EFFECTS OF C-TERMINAL TRUNCATION OF THE RECOMBINANT DELTA-OPIOID RECEPTOR ON PHOSPHOLIPASE-C AND ADENYLYL-CYCLASE COUPLING

Opioid receptors belong to the superfamily of guanine nucleotide bindi ng (G) protein-coupled receptors. There is now growing evidence in sup port of a stimulatory coupling of opioid receptors to phospholipase C (PLC), via a pertussis toxin-sensitive G protein, leading to the gener ation of the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5) P-3]. We have generated two C-terminal truncation mutants of the delta -opioid receptor lacking the final 15 or 37 amino acids and examined t heir coupling to PLC and adenylyl cyclase. D-[Pen(2.5)]-enkephalin (DP DPE) mediated Ins(1,4,5)P-3 formation and cyclic AMP inhibition was me asured in whole cells and assayed using radioreceptor mass assays. DPD PE produced a time-and dose-dependent increase in Ins(1,4,5)P-3 mass f ormation in Chinese hamster ovary (CHO) cells expressing the delta(wt) , delta(15), and delta(37) receptors. As the C terminus was truncated, the time to maximum stimulation (15 s in CHO delta(wt), 60 s in CHO d elta(15), and 120 s in CHO delta(37)) increased and removal of the C t erminus resulted in a prompt return to basal Ins(1,4,5)P-3 levels. Whe reas the dose-response curves to Ins(1,4,5)P-3 formation and cyclic AM P inhibition remained largely unaffected by C-terminal truncation, the re were large differences in the pEC/IC50 values, with cyclic AMP inhi bition being the more potent, perhaps indicating G(i alpha) coupling t o adenylyl cyclase and G(i beta/gamma) coupling to PLC. Collectively, these data indicate that the C terminus of the delta-opioid receptor i s unimportant in the acute coupling to adenylyl cyclase but may have a role to play in PLC coupling. We hypothesize that an intact C terminu s is required to allow normal ''strong'' coupling of receptor to G(i) and that truncation weakens this link as reflected in an increased tim e to peak. In addition, if the coupling is weak, the acute response to agonist stimulation rapidly uncouples.