ACTIVATING AND INACTIVATING MUTATIONS OF THE ALPHA-SUBUNIT OF GI2 PROTEIN HAVE OPPOSITE EFFECTS ON PROLIFERATION OF NIH 3T3 CELLS

Previous studies have demonstrated that mutations of highly conserved residues in the alpha-subunit of G(s) (alpha(s)) can inhibit either the intrinsic GTPase activity (glutamine-227 to leucine, Q227L) or the ability of the protein to be activated by GTP (glycine-226 to alanine, G226A). We stably transfected NIH 3T3 cells with cDNAs encoding G(i2) alpha-subunit (alpha(i2)) containing either wild-type sequence or the homologous mutations Q205L and G204A. High expression of wild-type alpha(i2) Q205L alpha(i2), and G204A alpha(i2) was confirmed in transfected cells by immunoblot analysis. The overexpression of all three alpha(i2) proteins was accompanied by an increase in beta-subunit expression. Q205L alpha(i2) was a poor substrate for ADP-ribosylation by pertussis toxin as compared with wild-type alpha(i2). Expression of Q205L alpha(i2) markedly decreased forskolin- or cholera toxin-stimulated intracellular cAMP levels in intact cells, confirming the constitutively activated state of the protein. In contrast, G204A alpha(i2) increased intracellular cAMP and was resistant to guanosine 5'-[gamma-thio]triphosphate-induced inhibition of ADP-ribosylation by pertussis toxin, as expected for an inactive alpha(i2). Transfection of wild-type, Q205L, or G204A alpha(i2) cDNA did not induce focus formation of NIH 3T3 cells. However, overexpression of Q205L alpha(i2) induced a decreased serum requirement, a reduced doubling time, and an 8- to 10-fold increase in [H-3]thymidine incorporation. Q205L alpha(i2) cells formed small colonies in soft agar, demonstrating some degree of anchorage-independent proliferation. Expression of G204A alpha(i2) slowed the growth of NIH 3T3 cells. We conclude that alpha(i2) plays an important role in regulation of fibroblast growth.