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The Ras-GRF1 exchange factor is strongly implicated in the control of neuronal Ras. The activity of Ras-GRF1 is regulated by increases in intracellular calcium and the release of Gbetagamma subunits from heterotrimeric G-proteins. Increases in Ras-GRF1 activity toward Ras that are stimulated by receptors coupled to G-proteins are associated with enhanced phosphorylation of Ras-GRF1 on one or more serine residues. Co-expression of Ras-GRF1 with subtype 1 human muscarinic receptors in COS-7 cells allowed mapping of a carbachol-stimulated phosphorylation site to a region composed of residues 916-976. Site-directed mutagenesis replaced each of the serine residues within this region with alanine and demonstrated that serine 916 is a major site of in vivo phosphorylation of Ras-GRF1 in both COS-7 cells and NIH-3T3 fibroblasts. Serine 916 was a substrate for protein kinase A both in vivo and in vitro, suggesting a novel link between the cAMP and Ras signaling systems. Carbachol-dependent phosphorylation of serine 916 occurred through a protein kinase A-independent pathway, however. Full-length Ras-GRF1 that contains an alanine 916 mutation was only partially activated by carbachol, suggesting that phosphorylation at residue 916 is necessary for full activation. Phosphorylation of serine 916 in response to forskolin treatment did not, however, increase the activity of Ras-GRF1, indicating that it is not sufficient for activation.

Expression of certain subtypes of human muscarinic receptors in NIH 3T3 cells provides an agonist-dependent model of cellular transformation by formation of foci in response to carbachol. Although focus formation correlates with the ability of the muscarinic receptors to activate phospholipase C, the actual mitogenic signal transduction pathway is unknown. Through cotransfection experiments and measurement of the activation state of native and epitope-tagged Ras proteins, the contributions of Ras and Ras GTPase-activating protein (Ras-GAP) to muscarinic receptor-dependent transformation were defined. Transforming muscarinic receptors were able to activate Ras, and such activation was required for transformation because focus formation was inhibited by coexpression of either Ras with a dominant-negative mutation or constructs of Ras-GAP that include the catalytic domain. Coexpression of the N-terminal region of GAP or of its isolated SH3 (Src homology 3) domain, but not its SH2 domain, was also sufficient to suppress muscarinic receptor-dependent focus formation. Point mutations at conserved residues in the Ras-GAP SH3 domain reversed its action, leading to an increase in carbachol-dependent transformation. The inhibitory effect of expression of the Ras-GAP SH3 domain occurs proximal to Ras activation and is selective for the mitogenic pathway activated by carbachol, as cellular transformation by either v-Ras or trkA/nerve growth factor is unaffected.

Summary: An internal control mechanism capable of controlling gonadotrophic hormone secretion in the human trophoblast has been identified by incubation of an established cell line with a precursor of progesterone. Biological, biochemical, and electron microscopic findings verify inhibition of gonadotrophic hormone synthesis, depletion of glycogen, activation of phosphorylase, the glycogen-metabolizing enzyme, and ultrastructural appearance of liquid-containing cytoplasmic granules in single cells, indicative of steroid synthesis.

The transverse magnetoresistance ??(T,H)??(T,H)-?(T,0) has been measured at fixed temperatures in the range 0.5K \leqslant T \leqslant 40K as a function of magnetic field H \leqslant 15T for polycrystalline samples of U6Fe and U6Co, and a single crystal of UPt3(\overrightarrowH||\overrightarrowa and \overrightarrowH||\overrightarrowc, \overrightarrowJ||\overrightarrowb). The sign of ??(T,H) changes from positive to negative as T increases through ?25K for U6Fe and U6Co, and \i?18K for UPt3. At low temperatures the data obey a novel universal form ??=AH2/B+H for all three materials, where A and B are T- and H-independent constants. Such behavior is postulated as typical of materials entering a heavy Fermi liquid state at low temperatures.