Mitogenic signalling by delta opioid receptors expressed in rat-1 fibroblasts involves activation of the p70s6k/p85s6k S6 kinase.

The regulation of mitogenic signalling pathways by G-protein-coupled receptors has been studied in Rat-1 fibroblasts stably transfected with the murine delta opioid receptor. We showed recently that stimulation of this receptor led to the activation of the p42 and p44 isoforms of mitogen-activated protein (MAP) kinase [Burt, Carr, Mullaney, Anderson and Milligan (1996) Biochem. J. 320, 227-235]. The present study has examined the role of the ribosomal S6 kinase p70(s6k) in mitogenic signalling by the delta opioid receptor. Treatment of Rat-1 fibroblasts expressing this receptor with the synthetic enkephalin [d-Ala,d-Leu]-enkephalin (DADLE) led to a dose-dependent increase in p70(s6k) enzyme activity. Activation of p70(s6k) was dependent on the level of delta opioid receptor expressed and was sustained above basal levels for several hours. Immunoblotting revealed that p70(s6k) was subject to increased phosphorylation, the extent of which coincided temporally with enzyme activation. Activation of p70(s6k) by DADLE, but not by platelet-derived growth factor, was blocked by pretreatment of cells with pertussis toxin. Activation of p70(s6k) was also partly blocked by wortmannin, indicating that phosphoinositide 3-OH kinase is required for full activation of p70(s6k) by opioid receptor agonists. Activation of the delta opioid receptor in transfected cells led to increased DNA synthesis. This increase was prevented by rapamycin, which also completely blocked activation of p70(s6k) by DADLE. In addition, prevention of the activation of p42 and p44 MAP kinases also blocked the induction of DNA synthesis by DADLE. These results suggest that the activation of both MAP kinases and p70(s6k) might be crucial to the induction of mitogenic responses by Gi-linked receptors such as the delta opioid receptor.