The constitutively active mutant Galpha13 transforms mouse fibroblast cells deficient in insulin-like growth factor-I receptor.
Insulin-like growth factor-I (IGF-I) receptor plays an important role in normal cell cycle progression and tumor growth, and it is thought to be essential for cellular transformation. To test this hypothesis, we stably transfected a GTPase-deficient mutant human Galpha13, which is highly oncogenic when overexpressed in vitro, into R- fibroblasts derived from IGF-I receptor-deficient mice. Northern blots of multiple clones revealed the expression of a 1.8-kilobase pair mutant Galpha13 transcript in transfected cells, in addition to the 6-kilobase pair endogenous mRNA. The transfection resulted in a doubling of the expression of Galpha13 protein in these cells as assessed by Western blot analysis. The transforming ability of the mutant Galpha13 was tested using the soft agar assay. Nontransfected R- cells cultured with 10% fetal bovine serum failed to form colonies after 3 weeks. Most of the mutant Galpha13-expressing clones formed significant numbers of colonies (11-50 colonies/1000 cells plated). Overexpression of the IGF-I receptor enabled R- cells to form colonies (27 colonies), and co-transfection of the mutant Galpha13 caused a further increase in colony formation (117-153 colonies) in three of five clones analyzed. Apparently Galpha13 works through pathways other than mitogen-activated protein kinase and c-Jun N-terminal kinase in transforming R- cells, because their activities were not significantly altered by the mutant Galpha13 expression. These results demonstrate that Galpha13 can induce cellular transformation through pathways apparently independent of the IGF-I receptor and that activation of the IGF-I receptor signaling pathways, although not essential for the transforming phenotype, enhances the effect of other pathways.
Liu
J L
JL
Section on Cellular and Molecular Physiology, Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
Blakesley
V A
VA
Gutkind
J S
JS
LeRoith
D
D
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000502:physiology; D006801:Humans; D051379:Mice; D016296:Mutagenesis; D012333:RNA, Messenger / Q000378:metabolism; D017526:Receptor, IGF Type 1 / Q000172:deficiency / Q000502:physiology*; D014162:Transfection
D012333:RNA, Messenger; D017526:Receptor, IGF Type 1; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019204:GTP-Binding Proteins