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.

With the development of targeted therapeutics, especially for small-molecule inhibitors, it is important to understand whether the observed in vivo efficacy correlates with the modulation of desired/intended target in vivo. We have developed a small-molecule inhibitor of all three vascular endothelial growth factor (VEGF) receptors (VEGFR), platelet-derived growth factor receptor, and c-Kit tyrosine kinases, pazopanib (GW786034), which selectively inhibits VEGF-induced endothelial cell proliferation. It has good oral exposure and inhibits angiogenesis and tumor growth in mice. Because bolus administration of the compound results in large differences in C max and C trough , we investigated the effect of continuous infusion of a VEGFR inhibitor on tumor growth and angiogenesis.

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 G i -coupled lysophosphatidic acid and ␣2A adrenergic receptors or overexpression of G␤1␥2 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 G i -coupled receptor stimulation. Inhibition of endogenous Src family kinase activity by cellular expression of a dominant negative kinase-inactive mutant of c-Src inhibits G␤1␥2 subunit-mediated and G icoupled 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 G i -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 G␤␥ subunitmediated activation of Src family nonreceptor tyrosine kinases can account for the G i -coupled receptor-mediated tyrosine phosphorylation events that direct recruitment of the Shc and Grb2 adapter proteins to the membrane.The low molecular weight G protein Ras functions as a signaling intermediate in many pathways involved in the regulation of cellular mitogenesis and differentiation. Ras activation by growth factor receptors that possess intrinsic tyrosine kinase activity follows ligand-induced phosphorylation of specific docking sites on the receptor itself or adapter proteins, such as Shc and insulin receptor substrate-1, which serve to recruit Ras guanine nucleotide exchange factors to the plasma membrane (1, 2). Recently, several receptors that couple to heterotrimeric G proteins, including the lysoph...

Inhibition of the vascular endothelial growth factor (VEGF) signaling pathway has emerged as one of the most promising new approaches for cancer therapy. We describe herein the key steps starting from an initial screening hit leading to the discovery of pazopanib, N(4)-(2,3-dimethyl-2H-indazol-6-yl)-N(4)-methyl-N(2)-(4-methyl-3-sulfonamidophenyl)-2,4-pyrimidinediamine, a potent pan-VEGF receptor (VEGFR) inhibitor under clinical development for renal-cell cancer and other solid tumors.