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 α 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 α 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.
|Original language||English (US)|
|Number of pages||7|
|State||Published - 1994|