MUSCARINIC RECEPTORS TRANSFORM NIH 3T3 CELLS THROUGH A RAS-DEPENDENT SIGNALING PATHWAY INHIBITED BY THE RAS-GTPASE-ACTIVATING PROTEIN SH3 DOMAIN

Expression of certain subtypes of human muscarinic receptors in NIH 3T 3 cells provides an agonist-dependent model of cellular transformation by formation of foci in response to carbachol. Although focus formati on 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 act ivation state of native and epitope-tagged Ras proteins, the contribut ions of Ras and Ras GTPase-activating protein (Ras-GAP) to muscarinic receptor-dependent transformation were defined. Transforming muscarini c receptors were able to activate Ras, and such activation was require d for transformation because focus formation was inhibited by coexpres sion of either Ras with a dominant-negative mutation or constructs of Ras-GAP that include the catalytic domain. Coexpression of the N-termi nal region of GAP or of its isolated SH3 (Src homology 3) domain, but not its SH2 domain, was also sufficient to suppress muscarinic recepto r-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 expressio n of the Ras-GAP SH3 domain occurs proximal to Ras activation and is s elective for the mitogenic pathway activated by carbachol, as cellular transformation by either v-Ras or trkA/nerve growth factor is unaffec ted.