Affinity crosslinking of 125I-labeled human beta-endorphin to cell lines possessing either mu- or delta-type opioid binding sites.
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.
Keren
O
O
Department of Psychiatry, New York University Medical Center, NY 10016.
Gioannini
T L
TL
Hiller
J M
JM
Simon
E J
EJ
D001665:Binding Sites; D002460:Cell Line; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D020881:Enkephalin, D-Penicillamine (2,5)-; D004745:Enkephalins / Q000378:metabolism; D006801:Humans; D011957:Receptors, Opioid / Q000378:metabolism*; D017465:Receptors, Opioid, delta; D017450:Receptors, Opioid, mu; D001615:beta-Endorphin / Q000378:metabolism*
D004745:Enkephalins; D011957:Receptors, Opioid; D017465:Receptors, Opioid, delta; D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D001615:beta-Endorphin; D020881:Enkephalin, D-Penicillamine (2,5)-