Affinity crosslinking of 125I-labeled human beta-endorphin to cell lines possessing either mu- or delta-type opioid...

The specific labeling of opioid receptor-related polypeptides was compared in two cell lines which differ in their opioid receptor population: SK-N-SH which contains predominantly mu-type opioid receptors, and NG-108-15, which contains exclusively delta-type opioid receptors. Labeling of opioid receptors was achieved by affinity cross-linking of membranes, using 125I-labeled human beta-endorphin, followed by solubilization in sodium dodecyl sulphate (SDS), SDS-gel electrophoresis and autoradiography. Different labeling patterns were obtained from these two cell lines. In SK-N-SH cells, 3 major proteins were labeled, corresponding to molecular weights of 92, 65 and 25 kDa, while in the NG-108-15 cells, 53-kDa and 25-kDa polypeptides were the major ones labeled. The radioactivity incorporated into the 92- and 65-kDa peptide bands derived from SK-N-SH cells was displaced by the mu-selective ligand Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGO) but not by the delta-selective ligand [D-Pen2,D-Pen5]enkephalin (DPDPE). The radioactivity incorporated into the NG-108-15-derived peptide bands was displaced by the delta-selective ligand, but not by the mu-selective ligand. This confirms our previous finding in mammalian brain which demonstrated that mu- and delta-opioid binding sites can be identified as distinct proteins which differ in molecular size.