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.

Src family nonreceptor tyrosine kinases are an integral component of the signal transduction apparatus employed by growth factor receptor tyrosine kinases. As such, their role in cellular growth control and malignant transformation has been the subject of intensive investigation. In contrast, classical G-protein-coupled receptor (GPCR) signaling involves activation of second messenger-regulated serine/threonine kinases or ion channels, and is primarily involved in neurotransmission and the shortterm regulation of intermediary metabolism. Over the past decade, this strictly dichotomous model of transmembrane signaling has been challenged by the discovery that GPCRs also exert control over cellular growth, proliferation, and differentiation, and do so by stimulating tyrosine phosphorylation cascades. Several mechanisms, from the direct association of Src family kinases with GPCRs or receptor-associated proteins, to the transactivation of receptor tyrosine kinases and focal adhesion complexes by G-protein-mediated signals, permit GPCRs to activate Src family kinases. Conversely, Src activity plays a central role in controlling GPCR trafficking and effects on cell proliferation and cytoskeletal rearrangement. It is now clear that GPCRs and Src family kinases do not belong to separate, exclusive clubs. Rather, these strange bedfellows are intimately involved in multilayered forms of crosstalk that influence a host of cellular processes.

Many receptors that couple to heterotrimeric guanine-nucleotide binding proteins (G proteins) have been shown to mediate rapid activation of the mitogen-activated protein kinases Erk1 and Erk2. In different cell types, the signaling pathways employed appear to be a function of the available repertoire of receptors, G proteins, and effectors. In HEK-293 cells, stimulation of either ␣1B-or ␣2A-adrenergic receptors (ARs) leads to rapid 5-10-fold increases in Erk1/2 phosphorylation. Phosphorylation of Erk1/2 in response to stimulation of the ␣2A-AR is effectively attenuated by pretreatment with pertussis toxin or by coexpression of a G␤␥ subunit complex sequestrant peptide (␤ARK1ct) and dominantnegative mutants of Ras (N17-Ras), mSOS1 (SOS-Pro), and Raf (⌬N-Raf). Erk1/2 phosphorylation in response to ␣1B-AR stimulation is also attenuated by coexpression of N17-Ras, SOS-Pro, or ⌬N-Raf, but not by coexpression of ␤ARK1ct or by pretreatment with pertussis toxin. The ␣1B-and ␣2A-AR signals are both blocked by phospholipase C inhibition, intracellular Ca 2؉ chelation, and inhibitors of protein-tyrosine kinases. Overexpression of a dominant-negative mutant of c-Src or of the negative regulator of c-Src function, Csk, results in attenuation of the ␣1B-AR-and ␣2A-AR-mediated Erk1/2 signals. Chemical inhibitors of calmodulin, but not of PKC, and overexpression of a dominant-negative mutant of the protein-tyrosine kinase Pyk2 also attenuate mitogenactivated protein kinase phosphorylation after both ␣1B-and ␣2A-AR stimulation. Erk1/2 activation, then, proceeds via a common Ras-, calcium-, and tyrosine kinase-dependent pathway for both G i -and G q/11 -coupled receptors. These results indicate that in HEK-293 cells, the G␤␥ subunit-mediated ␣2A-AR-and the G␣ q/11 -mediated ␣1B-AR-coupled Erk1/2 activation pathways converge at the level of phospholipase C. These data suggest that calcium-calmodulin plays a central role in the calcium-dependent regulation of tyrosine phosphorylation by G protein-coupled receptors in some systems.

The phosphotyrosine residues of receptor tyrosine kinases serve as unique binding sites for proteins involved in intracellular signaling, which contain SRC homology 2 (SH2) domains. Since overexpression or activation of the pp60C-src kinase has been reported in a number of human tumors, including primary human breast carcinomas, we examined the interactions of the SH2 and SH3 domains of human SRC with target proteins in human carcinoma cell lines. Glutathione S-transferase fusion proteins containing either the SH2, SH3, or the entire SH3/SH2 region of human SRC were used to affinity purify tyrosine-phosphorylated proteins from human breast carcinoma cell lines. We show here that in human breast carcinoma cell lines, the SRC SH2 domain binds to activated epidermal growth factor receptor (EGFR) and pl85HER2/neu. SRC SH2 binding to EGFR was also observed in a nontumorigenic cell line after hormone stimulation. Endogenous pp60c-src was found to tightly associate with tyrosinephosphorylated EGFR. Association of the SRC SH2 with the EGFR was blocked by tyrosyl phosphopeptides containing the sequences surrounding tyrosine-530, the regulatory site in the SRC C terminus, or sequences surrounding the maEjor sites of autophosphorylation in the EGFR. These results raise the possibility that association of pp6(C-src with these receptor tyrosine kinases is an integral part of the signaling events mediated by these receptors and may contribute to malignant transformation.Signaling mediated by receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR), requires receptor autophosphorylation on tyrosine (1). These phosphotyrosine residues serve as unique binding sites for proteins that contain SRC homology 2 (SH2) domains. This protein motif recognizes phosphotyrosine in a sequence-specific manner, and the in vivo specificity of proteins for their cognate phosphotyrosine residue in the receptor is maintained in in vitro binding assays using the isolated SH2 domains of these proteins. Such domains are found in a number of proteins involved in intracellular signaling including p85, the noncatalytic subunit of phosphatidylinositol 3-kinase; GAP, the GTPase-activating protein of p2lras; and phospholipase Cy (2). Constitutive activation of these signaling pathways is apparent in many malignancies. Human breast cancers often overexpress two closely related receptor tyrosine kinases, EGFR or p185HER2/neu, and amplification of these genes is correlated with poor clinical prognosis (3-5). It has recently been reported that many primary human breast tumors also show elevated activity of pp6c-src (6), suggesting that this protein may play a role in carcinoma of the breast. These results indicate that activation of downstream events mediated by both receptor and nonreceptor tyrosine kinases may be critical in some types of human neoplasia. Evidence for similar interactions between receptor and nonreceptor tyro-The publication costs of this article were defrayed in part by page charge payment. This article must the...