title: Gbetagamma subunits mediate Src-dependent phosphorylation of the epidermal growth factor receptor. A scaffold for G protein-coupled receptor-mediated Ras activation.
abstract: In many cells, stimulation of mitogen-activated protein kinases by both receptor tyrosine kinases and receptors that couple to pertussis toxin-sensitive heterotrimeric G proteins proceed via convergent signaling pathways. Both signals are sensitive to inhibitors of tyrosine protein kinases and require Ras activation via phosphotyrosine-dependent recruitment of Ras guanine nucleotide exchange factors. Receptor tyrosine kinase stimulation mediates ligand-induced receptor autophosphorylation, which creates the initial binding sites for SH2 domain-containing docking proteins. However, the mechanism whereby G protein-coupled receptors mediate the phosphotyrosine-dependent assembly of a mitogenic signaling complex is poorly understood. We have studied the role of Src family nonreceptor tyrosine kinases in G protein-coupled receptor-mediated tyrosine phosphorylation in a transiently transfected COS-7 cell system. Stimulation of Gi-coupled lysophosphatidic acid and alpha2A adrenergic receptors or overexpression of Gbeta1gamma2 subunits leads to tyrosine phosphorylation of the Shc adapter protein, which then associates with tyrosine phosphoproteins of approximately 130 and 180 kDa, as well as Grb2. The 180-kDa Shc-associated tyrosine phosphoprotein band contains both epidermal growth factor (EGF) receptor and p185(neu). 3-5-fold increases in EGF receptor but not p185(neu) tyrosine phosphorylation occur following Gi-coupled receptor stimulation. Inhibition of endogenous Src family kinase activity by cellular expression of a dominant negative kinase-inactive mutant of c-Src inhibits Gbeta1gamma2 subunit-mediated and Gi-coupled receptor-mediated phosphorylation of both EGF receptor and Shc. Expression of Csk, which inactivates Src family kinases by phosphorylating the regulatory carboxyl-terminal tyrosine residue, has the same effect. The Gi-coupled receptor-mediated increase in EGF receptor phosphorylation does not reflect increased EGF receptor autophosphorylation, assayed using an autophosphorylation-specific EGF receptor monoclonal antibody. Lysophosphatidic acid stimulates binding of EGF receptor to a GST fusion protein containing the c-Src SH2 domain, and this too is blocked by Csk expression. These data suggest that Gbetagamma subunit-mediated activation of Src family nonreceptor tyrosine kinases can account for the Gi-coupled receptor-mediated tyrosine phosphorylation events that direct recruitment of the Shc and Grb2 adapter proteins to the membrane.
authors.affiliation: Howard Hughes Medical Institute and the Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: L M
authors.initials: LM
authors.lastname: Luttrell
authors.forename: G J
authors.initials: GJ
authors.lastname: Della Rocca
authors.forename: T
authors.initials: T
authors.lastname: van Biesen
authors.forename: D K
authors.initials: DK
authors.lastname: Luttrell
authors.forename: R J
authors.initials: RJ
authors.lastname: Lefkowitz
mesh_terms: D000818:Animals; D019556:COS Cells; D066246:ErbB Receptors / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008934:Mitogens / Q000378:metabolism; D010766:Phosphorylation; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism*; D018909:src Homology Domains; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D008934:Mitogens; D014443:Tyrosine; D066246:ErbB Receptors; D016392:Proto-Oncogene Proteins pp60(c-src); D019061:src-Family Kinases; D019204:GTP-Binding Proteins; D018631:ras Proteins