Agonist activation of p42 and p44 mitogen-activated protein kinases following expression of the mouse δ opioid receptor in Rat-1 fibroblasts: effects of receptor expression levels and comparisons with G-protein activation Available to Purchase

Rat-1 fibroblasts were transfected with a cDNA encoding the mouse Δ opioid receptor. Two separate clones, D2 (which expressed some 6 pmol of the receptor/mg of membrane protein) and DOE (which expressed some 0.2 pmol/mg of membrane protein), were examined in detail. With membranes from both clones, the opioid agonist [D-Ala²]leucine enkephalin (DADLE) caused stimulation of high-affinity GTPase activity and of the binding of guanosine 5´-[γ-[³⁵S]thio]triphosphate, and inhibition of forskolin-amplified adenylate cyclase activity. DADLE also induced phosphorylation and activation of both the p42^MAPK (42 kDa isoform) and p44^MAPK (44 kDa isoform) members of the mitogen-activated protein kinase (MAP kinase) family. All of these effects of DADLE were prevented in both clones by pretreatment of the cells with pertussis toxin. The maximal response that could be produced by DADLE in direct assays of G-protein activation were substantially greater in clone D2 than in clone DOE, but in both clones essentially full phosphorylation of both p42^MAPK and p44^MAPK could be achieved. EC₅₀ values for DADLE stimulation of GTPase activity and for activation of p44^MAPK were substantially lower in clone D2 than in clone DOE. Moreover, in both clones the EC₅₀ value for DADLE stimulation of p44^MAPK was substantially lower than that for stimulation of GTPase activity, and the Hill coefficients for agonist activation of p44^MAPK (h > 1) displayed marked co-operativity whereas those for G-protein activation did not (h 0.8–1.0). DADLE activation of p44^MAPK showed more sustained kinetics in clone D2 than in clone DOE. By contrast, lysophosphatidic acid, acting at an endogenously expressed G-protein-coupled receptor, also activated p44^MAPK in both clones in a pertussis toxin-sensitive manner, but both the kinetics and the concentration–response curve for activation of p44^MAPK by this ligand were similar. As with other systems, maintained cellular levels of a cAMP analogue prevented the effects of both G-protein-coupled receptors on activation of p44^MAPK. These results demonstrate for the first time that an opioid receptor, at least when expressed in Rat-1 fibroblasts, is able to initiate activation of the MAP kinase cascade in a G_i-dependent manner, and show that only a very small proportion of the cellular G_i population is required to be activated to result in full phosphorylation of the p42^MAPK and p44^MAPK MAP kinases.