Ras mutants with the ability to interact with different effectors have played a critical role in the identification of Ras-dependent signaling pathways. We used two mutants, Ras S35 and Ras G37 , which differ in their ability to bind Raf-1, to examine Ras-dependent signaling in thyroid epithelial cells. abolished TSH-stimulated changes in cell morphology and thyroglobulin expression, while Ras G37 had no effect on these activities. Together, the data indicate that cross talk between Ras and PKA discriminates between distinct Ras effector pathways.Ras proteins are important signaling intermediates that convey signals initiated at the cell surface to effector pathways in the cytoplasm. Ras exerts effects on cell transformation and proliferation, the actin cytoskeleton, differentiation, and apoptosis. It is likely that these effects are mediated by multiple effectors, including Raf-1, RalGDS (2), phosphatidylinositol 3-kinase (PI3K) (48), and other Ras-binding proteins (reviewed in reference 38), as well as members of the Rho family (26,44,45).The elucidation of multiple Ras effector pathways was greatly facilitated by the isolation of Ras mutants which interact with single downstream effectors (60). Ras S35 binds preferentially to Raf, while Ras G37 binds to RalGDS (49,53,60 While the differential effects of Ras S35 and Ras G37 on Raf and MAPK activity are consistently observed (23,25,27,46,49,60), other effects of these mutants vary. Both Ras S35 and Ras G37 transform some NIH 3T3 strains, inducing growth in medium with low serum levels, anchorage-independent proliferation, and tumor formation in nude mice (27). The activity of Ras G37 in these assays suggests that Ras stimulates some of the same biological effects through Raf-independent pathways and that the effector pathways used by Ras vary in different cell types. Consistently, Ras V12 stimulates transformation in most cells, while activated forms of Raf transform fibroblasts but not epithelial cells (41). If Ras signals through multiple effectors, the selection of a particular effector pathway should be regulated. One potential way to achieve such regulation is through cross talk between Ras and other signaling pathways.One of the best-studied examples of cross talk is that between Ras and protein kinase A (PKA). In many cells, cyclic AMP (cAMP) inhibits Ras signaling through Raf and the MAPK cascade (reviewed in reference 6). cAMP activates PKA, which phosphorylates Raf-1 at multiple serine residues. PKA-mediated phosphorylation reduces the affinity of Raf for Ras (17,63) and decreases Raf kinase activity (10,17,57), effects which may inhibit Ras-mediated proliferation. Ras and cAMP also collaborate to produce similar effects. In PC12 cells, Ras and cAMP induce neurite extension (13, 54) and promote cell survival (64). Ras and cAMP stimulate proliferation in thyroid cells (7,36,39), and Ras activity is required for the mitogenic effects of thyrotropin (TSH) (32). Despite the requirement for Ras, TSH downregulates signaling through Raf and the MAPK cascade...

Thyroid-stimulating hormone stimulates proliferation through both the cAMP-dependent protein kinase and Ras but not through Raf-1 and mitogen-activated and extracellular signal-related kinase kinase. We now report that thyroid-stimulating hormone represses mitogen-activated protein kinase activity and that microinjection of an effector domain mutant Ha-Ras protein, Ras(12V,37G), defective in Raf-1 binding and mitogenactivated protein kinase activation, stimulates DNA synthesis in quiescent and thyroid-stimulating hormonetreated thyrocytes. A yeast two-hybrid screen identified RalGDS as a Ras(12V,37G) binding protein and therefore a potential effector of Ras in these cells. Associations between Ras and RalGDS were observed in extracts prepared from thyroid cells. Microinjection of a mutant RalA(28N) protein thought to sequester RalGDS family members reduced DNA synthesis stimulated by Ras as well as cAMP-mediated DNA synthesis in two cell lines which respond to cAMP with mitogenesis. These results support the idea that RalGDS may be an effector of Ras in cAMP-mediated growth stimulation.The elucidation of the critical role played by Ras in growth control initiated an intensive effort to identify Ras binding effector molecules. The first Ras binding protein identified was p120GAP (1), which functions both as an effector and downregulator of Ras. The best characterized Ras effector is the cytoplasmic serine/threonine protein kinase Raf-1 (2, 3). Direct interaction between the N-terminal domain of Raf-1 and the effector loop of Ras was first demonstrated using the yeast two-hybrid system (4, 5), while later studies reported the coprecipitation of Ras and Raf (6, 7). A number of other proteins bind directly to GTP-bound Ras, including the p110 catalytic subunit of phosphatidylinositol 3-kinase (8), AF6 (9), Rin-1 (10), NF1 (11), MEKK1 (12), protein kinase C (13), a herpesvirus-encoded protein (14), and a guanine nucleotide dissociation stimulator for the Ras-related protein Ral (RalGDS) and a closely related protein (RGL) (15-18). While interaction of Ras with Raf-1, p110 (8), and RalGDS (19) results in increased enzyme activity, the biological outcomes of these other Rasprotein interactions are less understood. TSH 1 -stimulated DNA synthesis requires both the cAMP-dependent protein kinase and Ras (20). Although Ras-dependent, mitogenic signaling induced by TSH does not require Raf-1 or MEK (21), suggesting that Ras utilizes alternate effectors in the presence of cAMP. To identify such effectors, an extensive two-hybrid screen of a thyroid cell cDNA library was performed with an effector domain mutant of Ras which does not bind Raf-1 (22) but stimulates DNA synthesis in thyrocytes. This screen repeatedly identified RalGDS as a potential Ras effector. Consistent with such a role, interaction between Ras and RalGDS was detected in extracts prepared from thyroid cells. Further, microinjection of a mutant RalA(28N) protein thought to sequester RalGDS (19, 23) specifically reduced cAMP-mediated DNA synthesis in both rat ...

These findings indicate there is an abnormality in the residua of the cortical subplate in the anterior region of the adult parahippocampal gyrus in schizophrenia subjects.

Therapeutic treatment of parkinsonian monkeys by chronic administration of levodopa (l-DOPA) leads to the development of dyskinesias and other motor fluctuations. It is unclear whether there are alterations in the dopamine system that are related to the induction of dyskinesias by l-DOPA, but recent attention has focused on the D1 receptor system. The present study assessed the consequences of chronic l-DOPA treatment in monkeys made parkinsonian with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on indices of the pre- and post-synaptic dopamine (DA) system. Treatment with therapeutic doses of l-DOPA led to the induction of dyskinesias in the MPTP-treated monkeys. High-pressure liquid chromatography was used for measurement of tissue levels of DA and its metabolites, and quantitative autoradiography was used to examine the regional integrity of the presynaptic DA system (by measuring [3H]mazindol binding to DA uptake sites). Quantitative autoradiography was used to measure the number of postsynaptic D1 receptors (using [3H] SCH 23390) in the striatum and pallidum of normal, MPTP alone, and MPTP monkeys treated chronically with l-DOPA. In both MPTP-treated monkeys, levels of DA and metabolites as well as [3H]mazindol binding were greatly reduced in the caudate and putamen, slightly more in dorsal than in ventral areas. However, the lack of increase in striatal DA levels along with higher [3H]mazindol binding in MPTP-plus-l-DOPA-treated monkeys suggested differences in the way DA was used after l-DOPA treatment In MPTP-treated monkeys, a significant increase (141-170% of normals) of D1 receptor numbers was observed in putamen and dorsal caudate. With l-DOPA treatment, the number of D1 receptor numbers was further elevated in caudal putamen (119-123%), dorsal caudate (110-130%), and in the internal segment of the globus pallidus (GPi; 164% of normals) of MPTP-treated monkeys as compared with MPTP treatment alone. This suggested that in MPTP-treated monkeys made dyskinetic by chronic pulsatile delivery of l-DOPA, there was enhanced production of D1 receptors in the direct striatal output to the GPi.

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The inducible nature of the immediate-early genes (IEGs) c-fos and zif268 allows their products to be used as activity markers in the brain. The utility of such markers in general is restricted because they can resolve only neurons activated by a single stimulus. To overcome this limitation, we have developed a double-label technique that exploits the dissimilar time course of zif268 mRNA and protein induction, allowing them to be separately induced by two different stimuli and independently stained to provide a visual display of neurons that are responsive to each stimulus. Two powerful features of this new imaging technique-the possibility of staining separate populations of activated neurons and the ability to visualize them at the cellular level-should extend IEG applications in biological activity mapping.

We have quantified by receptor autoradiography the number of NK1 receptors, using [125I] Bolton-Hunter labeled substance P, in striatum and pallidum (internal (GPi) or external (GPe) segment) of patients suffering from Alzheimer's (AD) and Parkinson's disease (PD). When compared to non-neurologic controls, a significant increase in the number of NK1 sites has been observed in the striatum of PD patients. No significant differences were observed for the GPi and GPe. We observed no significant differences from controls in the number of NK1 sites in the striatum and pallidum of AD cases. However, the number of NK1 sites in the striatum of AD patients was significantly lower than that of PD patients. These results show that the expression of NK1 receptors in the basal ganglia is affected in PD.