INTERACTION OF DELTA-OPIOID RECEPTORS WITH MULTIPLE G-PROTEINS - A NONRELATIONSHIP 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 delta-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 delta-opioid receptors) and to ascertain whether there is any correlation between delta-opioid ago nist potency to inhibit adenylyl cyclase and to activate G proteins. I nteractions between receptors and G proteins were investigated using a gonist-stimulated incorporation of the photoreactive GTP analog azidoa nilido[alpha-P-32]GTP ([alpha-P-32]AA-GTP) into G protein alpha subuni ts, with subsequent separation by urea/sodium dodecyl sulfate-polyacry lamide gel electrophoresis. In NG108-15, NS20Y, and N1E115 cell membra nes, four alpha subunits (G(i2 alpha), one isoform of G(i3 alpha), and both isoforms of G(o alpha)) in the 39-41-kDa region were labeled wit h [alpha-P-32]AA-GTP. The delta-opioid agonist [D-Ala(2),D-Leu(5)]-enk ephalin (DADLE) produced a dose-dependent, naloxone-reversible increas e of [alpha-P-32]AA-GTP incorporation into all four alpha subunit subt ypes, in all cell lines tested. In addition, with the single exception of G(i3 alpha) in NG108-15 cells, the maximal increases in incorporat ion of the photoaffinity label into all G(alpha) subunits induced by D ADLE were similar. The B-max values determined for delta-opioid recept ors in NG108-15, NS20Y, and N1E115 cell membranes were 570, 370, and 1 20 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 ED(50) values to produce labe ling of the G(alpha) subunits between cell lines differed by > 100-fol d. In fact, only in NS20Y cells were the IC50 and ED(50) values compar able. Firstly, these results suggest that simultaneous coupling of the delta-opioid receptor to multiple G protein alpha subunits occurs in a variety of cell lines that express a range of receptor densities. Se condly, the magnitudes with which delta-opioid receptors interact with available G(alpha) subunits in response to agonist are approximately the same. Finally, there appears to be no relationship between the pot ency of agonists to inhibit adenylyl cyclase and that required for act ivation of G proteins.