When expressed in PC12 cells, the platelet-derived growth factor ␤ receptor (␤PDGF-R) mediates cell differentiation. Mutational analysis of the ␤PDGF-R indicated that persistent receptor stimulation of the Ras/Raf/mitogenactivated protein (MAP) kinase pathway alone was insufficient to sustain PC12 cell differentiation. PDGF receptor activation of signal pathways involving p60 c-src or the persistent regulation of phospholipase C␥ was required for PC12 cell differentiation. ␤PDGF-R regulation of phosphatidylinositol 3-kinase, the GTPase-activating protein of Ras, and the tyrosine phosphatase, Syp, was not required for PC12 cell differentiation. In contrast to overexpression of oncoproteins involved in regulating the MAP kinase pathway, growth factor receptor-mediated differentiation of PC12 cells requires the integration of other signals with the Ras/Raf/MAP kinase pathway.The platelet-derived growth factor receptor (PDGF-R) is a transmembrane polypeptide encoding an intrinsic tyrosine kinase in its intracellular domain. Two distinct PDGF-R genes encode either an ␣ (7, 42, 47) or a ␤ (6, 19, 73) subunit. Binding of PDGF (22) induces dimerization (23) and trans phosphorylation of the ␤PDGF-R on specific tyrosine residues (35). The phosphorylated receptor initiates a series of intracellular signals which ultimately lead to cell growth (12), differentiation (20), and chemotaxis (70) depending on the cellular context.A number of tyrosines on the intracellular domain of the ␤PDGF-R are phosphorylated upon activation of the receptor and serve as recognition sites for proteins which contain Src homology 2 (SH2) domains (57). For example, the ␤PDGF-R has been shown to associate with Src family tyrosine kinases p60 c-src , p59 fyn , and p62 c-yes (39) via juxtamembrane tyrosines 579 and 581, which are in vivo phosphorylation sites (50). The SH2 domain-containing proteins p46 and p52 (Shc proteins) bind to the ␤PDGF-R at multiple phosphotyrosines including tyrosine 581 and indirectly via association with other tyrosinephosphorylated proteins (74). Phosphorylation of tyrosines 740 and 751 is critical for association of the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3-K) with the ␤PDGF-R (2, 8, 29-31). Phosphorylated tyrosine 751 also binds Nck via its SH2 binding domain (52). The GTPase-activating protein (GAP) of Ras is tyrosine phosphorylated in response to PDGF and binds to the receptor at phosphorylated 33,49). The SH2-containing phosphotyrosine phosphatase, Syp, associates with phosphorylated tyrosine 1009 (34, 41). In addition, there is evidence which suggests that the adaptor protein, Grb2, associates with the ␤PDGF-R via tyrosine 716 (1) and indirectly through Syp (44). Phosphorylated tyrosine 1009 may also influence the binding of phospholipase C␥ (PLC␥) to tyrosine 1021 (28, 59). Mutational analysis of the ␤PDGF-R has demonstrated that phosphorylated tyrosines 740 and 751, which mediate association with PI3-K, and tyrosine 1021, which mediates association with PLC␥, are necessary for the transducti...

Persistent stimulation of specific protein kinase pathways has been proposed as a key feature of receptor tyrosine kinases and intracellular oncoproteins that signal neuronal differentiation of rat pheochromocytoma (PC12) cells. Among the protein serine/threonine kinases identified to date, the p42/44 mitogen-activated protein (MAP) kinases have been highlighted for their potential role in signalling PC12 cell differentiation. We report here that retrovirus-mediated expression of GTPase-deficient, constitutively active forms of the heterotrimeric Gq family members, G alpha qQ209L and G alpha 16Q212L, in PC12 cells induces neuronal differentiation as indicated by neurite outgrowth and the increased expression of voltage-dependent sodium channels. Differentiation was not observed after cellular expression of GTPase-deficient forms of alpha i2 or alpha 0, indicating selectivity for the Gq family of G proteins. As predicted, overexpression of alpha qQ209L and alpha 16Q212L constitutively elevated basal phospholipase C activity approximately 10-fold in PC12 cells. Significantly, little or no p42/44 MAP kinase activity was detected in PC12 cells differentiated with alpha 16Q212L or alpha qQ209L, although these proteins were strongly activated following expression of constitutively active cRaf-1. Rather, a persistent threefold activation of the cJun NH2-terminal kinases (JNKs) was observed in PC12 cells expressing alpha qQ209L and alpha 16Q212L. This level of JNK activation was similar to that achieved with nerve growth factor, a strong inducer of PC12 cell differentiation. Supportive of a role for JNK activation in PC12 cell differentiation, retrovirus-mediated overexpression of cJun, a JNK target, in PC12 cells induced neurite outgrowth. The results define a p42/44 MAP kinase-independent mechanism for differentiation of PC12 cells and suggest that persistent activation of the JNK members of the proline-directed protein kinase family by GTPase-deficient G alpha q and G alpha 16 subunits is sufficient to induce differentiation of PC12 cells.

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Small cell lung carcinoma (SCLC) accounts for 20 -25% of primary lung cancers and is rapidly growing, widely metastatic, and rarely curable. Autocrine stimulation of multiple G protein-coupled neuropeptide receptor systems contributes to the transformed growth of SCLC. The ability of neuropeptide receptors to stimulate phospholipase C and mobilize intracellular Ca 2؉ indicates that G q family members of heterotrimeric G proteins are a convergence point mediating autocrine signaling by multiple neuropeptides in SCLC. Expression of a GTPase-deficient, constitutive active form of an ␣ q family member, ␣ 16 Q212L, in SCLC markedly inhibited growth of the cells in soft agar and tumor formation in nude mice. SCLC lines expressing ␣ 16 Q212L exhibited 2-4-fold elevated basal phospholipase C activity, but neuropeptide and hormone-regulated intracellular Ca 2؉ mobilization was nearly abolished. The data suggest that Ca 2؉ mobilization is an obligatory signal in neuropeptide-stimulated growth of SCLC. In addition, the proline-directed c-Jun NH 2 -terminal kinases/stress-activated protein kinases, which are members of the mitogen-activated protein kinase family, were stimulated ϳ2-fold in parental SCLC in response to exogenous neuropeptides and muscarinic agonists and were constitutively activated to the same degree in ␣ 16 Q212L-expressing SCLC. Thus, ␣ 16 Q212L expression induced desensitization of neuropeptide-stimulated Ca 2؉ signaling and persistent activation of the c-Jun NH 2 -terminal kinase/ stress-activated protein kinase pathway. We propose that the induction of discordant signaling by selective perturbation of receptor-regulated effector systems leads to the inhibition of SCLC cell growth. Small cell lung carcinoma (SCLC)1 displays neuroendocrine features exemplified by the presence of cytoplasmic neurosecretory granules containing a wide variety of mitogenic neuropeptides including gastrin-releasing peptide, arginine vasopressin, neurotensin, cholecystokinin, and many others (1, 2). Significantly, SCLC also expresses receptors for these neuropeptides, thereby establishing autocrine-stimulated cell growth (3). The number and variability of neuropeptides released from individual small cell carcinomas hampers effective blockade of mitogenic signaling at the level of ligand/receptor binding using specific neuropeptide antagonists. This redundancy at the level of receptor signaling highlights the importance of defining the intracellular components involved in mitogenic signal transduction in SCLC where convergence of multiple neuropeptide receptor systems into common pathways would be anticipated.Molecular cloning of the receptors for gastrin-releasing peptide, vasopressin, and gastrin/cholecystokinin reveals that they are members of the superfamily of seven membrane-spanning receptors (4 -8). As a class, these receptors initiate signaling in response to ligand binding by interacting with heterotrimeric G proteins. Although the repertoire of G proteins potentially involved in neuropeptide signaling in SCLC is quite...