A G-PROTEIN BETA-GAMMA DIMER-MEDIATED PATHWAY CONTRIBUTES TO MITOGEN-ACTIVATED PROTEIN-KINASE ACTIVATION BY THYROTROPIN-RELEASING-HORMONE RECEPTORS IN TRANSFECTED COS-7 CELLS

Activation of mitogen-activated protein kinase (MAPK) is induced by ad ding thyrotropin-releasing hormone (TRH) to COS-7 cells cotransfected with TRH receptors and an epitope-tagged MAPK. Long term treatment of the cells with pertussis toxin has no effect on TRH-induced MAPK activ ation. Incubation of the cells with the protein kinase C (PKC) inhibit or GF109203X causes an almost complete inhibition of MAPK activation b y the PKC activator phorbol-12-myristate-13-acetate. In contrast, only similar to 50% of the TRH-induced MAPK activity is inhibited by GF109 203X, indicating that activation of MAPK by TRH is only partially depe ndent on PKC. The inhibitory effect of GF109203X is additive with that of p21(N17ras), a dominant negative mutant of p21(ras) that exerts li ttle effect on PKC-dependent MAPK activation by phorbol-12-myristate-1 3-acetate. The TRH-induced activation of MAPK also is inhibited partia lly by overexpression of transducin alpha subunits (alpha t), an agent known to sequester free G protein beta gamma dimers. However, the inh ibitory potency of alpha t on TRH-induced activation is about half of that obtained in cells transfected with m2 muscarinic receptors, which activate MAPK exclusively through beta gamma dimers. The effect of al pha t is also additive with that of GF109203X but not with that of p21 (N17ras). MAPK activation is not induced by the constitutively active form of G(alpha q) due to an inhibitory effect of its expression at a step downstream of that at which PKC-dependent and -independent routes to MAPK converge. Our results demonstrate that TRH receptors activate MARK by a pathway only partially dependent on PKC activity. Furthermo re, they indicate that beta gamma dimers of a pertussis and cholera to xin-insensitive G protein are involved in the PKC-independent fraction of the dual signaling route to MAPK initiated in the TRH receptor.