Identification of three separate guanine nucleotide-binding proteins that interact with the δ-opioid receptor in NG108-15 neuroblastoma x glioma hybrid cells

Five separate guanine nucleotide-binding proteins (G proteins) were immunologically identified in membranes from neuroblastoma x glioma NG108-15 hybrid cells. These α subunit proteins were G(i2α), two isoforms of G(i3α), and two isoforms of G(oα). The G proteins that interacted with δ- opioid receptors in these membranes were identified using cholera toxin (CTX)-induced ADP-ribosylation and antisera selective for various G protein α subunits. In the presence of δ-opioid agonists, CTX induced the incorporation of [³²P]ADP-ribose into three pertussis toxin substrates. Using antisera generated against peptide sequences from G(α) subunits, these three pertussis toxin substrates were identified as G(i2α), G(o2α), and one isoform of G(i3α), which has yet to be identified. This CTX-induced labeling was demonstrated to be mediated via the δ-opioid receptor in these hybrid cells by the observation that δ agonists D-Ala²-D-Leu⁵-enkephalin (DADLE) and D-Pen²-D-Pen⁵-enkephalin, as well as the nonselective agonists etorphine and bremazocine, were active, but the μ agonist PL017 and the κ agonist U-50-488H did not show this activity. This incorporation into all three substrates induced by DADLE was dose dependent, with EC₅₀ (95% confidence interval) values ranging from 12 (3-52) to 183 (65-520) nM, which compared with the K(d) value of 10 ± 1.5 nM for this agonist, a dose that produces maximal inhibition of adenylate cyclase activity. Furthermore, pretreatment of the cells with pertussis toxin or treatment of the membranes with the antagonist naloxone blocked the incorporation induced by DADLE. Incorporation of [³²P]ADP-ribose into all three substrates decreased 35- 83% in membranes in which the receptors had been down-regulated by chronic treatment of the cells with DADLE. Thus, a single opioid receptor type can interact with three separate G proteins.