The Ras-dependent activation of mitogen-activated protein (MAP) kinase pathways by many receptors coupled to heterotrimeric guanine nucleotide binding proteins (G proteins) requires the activation of Src family tyrosine kinases. Stimulation of beta2 adrenergic receptors resulted in the assembly of a protein complex containing activated c-Src and the receptor. Src recruitment was mediated by beta-arrestin, which functions as an adapter protein, binding both c-Src and the agonist-occupied receptor. beta-Arrestin 1 mutants, impaired either in c-Src binding or in the ability to target receptors to clathrin-coated pits, acted as dominant negative inhibitors of beta2 adrenergic receptor-mediated activation of the MAP kinases Erk1 and Erk2. These data suggest that beta-arrestin binding, which terminates receptor-G protein coupling, also initiates a second wave of signal transduction in which the "desensitized" receptor functions as a critical structural component of a mitogenic signaling complex.

Many receptors that couple to heterotrimeric G proteins have been shown to mediate the rapid activation of MAP 1 kinases. Among these are receptors for several substances either present in the general circulation, released as neurotransmitters, or produced locally by vascular endothelium or activated platelets. These include catecholamines, acetylcholine, pituitary glycopeptide hormones, adenosine, angiotensins, bombesin, endothelins, LPA, and ␣-thrombin (1). Receptors for these substances, activated in response to systemic or locally generated ligands, may in turn play significant roles in the endocrine or paracrine regulation of cell proliferation.Heterogeneity exists in the mechanisms whereby G proteincoupled receptors activate MAP kinases. Depending upon receptor and cell type, MAP kinase activation may be mediated by pertussis toxin-sensitive or -insensitive G proteins and be either PKC-or Ras-dependent. In COS-7 cells, for example, activation of MAP kinase via the pertussis toxin-insensitive, Gq-coupled, ␣1B adrenergic and M1 muscarinic acetylcholine receptors is significantly inhibited by PKC depletion but insensitive to expression of a dominant-negative mutant of Ras. In contrast, activation of MAP kinase via the pertussis toxinsensitive Gi-coupled ␣2A adrenergic and M2 muscarinic acetylcholine receptors is PKC-independent but requires Ras activation and is sensitive to inhibitors of tyrosine protein kinases (2). Similarly, LPA, a potent stimulator of mitogenesis in quiescent fibroblasts that signals via a G protein-coupled receptor coupling to both pertussis toxin-sensitive and -insensitive G proteins (3-5), activates MAP kinase via a pertussis toxin-sensitive pathway involving Ras and Raf activation (6, 7). LPA-mediated MAP kinase activation is sensitive to tyrosine kinase inhibitors (7, 8) but independent of its effects on phosphatidylinositol hydrolysis and its ability to inhibit adenylyl cyclase (4,8). In COS-7 cells, Ras-dependent MAP kinase activation via ␣2A adrenergic (9), M2 muscarinic acetylcholine, D2 dopamine, and A1 adenosine receptors (10) is mediated largely by G␤␥ subunits derived from pertussis toxin-sensitive G proteins. Indeed, overexpression of G␤␥ subunits, but not constitutively activated G␣i1 or G␣i2 mutants, is sufficient to activate MAP kinase (9 -11) in these cells.