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.

Activated alpha subunit of Go protein induces oocyte maturation.

The capability of various activated guanine nucleotide binding regulatory protein (G protein) alpha subunits to induce meiotic maturation was studied. Activated Go protein alpha subunit (alpha o*) but not the three inhibitory G protein alpha subunits triggered meiotic maturation in Xenopus oocytes. The effect was concentration dependent with a half-maximal effect in the 100-200 pM range. Injection of alpha o* stimulated protein kinase C activity. Coinjection of the peptide containing residues 19-36 of protein kinase C [PKC-(19-36)], a specific protein kinase C inhibitor, blocked the alpha o*- but not progesterone-induced maturation. Cycloheximide and the injection of antisense oligonucleotides specific to the c-mos transcript blocked alpha o-induced maturation. Immunoprecipitation with a mos protein-specific monoclonal antibody showed that alpha o-injected oocytes had phosphorylated mos protein. When PKC-(19-36) was coinjected with alpha o*, phosphorylated mos protein was not observed. These observations indicate that alpha o*, through protein kinase C and the translation of c-mos, can trigger meiotic division of Xenopus oocytes. Our results raise the possibility that persistently activated G proteins through cellular protooncogenes may regulate cell-cycle resumption.