Affinity cross-linked δ-opioid receptor in NG108-15 cells is low molecular weight (25 kDa) and coupled to GTP-binding proteins

The affinity cross-linking of the δ-opioid receptor in neuroblastoma X glioma NG108-15 cells was undertaken using (3-[125Iiodotyrosyl27)human-β-endorphin ([125I]β-endorphin) and disuccinimidyl suberate (DSS)or bis(sulfosuccinimidyl) suberatc (BS3) in order to estimate molecular size. Following sodium dodccyi sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, two radioactive bands were observed. Labeling of a major band of 29 kDa diminished in the presence of unlabeled selective δ-opioid agonist, [D-Pen2,D-Pen5]enkephalin (DPDPE), in a concentration-dependent manner, while labeling of a minor band of 58 kDa was hardly affected. The labeling intensity of the 29 kDa band decreased by addition of guanosine 5'-(3-o-lhio)triphosphate (GTPγS) or by pretreatment of cells with pertussis toxin. These results, taking the molecular weight of covalently bound β-endorphin (3.6 kDa) into consideration, suggest that the δ-opioid receptor in NG108-15 cell membrane is a 25 kDa protein which is coupled to pertussis toxin-sensitive guanosine triphosphate-binding proteins (G-proteins).