Characterization of beta-125I-endorphin cross-linked proteins in NG108-15 cell membranes. A 25-kilodalton protein with properties of delta-opioid-binding site.
Cross-linking of beta-125I-endorphin to NG108-15 cell membranes labeled bands with molecular masses of 55, 35, and 25 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We applied several criteria to evaluate the relevance of these cross-linked bands to delta-opioid receptors, including selectivity, stereospecificity, affinity, G-protein coupling, down-regulation, and correlation with opioid receptor level in different well-characterized cell lines. Only the 25 kDa protein adequately fulfilled all these criteria. Thus, cross-linking to the 25-kDa band was selectively inhibited by ligands with delta-opioid affinity, but not by mu-opioid, kappa-opioid, or optically inactive opioid ligands or by non-opioid ligands. Based on inhibition of cross-linking, we calculated an affinity of [D-Ala2,D-Leu5]enkephalin binding to the 25-kDa and (Kd = 6 nM) that is similar to that reported for [D-Ala2,D-Leu5]enkephalin binding to NG108-15 membranes; this affinity decreased approximately 10-fold in the presence of Na+/guanyl-5'-yl imidodiphosphate. Chronic agonist treatment of NG108-15 cells reduced cross-linking to the 25-kDa band, but not to others, in a manner parallel to down-regulation of opioid receptors. Finally, the amount of the 25-kDa band was roughly proportional to the level of opioid receptors present in N18TG2, NS20Y, ST7-3, and ST8-4 cells. The 25-kDa band was absent in PC12h, NIH3T3, and C6BU1 cells as well as in liver, all of which had no detectable opioid binding.
Ko
J L
JL
Department of Pharmacology, University of Minnesota Medical School, Minneapolis 55455.
Lee
N M
NM
Loh
H H
HH
D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003432:Cross-Linking Reagents; D015536:Down-Regulation; D004723:Endorphins / Q000378:metabolism*; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D020881:Enkephalin, D-Penicillamine (2,5)-; D016308:Enkephalin, Leucine-2-Alanine / Q000378:metabolism; D004745:Enkephalins / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D016716:PC12 Cells; D010455:Peptides / Q000378:metabolism; D011957:Receptors, Opioid / Q000378:metabolism
D003432:Cross-Linking Reagents; D004723:Endorphins; D004745:Enkephalins; D008565:Membrane Proteins; D010455:Peptides; D011957:Receptors, Opioid; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D016308:Enkephalin, Leucine-2-Alanine; D020881:Enkephalin, D-Penicillamine (2,5)-; D019204:GTP-Binding Proteins