Am. Aragay et al., G(12) REQUIREMENT FOR THROMBIN-STIMULATED GENE-EXPRESSION AND DNA-SYNTHESIS IN 1321N1 ASTROCYTOMA-CELLS, The Journal of biological chemistry, 270(34), 1995, pp. 20073-20077
Thrombin stimulation of 1321N1 astrocytoma cells leads to Ras-dependen
t AP-1-mediated transcriptional activation and to DNA replication. In
contrast to what has been observed in most cell systems, in 1321N1 cel
ls these responses are pertussis toxin-insensitive. The pertussis toxi
n-insensitive G-protein G(12) has been implicated in cell growth and t
ransformation in different cell systems. We have examined the potentia
l 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 G(12) 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 inhibit
ion of thrombin-stimulated DNA synthesis. In contrast, microinjection
of nonimmune IgG or an antibody directed against the C terminus of G a
lpha(0) did not reduce the mitogenic response to thrombin. Furthermore
, microinjection of the anti-G alpha(12) antibody had no effect on fib
roblast growth factor-stimulated DNA synthesis. These results demonstr
ate a specific role for G alpha(12) in the mitogenic response to throm
bin in human astroglial cells.