Interaction of δ-opioid receptors with multiple G proteins: A non-relationship between agonist potency to inhibit adenylyl cyclase and to activate G proteins

The purpose of the present investigation was to determine whether the coupling of δ-opioid receptors to multiple G proteins in NG108-15 neuroblastoma X glioma cells is a characteristic limited to only this cell line (because of the high density of δ-opioid receptors) and to ascertain whether there is any correlation between δ-opioid agonist potency to inhibit adenylyl cyclase and to activate G proteins. Interactions between receptors and G proteins were investigated using agonist-stimulated incorporation of the photoreactive GTP analog azidoanilido[α-³²P]GTP ([α-³²P]AA-GTP) into G protein α subunits, with subsequent separation by urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In NG108-15, NS20Y, and N1E115 cell membranes, four a subunits (G_i2α, one isoform of G_i3α, and both isoforms of G_oα) in the 39-41-kDa region were labeled with [α-³²P]AA-GTP. The δ-opioid agonist [D-Ala²,D-Leu⁵]-enkephalin (DADLE) produced a dose-dependent, naloxone-reversible increase of [α-³²P]AA-GTP incorporation into all four α subunit subtypes, in all cell lines tested. In addition, with the single exception of G_i3α, in NG108-15 cells, the maximal increases in incorporation of the photoaffinity label into all G_α subunits induced by DADLE were similar. The B_max values determined for δ-opioid receptors in NG108-15, NS20Y, and N1E115 cell membranes were 570, 370, and 120 fmol/mg of protein, respectively. Finally, although the IC₅₀ values to inhibit intracellular cAMP production and affinity for DADLE were similar across the three cell lines, the ED₅₀ values to produce labeling of the G_α subunits between cell lines differed by >100-fold. In fact, only in NS20Y cells were the IC₅₀ and ED₅₀ values comparable. Firstly, these results suggest that simultaneous coupling of the δ-opioid receptor to multiple G protein a subunits occurs in a variety of cell lines that express a range of receptor densities. Secondly, the magnitudes with which δ-opioid receptors interact with available G_α subunits in response to agonist are approximately the same. Finally, there appears to be no relationship between the potency of agonists to inhibit adenylyl cyclase and that required for activation of G proteins.