G12 requirement for thrombin-stimulated gene expression and DNA synthesis in 1321N1 astrocytoma cells.
Thrombin stimulation of 1321N1 astrocytoma cells leads to Ras-dependent AP-1-mediated transcriptional activation and to DNA replication. In contrast to what has been observed in most cell systems, in 1321N1 cells these responses are pertussis toxin-insensitive. The pertussis toxin-insensitive G-protein G12 has been implicated in cell growth and transformation in different cell systems. We have examined the potential role of this protein in AP-1-mediated transcriptional activation and DNA synthesis in 1321N1 cells. Transient expression of an activated (GTPase-deficient) mutant of G alpha 12 increased AP-1-dependent gene expression. This response was inhibited by co-expression of a dominant negative Ala-15 Ras protein. To determine whether the pertussis toxin-insensitive G12 protein is involved in the thrombin-stimulated DNA synthesis, an inhibitory antibody against the C-terminal sequence of G alpha 12 subunit was microinjected into 1321N1 cells. Microinjection of the anti-G alpha 12 resulted in a concentration-dependent inhibition of thrombin-stimulated DNA synthesis. In contrast, microinjection of nonimmune IgG or an antibody directed against the C terminus of G alpha o did not reduce the mitogenic response to thrombin. Furthermore, microinjection of the anti-G alpha 12 antibody had no effect on fibroblast growth factor-stimulated DNA synthesis. These results demonstrate a specific role for G alpha 12 in the mitogenic response to thrombin in human astroglial cells.
Aragay
A M
AM
Division of Biology, California Institute of Technology, Pasadena 91125, USA.
Collins
L R
LR
Post
G R
GR
Watson
A J
AJ
Feramisco
J R
JR
Brown
J H
JH
Simon
M I
MI
D000595:Amino Acid Sequence; D000906:Antibodies / Q000008:administration & dosage; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism; D001254:Astrocytoma / Q000235:genetics / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D004247:DNA / Q000096:biosynthesis*; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology; D037342:Pertussis Toxin; D013917:Thrombin / Q000494:pharmacology*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D018631:ras Proteins / Q000378:metabolism
D000906:Antibodies; D010446:Peptide Fragments; D018808:Transcription Factor AP-1; D010566:Virulence Factors, Bordetella; D004247:DNA; D037342:Pertussis Toxin; D013917:Thrombin; D019204:GTP-Binding Proteins; D018631:ras Proteins