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 Gs (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 Gi2 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 [3H]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.
Hermouet
S
S
Molecular Pathophysiology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
Merendino
J J
JJ
Gutkind
J S
JS
Spiegel
A M
AM
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002455:Cell Division; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism*; D004261:DNA Replication; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000502:physiology; D015870:Gene Expression; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D051381:Rats; D014162:Transfection
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D000242:Cyclic AMP; D019204:GTP-Binding Proteins