thomas S.M. Brugge J.S. Annu. Rev. Cell Dev. Biol. 1997; 13: 513-609Crossref PubMed Scopus (2175) Google Scholar). Various G protein-mediated physiological functions are sensitive to tyrosine kinase inhibitors. Many GPCRs are able to increase the activity of Src family tyrosine kinases (10Chen J. Iyengar R. Science. 1994; 263: 1278-1281Crossref PubMed Scopus (107) Google Scholar, 11Wan Y. Kurosaki T. Huang X.Y. Nature. 1996; 380: 541-544Crossref PubMed Scopus (259) Google Scholar, 12Wan Y. Bence K. Hata A. Kurosaki T. Veillette A. Huang X.Y. J. Biol. Chem. 1997; 272: 17209-17215Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 13Luttrell L.M. Hawes B.E. van Biesen T. Luttrell D.K. Lansing T.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 19443-19450Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar, 15Schieffer B. Paxton W.G. Chai Q. Marrero M.B. Bernstein K.E. J. Biol. Chem. 1996; 271: 10329-10333Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 16Simonson M.S. Wang Y. Herman W.H. J. Biol. Chem. 1996; 271: 77-82Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 23Ishida M. Marrero M.B. Schieffer B. Ishida T. Bernstein K.E. Berk B.C. Circ. Res. 1995; 77: 1053-1059Crossref PubMed Scopus (167) Google Scholar, 24Ptasznik A. Traynor-Kaplan A. Bokoch G.M. J. Biol. Chem. 1995; 270: 19969-19973Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 25Rodriguez-Fernandez J.L. Rozengurt E. J. Biol. Chem. 1996; 271: 27895-27901Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 26Schieffer B. Drexler H. Ling B.N. Marrero M.B. Am. J. Physiol. 1997; 272: C2019-C2030Crossref PubMed Google Scholar, 27Postma F.R. Hengeveld T. Alblas J. Giepmans B.N. Zondag G.C. Jalink K. Moolenaar W.H. J. Cell Biol. 1998; 140: 1199-1209Crossref PubMed Scopus (105) Google Scholar). Although the mechanism by which other G proteins activate Src family tyrosine kinases is not known, we have shown previously that Gαs and Gαi can directly interact with and activate Src (18Gu C. Ma Y.C. Benjamin J. Littman D. Chao M.V. Huang X.Y. J. Biol. Chem. 2000; 275: 20726-20733Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 28Ma Y.C. Huang J. Ali S. Lowry W. Huang X.Y. Cell. 2000; 102: 635-646Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). During our investigation of the role of tyrosine kinases in G protein signaling, we notice that in mouse embryonic fibroblast (MEF) cells deficient in Src family tyrosine kinases (SYF cells) a constitutively active mutant of Gαs (GαsQ227L) is able to increase the kinase activity of ERK MAPK. Indeed stimulation of the endogenous Gs-coupled β2-AR also activates ERK MAPK in SYF cells. However, the internalization of β2-AR is blocked in SYF cells. Thus, these observations implicate that receptor internalization might not be necessary for the activation of the ERK MAPK pathway by β2-AR. To confirm this, we used MEF cells deficient in β-arrestin 2 because β-arrestin 2 has been shown to be essential for β2-AR internalization (29Kohout T.A. Lin F.S. Perry S.J. Conner D.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1601-1606PubMed Google Scholar). We found that although β-arrestin 2 deficiency impairs β2-AR internalization, it does not affect β2-AR signaling to the ERK MAPK pathway. Together, these data demonstrate that Src plays different roles in β2-AR signaling to MAPK and in receptor internalization and that receptor internalization is not required for β2-AR signaling to ERK MAPK in MEF cells. Cell Lines and Plasmid Constructs—The MEF cells deficient in Src family tyrosine kinases (SYF cells) were purchased from ATCC. The SYF/Src cells were established by stably transfecting human c-Src into SYF cells (28Ma Y.C. Huang J. Ali S. Lowry W. Huang X.Y. Cell. 2000; 102: 635-646Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). The MEF cells deficient in either β-arrestin 2 or deficient in β-arrestin 1 and 2 were kindly provided by Dr. R. Lefkowitz (Duke University) (29Kohout T.A. Lin F.S. Perry S.J. Conner D.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1601-1606PubMed Google Scholar). All plasmid cDNAs for human G proteins were purchased from Guthrie Research Institute. The cDNA for the human β2-AR was kindly provided by Dr. C. Malbon (State University of New York at Stony Brook) and subcloned into the pcDNA3 vector. A GFP tag was inserted into the 3′ end of β2-AR. ERK MAPK Assay—The p44/42 MAP kinase assay was performed using kits from Cell Signaling Technology as described previously (11Wan Y. Kurosaki T. Huang X.Y. Nature. 1996; 380: 541-544Crossref PubMed Scopus (259) Google Scholar). Whole cell lysates were prepared from MEF, SYF, SYF/Src, β-arrestin --/-, or β-arrestin 1-/-2-/- fibroblast cells. Cells were either treated or not with isoproterenol (10 μm) for 5 min. A monoclonal antibody to the phospho-p44/42 ERK MAPK (cross-linked to agarose beads) was added to immunoprecipitate the active ERK MAPK from cell lysates. Substrates (200 μm ATP and 2 μg glutathione S-transferase-Elk-1 fusion protein) were added, and the reaction was allowed to proceed at 30 °C for 30 min. After SDS-PAGE, the ERK MAPK activity (the phosphorylation of glutathione S-transferase-Elk-1 by ERK MAPK) was measured by Western blotting with an anti-phospho-Elk-1 antibody (Cell Signaling Technology). Receptor Internalization—Cells were transiently transfected with a plasmid carrying a GFP-tagged human β2-adrenergic receptor (pcDNA3-β2-AR-GFP). Twenty-four h after transfection, cells were split onto poly-d-lysine-coated glass coverslips. Forty-eight h after transfection, cells were treated with or without 10 μm isoproterenol at 37 °C for 30 min, rinsed quickly three times with phosphate-buffered saline, and further incubated in 3.7% formaldehyde for 20 min at room temperature. After being rinsed again three times in phosphate-buffered saline, the coverslips were mounted on a microscope slide with Vecta Shield mounting medium (Vector Laboratories) before imaging by fluorescence microscopy. Fluorescence microscopy was performed on a Zeiss Axiovert 35 microscope. Src Family Tyrosine Kinases Are Not Required for GαsQ227L Stimulation of ERK MAPK in MEF Cells—To genetically test the role of Src family tyrosine kinases in Gs signaling to the ERK MAPK pathway, we investigated the stimulation of ERK MAPK by the constitutively active mutant of Gαs (GαsQ227L) in Src family tyrosine kinase knock-out MEF cells. We used the SYF cells that were derived from Src, Yes, and Fyn triple knock-out mouse embryos (30Klinghoffer R.A. Sachsenmaier C. Cooper J.A. Soriano P. EMBO J. 1999; 18: 2459-2471Crossref PubMed Scopus (647) Google Scholar). Because Src, Yes, and Fyn are the three ubiquitously expressed members of the Src family tyrosine kinases, no Src family tyrosine kinase activity was detected in these SYF cells (30Klinghoffer R.A. Sachsenmaier C. Cooper J.A. Soriano P. EMBO J. 1999; 18: 2459-2471Crossref PubMed Scopus (647) Google Scholar). We found that transient expression of GαsQ227L in the control wild-type MEF cells led to increased activity of ERK MAPK (Fig. 1A). In SYF cells, expression of GαsQ227L also increased the ERK MAPK activity to a similar degree (Fig. 1B). These genetic data demonstrated that Src family tyrosine kinases are not required for GαsQ227L signaling to the ERK MAPK pathway. As controls, we also examined the requirement for Src family tyrosine kinases by some other G proteins signaling to ERK MAPK in MEF cells. As shown in Fig. 1C, transient expression of constitutively active mutants of Gα12 (Gα12Q231L), Gα13 (Gα13Q226L), Gαq (GαqQ209L), Gαi2 (Gαi2Q205L), and Gβ1γ2 led to the activation of ERK MAPK to different degrees. Interestingly, in SYF cells, except for Gα13Q226L, the other G protein mutants (Gαi2Q205L, Gα12Q231L, GαqQ209L, and Gβ1γ2) failed to stimulate ERK MAPK (Fig. 1D). These data suggest that Gα13Q226L does not require Src family tyrosine kinases signaling to the ERK MAPK pathway in MEF cells, whereas Gαi2Q205L, Gα12Q231L, GαqQ209L, and Gβ1γ2 require Src family tyrosine kinases for the activation of ERK MAPK in MEF cells. In this work, we focused on the Gs signaling to ERK MAPK. Thus, we did not examine further the Src-dependent signaling by other G proteins to the ERK MAPK pathway in MEF cells. Src Family Tyrosine Kinases Are Not Required for β2-Adrenergic Receptor Stimulation of ERK MAPK in MEF Cells—The above study examined the role of Src family tyrosine kinases in signaling to the ERK MAPK pathway by exogenously expressing constitutively active Gs mutant proteins. Next we examined the requirement of endogenous Gs protein-coupled receptors for Src family tyrosine kinases. We used the β2-adrenergic receptor as a representative for Gs-coupled receptors. Treatment of MEF cells with the β2-AR agonist isoproterenol increased the cAMP levels in MEF and SYF cells, indicating the presence of endogenous β2-adrenergic receptors in these cells (Fig. 2A). As shown in Fig. 2B, treatment of SYF cells with isoproterenol led to an increased activity of ERK MAPK, indicating that Src family tyrosine kinases are not essential for β2-AR signaling to ERK MAPK. Isoproterenol also increased the activity of ERK MAPK in SYF/Src cells (re-expression of Src in SYF cells) (Fig. 2B). Similar results also were observed with another β2-AR agonist, terbutaline (data not shown). This is consistent with the above data that showed that Gαs-mediated activation of ERK MAPK is independent of Src family tyrosine kinases. Hence, the endogenous β2-AR still was able to stimulate ERK MAPK in SYF cells. β-Arrestin 2 Is Required for β2-Adrenerigc Receptor Internalization but Not for β2-Adrenergic Receptor Signaling to ERK MAPK in MEF Cells—It has been reported that β2-AR internalization might initiate a second wave of intracellular signaling, including the activation of the ERK MAPK pathway in HEK 293 cells (19Luttrell L.M. Ferguson S.S. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Science. 1999; 283: 655-661Crossref PubMed Scopus (1264) Google Scholar). To investigate whether that is the case in MEF cells, we genetically examined the role of β-arrestin proteins in β2-AR stimulation of ERK MAPK. Radioligand binding studies with β-arrestin 2-/-, β-arrestin 1-/-, and β-arrestin 1-/-2-/- MEF cells showed that the internalization of β2-AR in β-arrestin 2-/- cells and β-arrestin 1-/-2-/- cells was significantly impaired (29Kohout T.A. Lin F.S. Perry S.J. Conner D.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1601-1606PubMed Google Scholar). On the other hand, the β2-AR internalization was normal in β-arrestin 1-/-cells (29Kohout T.A. Lin F.S. Perry S.J. Conner D.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1601-1606PubMed Google Scholar). In the control wild-type MEF cells, transient expression of a C-terminal GFP-tagged β2-AR showed membrane staining as well as cytoplasmic staining (Fig. 3A). Thirty min after the addition of isoproterenol to these cells, an increased level of internalization of these GFP-tagged β2-adrenergic receptors was observed (Fig. 3B). On the other hand, in β-arrestin 2-/- cells, treatment with isoproterenol did not lead to internalization of the GFP-tagged β2-ARs. These results are consistent with the previous report that β-arrestin 2 plays a critical role in β2-AR internalization (29Kohout T.A. Lin F.S. Perry S.J. Conner D.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1601-1606PubMed Google Scholar) (Fig. 3, C and D). Next we investigated the potential role of β-arrestin proteins and thus receptor internalization in β2-AR-initiated activation of ERK MAPK. As shown in Fig. 4, addition of isoproterenol to the β-arrestin 1-/-2-/- cells led to increased ERK MAPK activity (same as in the wild-type MEF cells and β-arrestin 2-/- cells) (Fig. 4). Moreover, similar results also were observed with another β2-AR agonist, terbutaline (data not shown). Together, these results demonstrate that in MEF cells, β-arrestin proteins and thus receptor internalization are not essential for β2-AR signaling to the ERK MAPK pathway. Src Family Tyrosine Kinases Are Required for β2-Adrenergic Receptor Internalization—Because we did not find an essential role for receptor internalization in the activation of the ERK MAPK pathway by β2-AR in MEF cells and because Src has been reported to form a complex with β-arrestin 2, we examined the genetic requirement for Src family tyrosine kinases in β2-AR internalization. In contrast to what was observed in the control wild-type MEF cells (Fig. 3, A and B), isoproterenol did not induce the internalization of the GFP-tagged β2-AR in SYF cells (Fig. 5, A and B). To confirm that this defect in receptor internalization was caused by the absence of Src family tyrosine kinases, we re-introduced Src into SYF cells. Re-expression of Src in these SYF cells rescued the receptor internalization phenotype (Fig. 5, C and D). These data indicate that even though Src family tyrosine kinases are not required for β2-AR signaling to the ERK MAPK pathway they are essential for β2-AR internalization. Therefore, Src family tyrosine kinases play distinct roles in β2-AR signaling to ERK MAPK and in receptor internalization. Role of Src Family Tyrosine Kinases in β2-Adrenergic Receptor Signaling to ERK MAPK—Although prolonged exposure to cAMP leads to the inhibition of ERK MAPK in some types of cells, ligand stimulation of β2-AR is able to transiently stimulate ERK MAPK in most reported cases (17Wan Y. Huang X.Y. J. Biol. Chem. 1998; 273: 14533-14537Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 31Wu J. Dent P. Jelinek T. Wolfman A. Weber M.J. Sturgill T.W. Science. 1993; 262: 1065-1069Crossref PubMed Scopus (824) Google Scholar, 32Cook S.J. McCormick F. Science. 1993; 262: 1069-1072Crossref PubMed Scopus (865) Google Scholar, 33Faure M. Voyno-Yasenetskaya T.A. Bourne H.R. J. Biol. Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google Scholar, 34Lazou A. Bogoyevitch M.A. Clerk A. Fuller S.J. Marshall C.J. Sugden P.H. Circ. Res. 1994; 75: 932-941Crossref PubMed Scopus (85) Google Scholar, 35Crespo 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, 36Verheijen M.H. Defize L.H. J. Biol. Chem. 1997; 272: 3423-3429Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 37Vossler M.R. Yao H. York R.D. Pan M.G. Rim C.S. Stork P.J. Cell. 1997; 89: 73-82Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar, 38Schmitt J.M. Stork P.J. J. Biol. Chem. 2000; 275: 25342-25350Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 39Maudsley S. Pierce K.L. Zamah A.M. Miller W.E. Ahn S. Daaka Y. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 2000; 275: 9572-9580Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, 40Friedman J. Babu B. Clark R.B. Mol. Pharmacol. 2002; 62: 1094-1102Crossref PubMed Scopus (68) Google Scholar). In S49 mouse lymphoma cells, we previously reported a Gs-adenylyl cyclase-cAMP-protein kinase A-dependent pathway leading to ERK MAPK activation (17Wan Y. Huang X.Y. J. Biol. Chem. 1998; 273: 14533-14537Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). This classical Gs-protein kinase A signaling pathway also has been implicated in activating the ERK MAPK pathway through β2-AR stimulation in other cells such as the HEK 293 cells (38Schmitt J.M. Stork P.J. J. Biol. Chem. 2000; 275: 25342-25350Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Furthermore, this Gs signaling pathway via protein kinase A to ERK MAPK also has been reported for other Gs-coupled receptors (36Verheijen M.H. Defize L.H. J. Biol. Chem. 1997; 272: 3423-3429Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 41Jiang Y. Cypess A.M. Muse E.D. Wu C.R. Unson C.G. Merrifield R.B. Sakmar T.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10102-10107Crossref PubMed Scopus (46) Google Scholar). Because this pathway is independent of Ras, it is different from the growth factor receptor tyrosine kinase signaling to the ERK MAPK pathway. However, there are disagreements on whether Src family tyrosine kinases are involved in β2-AR signaling to the ERK MAPK pathway. In HEK 293 cells, β-arrestin was reported to form a complex with Src and brought Src to the β2-AR, leading to the receptor desensitization/internalization process, which initiates a second wave of signaling including the ERK MAPK pathway (19Luttrell L.M. Ferguson S.S. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Science. 1999; 283: 655-661Crossref PubMed Scopus (1264) Google Scholar). On the other hand, we show here that in MEF cells Src is not essential for β2-AR stimulation of ERK MAPK. Moreover, treatment of HEK 293 cells with Src family kinase inhibitors did not block Gs-coupled A2A-adenosine receptor signaling to the ERK MAPK pathway (42Klinger M. Kudlacek O. Seidel M.G. Freissmuth M. Sexl V. J. Biol. Chem. 2002; 277: 32490-32497Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The different roles of Src in β2-AR stimulation of ERK MAPK in these reports might reflect either the different cell types used or the methods used. Interestingly, we noticed that the basal activity of ERK MAPK in SYF cells was consistently higher than in MEF cells and SYF/Src cells (Fig. 2B; also Fig. 1, A-D). This difference in basal activity of ERK MAPK is not caused by a difference in ERK MAPK protein expression levels in MEF and SYF cells. These data imply that the basal ERK MAPK activity in MEF cells was suppressed by c-Src. Inhibition of ERK by activated c-Src was reported recently in fibroblast cells (43Schmitt J.M. Stork P.J. Mol. Cell. 2002; 9: 85-94Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). However, because MEF cells were serum-starved, c-Src was likely in the down-regulated (inactive) state. Thus, it seems that the down-regulated c-Src has an unexpected function here. This might be reminiscent of the MAPK Kss1 in the yeast Saccharomyces cerevisiae. Kss1 in its inactive form is a potent negative regulator of invasive growth (44Cook J.G. Bardwell L. Thorner J. Nature. 1997; 390: 85-88Crossref PubMed Scopus (222) Google Scholar, 45Madhani H.D. Styles C.A. Fink G.R. Cell. 1997; 91: 673-684Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). The nature of this potential negative regulation of basal ERK MAPK by c-Src needs further investigation. Role of Src Family Tyrosine Kinases in β2-Adrenergic Receptor Internalization—For β2-AR internalization, ligand stimulation of β2-AR leads to the activation of G protein-coupled receptor kinases and the phosphorylation of some C-terminal Ser/Thr residues of β2-AR by G protein-coupled receptor kinases. This phosphorylation in turn promotes the association of β2-AR with β-arrestin 2 and subsequent internalization (4Marchese A. Chen C. Kim Y.M. Benovic J.L. Trends Biochem. Sci. 2003; 28: 369-376Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 5Kohout T.A. Lefkowitz R.J. Mol. Pharmacol. 2003; 63: 9-18Crossref PubMed Scopus (364) Google Scholar). The essential role of β-arrestin 2 in β2-AR internalization was confirmed in MEF cells from β-arrestin 2 knock-out mice (29Kohout T.A. Lin F.S. Perry S.J. Conner D.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1601-1606PubMed Google Scholar). β-Arrestins can interact directly with clathrin (46Goodman 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 (1179) Google Scholar), which could promote receptor internalization through clathrin-coated pits (4Marchese A. Chen C. Kim Y.M. Benovic J.L. Trends Biochem. Sci. 2003; 28: 369-376Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 5Kohout T.A. Lefkowitz R.J. Mol. Pharmacol. 2003; 63: 9-18Crossref PubMed Scopus (364) Google Scholar). Following the above β2-AR internalization scheme, it was surprising to see the inhibition of β2-AR internalization in SYF cells. Our data strongly indicate that Src family tyrosine kinases are involved in the β2-AR internalization process. There was some suggestive evidence that Src family tyrosine kinases might act upstream of G protein-coupled receptor kinases in β2-AR internalization. First, overexpression of a dominant-negative mutant of c-Src, treatment with PP2 inhibitor, and antisense oligodeoxynucleotides in human epidermoid A431 carcinoma cells inhibited ligand-induced β2-AR internalization (47Fan G. Shumay E. Malbon C.C. Wang H. J. Biol. Chem. 2001; 276: 13240-13247Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). It was proposed that ligand stimulation of β2-AR leads to the phosphorylation of tyrosine residue 350 at the C-terminal tail of the β2-AR by a yet to be identified tyrosine kinase. Src then uses its Src homology 2 domain to bind this phosphorylated tyrosine 350. This association to the β2-AR also activates Src (47Fan G. Shumay E. Malbon C.C. Wang H. J. Biol. Chem. 2001; 276: 13240-13247Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Second, it was reported that Src might phosphorylate and activate G protein-coupled receptor kinases (48Sarnago S. Elorza A. Mayor Jr., F. J. Biol. Chem. 1999; 274: 34411-34416Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The detailed mechanism by which Src family tyrosine kinases participate in the β2-AR internalization needs further investigation (49Ma Y.C. Huang X.Y. Trends Cardiovasc. Med. 2002; 12: 46-49Crossref PubMed Scopus (31) Google Scholar). Nevertheless, our data clearly show that Src is essential for β2-AR internalization in MEF cells. Role of Receptor Internalization in β2-Adrenergic Receptor Signaling to ERK MAPK—Previous studies that used a dominant-negative dynamin mutant and/or mutant arrestin proteins to examine the potential role of receptor internalization in the β2-AR signaling to ERK MAPK yielded inconsistent results. In HEK 293 cells and COS-7 cells, although some researchers reported that receptor internalization was required for β2-AR initiated activation of the ERK MAPK pathway (19Luttrell L.M. Ferguson S.S. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Science. 1999; 283: 655-661Crossref PubMed Scopus (1264) Google Scholar, 50Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar, 51Pierce K.L. Maudsley S. Daaka Y. Luttrell L.M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1489-1494Crossref PubMed Scopus (198) Google Scholar), others reported no requirement for receptor internalization in β2-AR signaling to ERK MAPK (40Friedman J. Babu B. Clark R.B. Mol. Pharmacol. 2002; 62: 1094-1102Crossref PubMed Scopus (68) Google Scholar, 52DeGraff J.L. Gagnon A.W. Benovic J.L. Orsini M.J. J. Biol. Chem. 1999; 274: 11253-11259Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Our genetic data clearly demonstrate that the receptor internalization is not essential for β2-AR signaling to ERK MAPK in MEF cells. For GPCRs as a whole, whether the receptor internalization is needed for the activation of the ERK MAPK pathway remains to be fully clarified. Some researchers suggested that receptor internalization seems to be needed for activation of the ERK MAPK pathway by some GPCRs (51Pierce K.L. Maudsley S. Daaka Y. Luttrell L.M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1489-1494Crossref PubMed Scopus (198) Google Scholar, 53Vogler O. Nolte B. Voss M. Schmidt M. Jakobs K.H. van Koppen C.J. J. Biol. Chem. 1999; 274: 12333-12338Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 54Ignatova E.G. Belcheva M.M. Bohn L.M. Neuman M.C. Coscia C.J. J. Neurosci. 1999; 19: 56-63Crossref PubMed Google Scholar), whereas others believed that receptor internalization is not essential for ERK MAPK activation (52DeGraff J.L. Gagnon A.W. Benovic J.L. Orsini M.J. J. Biol. Chem. 1999; 274: 11253-11259Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 55Schramm N.L. Limbird L.E. J. Biol. Chem. 1999; 274: 24935-24940Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 56Yang W. Wang D. Richmond A. J. Biol. Chem. 1999; 274: 11328-11333Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 57Budd D.C. Rae A. Tobin A.B. J. Biol. Chem. 1999; 274: 12355-12360Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 58Whistler J.L. von Zastrow M. J. Biol. Chem. 1999; 274: 24575-24578Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Quite a few reports used dominant-negative mutants of dynamin to evaluate the role of receptor endocytosis in ERK MAPK activation, but caution needs to be exercised when interpreting the results because it has now been reported that dynamin is required for the activation of ERK MAPK by its upstream kinase MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase) in cells (59Kranenburg O. Verlaan I. Moolenaar W.H. J. Biol. Chem. 1999; 274: 35301-35304Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). It might be helpful to use genetic tools such as loss-of-function mutants or deletions of specific signaling components to demonstrate whether receptor internalization is indeed essential for the signaling of a particular GPCR to ERK MAPK. In summary, we have shown that Src family tyrosine kinases are not required for GαsQ227L stimulation of ERK MAPK in MEF cells. Ligand stimulation of the endogenous Gs-coupled β2-AR also activates ERK MAPK in SYF cells, whereas the internalization of β2-AR is blocked in SYF cells. Furthermore, we found that although the β-arrestin 2 deficiency impairs β2-AR internalization, it has no effect on β2-AR signaling to the ERK MAPK pathway. Therefore, Src and β-arrestin 2 play different roles in β2-AR signaling to ERK MAPK and in receptor internalization, and receptor internalization is not required for β2-AR signaling to ERK MAPK in MEF cells. We thank Dr. R. Lefkowitz for the MEF cells deficient in β-arrestin proteins. We thank T. Maack, D. McGarrigle, and X. Zhou for reading the manuscript." @default.
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• W2020589005 title "Distinct Roles for Src Tyrosine Kinase in β2-Adrenergic Receptor Signaling to MAPK and in Receptor Internalization" @default.
• W2020589005 cites W1594412667 @default.
• W2020589005 cites W1600400967 @default.
• W2020589005 cites W1663047717 @default.
• W2020589005 cites W1918531995 @default.
• W2020589005 cites W1963817582 @default.
• W2020589005 cites W1967282700 @default.
• W2020589005 cites W1976849281 @default.
• W2020589005 cites W1976874658 @default.
• W2020589005 cites W1979669507 @default.
• W2020589005 cites W1991535761 @default.
• W2020589005 cites W1993876642 @default.
• W2020589005 cites W1995685525 @default.
• W2020589005 cites W1996405087 @default.
• W2020589005 cites W199941779 @default.
• W2020589005 cites W1999530386 @default.
• W2020589005 cites W2006733565 @default.
• W2020589005 cites W2008812258 @default.
• W2020589005 cites W2015802741 @default.
• W2020589005 cites W2020960374 @default.
• W2020589005 cites W2022576341 @default.
• W2020589005 cites W2025472785 @default.
• W2020589005 cites W2026933806 @default.
• W2020589005 cites W2030854707 @default.
• W2020589005 cites W2035504348 @default.
• W2020589005 cites W2042154282 @default.
• W2020589005 cites W2045876058 @default.
• W2020589005 cites W2051152907 @default.
• W2020589005 cites W2055737181 @default.
• W2020589005 cites W2062068441 @default.
• W2020589005 cites W2063599982 @default.
• W2020589005 cites W2063840368 @default.
• W2020589005 cites W2068522523 @default.
• W2020589005 cites W2079051863 @default.
• W2020589005 cites W2079238835 @default.
• W2020589005 cites W2079246839 @default.
• W2020589005 cites W2080875376 @default.
• W2020589005 cites W2082889823 @default.
• W2020589005 cites W2089555312 @default.
• W2020589005 cites W2092121369 @default.
• W2020589005 cites W2094173028 @default.
• W2020589005 cites W2094469376 @default.
• W2020589005 cites W2095066587 @default.
• W2020589005 cites W2101257013 @default.
• W2020589005 cites W2107258432 @default.
• W2020589005 cites W2109791066 @default.
• W2020589005 cites W2112072577 @default.
• W2020589005 cites W2116949924 @default.
• W2020589005 cites W2117695239 @default.
• W2020589005 cites W2121430531 @default.
• W2020589005 cites W2151312020 @default.
• W2020589005 cites W2155331383 @default.
• W2020589005 cites W2159360156 @default.
• W2020589005 cites W2160951360 @default.
• W2020589005 cites W2171008621 @default.
• W2020589005 cites W2180795626 @default.
• W2020589005 cites W3102875255 @default.
• W2020589005 cites W4211258630 @default.
• W2020589005 cites W4292820908 @default.
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