Interaction of delta-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 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 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[alpha-32P]GTP ([alpha-32P]AA-GTP) into G protein alpha subunits, with subsequent separation by urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In NG108-15, NS20Y, and N1E115 cell membranes, four alpha subunits (Gi2 alpha, one isoform of Gi3 alpha, and both isoforms of Go alpha) in the 39-41-kDa region were labeled with [alpha-32P]AA-GTP. The delta-opioid agonist [D-Ala2,D-Leu5]-enkephalin (DADLE) produced a dose-dependent, naloxone-reversible increase of [alpha-32P]AA-GTP incorporation into all four alpha subunit subtypes, in all cell lines tested. In addition, with the single exception of Gi3 alpha in NG108-15 cells, the maximal increases in incorporation of the photoaffinity label into all G alpha subunits induced by DADLE were similar. The Bmax values determined for delta-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 EC50 values to produce labeling of the G alpha 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 delta-opioid receptor to multiple G protein alpha subunits occurs in a variety of cell lines that express a range of receptor densities. Secondly, 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 potency of agonists to inhibit adenylyl cyclase and that required for activation of G proteins.
Prather
P L
PL
Department of Pharmacology, School of Medicine, University of Minnesota, Minneapolis 55455.
Loh
H H
HH
Law
P Y
PY
D000067956:Adenylyl Cyclase Inhibitors; D000345:Affinity Labels; D001386:Azides / Q000378:metabolism; D016308:Enkephalin, Leucine-2-Alanine / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D010777:Photochemistry; D017465:Receptors, Opioid, delta / Q000187:drug effects / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000067956:Adenylyl Cyclase Inhibitors; D000345:Affinity Labels; D001386:Azides; D017465:Receptors, Opioid, delta; C031907:GTP gamma-4-azidoanilide; D016308:Enkephalin, Leucine-2-Alanine; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins