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 binding (G) protein-coupled receptors. There is now growing evidence in support of a stimulatory coupling of opioid receptors to phospholipase C (PLC), via a pertussis toxin-sensitive G protein, leading to the generation of the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. We have generated two C-terminal truncation mutants of the delta-opioid receptor lacking the final 15 or 37 amino acids and examined their coupling to PLC and adenylyl cyclase. D-[Pen(2,5)]-enkephalin (DPDPE) mediated Ins(1,4,5)P3 formation and cyclic AMP inhibition was measured in whole cells and assayed using radioreceptor mass assays. DPDPE produced a time- and dose-dependent increase in Ins(1,4,5)P3 mass formation in Chinese hamster ovary (CHO) cells expressing the delta(wt), delta15, and delta37 receptors. As the C terminus was truncated, the time to maximum stimulation (15 s in CHO delta(wt), 60 s in CHO delta15, and 120 s in CHO delta37) increased and removal of the C terminus resulted in a prompt return to basal Ins(1,4,5)P3 levels. Whereas the dose-response curves to Ins(1,4,5)P3 formation and cyclic AMP inhibition remained largely unaffected by C-terminal truncation, there were large differences in the pEC/IC50 values, with cyclic AMP inhibition being the more potent, perhaps indicating G(i alpha) coupling to adenylyl cyclase and G(i beta/gamma) coupling to PLC. Collectively, these data indicate that the C terminus of the delta-opioid receptor is unimportant in the acute coupling to adenylyl cyclase but may have a role to play in PLC coupling. We hypothesize that an intact C terminus is required to allow normal "strong" coupling of receptor to Gi and that truncation weakens this link as reflected in an increased time to peak. In addition, if the coupling is weak, the acute response to agonist stimulation rapidly uncouples.
Hirst
R A
RA
University Department of Anaesthesia, Leicester Royal Infirmary, England, UK.
Smart
D
D
Devi
L A
LA
Lambert
D G
DG
D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D000242:Cyclic AMP / Q000037:antagonists & inhibitors; D004174:Diprenorphine / Q000378:metabolism; D020881:Enkephalin, D-Penicillamine (2,5)-; D004745:Enkephalins / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis; D051379:Mice; D009154:Mutation; D017465:Receptors, Opioid, delta / Q000819:agonists / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000819:agonists / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism*
D000067956:Adenylyl Cyclase Inhibitors; D004745:Enkephalins; D017465:Receptors, Opioid, delta; D011994:Recombinant Proteins; D004174:Diprenorphine; D015544:Inositol 1,4,5-Trisphosphate; D020881:Enkephalin, D-Penicillamine (2,5)-; D000242:Cyclic AMP; D010738:Type C Phospholipases