The Q205LGo-alpha subunit expressed in NIH-3T3 cells induces transformation.
The growth functions of the heterotrimeric G protein G(o) was studied by expression in heterologous systems. The alpha-subunit of G(o) was mutated to convert Gln-205 to Leu (Q205L). Mutation of this conserved glutamine residue in G protein alpha-subunits is thought to persistently activate G proteins by inhibiting their GTPase activity. The wild type and mutant G(o)-alpha subunits were expressed in NIH-3T3 fibroblasts. These cells do not contain any measurable amounts of G(o)-alpha mRNA or protein. Transfection of wild type or Q205LG(o)-alpha subunit cDNA under the control of a dexamethasone-inducible promoter results in dexamethasone-dependent transcription of the mRNA and expression of the protein. The Q205LG(o)-alpha, but not wild type G(o)-alpha, stimulates mitogenesis in NIH-3T3 fibroblasts without significantly stimulating phospholipase C activity. Continuous expression of mutant G(o)-alpha induces focus formation, whereas transfections with vector alone or vector containing the native G(o)-alpha cDNA were without significant transforming effect in NIH-3T3 cells. Q205L G(o)-alpha did not induce focus formation in RAT-1 fibroblasts. Q205LG(o)-alpha-transformed NIH-3T3 cells are capable of anchorage-independent growth, as assessed by colony formation in soft agar. Q205LG(o)-alpha transformed cells induced tumors when injected into Nu/Nu mice. These results indicate that mutant G(o)-alpha subunits whose GTPase activity is presumably inhibited can induce the neoplastic transformation of NIH-3T3 cells in a phospholipase C-independent manner.
Kroll
S D
SD
Department of Pharmacology and Physiology, Mount Sinai School of Medicine, City University of New York, New York 10029.
Chen
J
J
De Vivo
M
M
Carty
D J
DJ
Buku
A
A
Premont
R T
RT
Iyengar
R
R
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D003001:Cloning, Molecular; D003907:Dexamethasone / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D005973:Glutamine; D007700:Kinetics; D007930:Leucine; D046911:Macromolecular Substances; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism; D009368:Neoplasm Transplantation; D009838:Oligodeoxyribonucleotides; D051381:Rats; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D005973:Glutamine; D003907:Dexamethasone; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D007930:Leucine