TY - JOUR
T1 - Characterization of β-125I-endorphin cross-linked proteins in NG108-15 cell membranes
T2 - A 25-kilodalton protein with properties of δ-opioid-binding site
AU - Ko, J. L.
AU - Lee, N. M.
AU - Loh, Horace H
N1 - Copyright:
Copyright 2004 Elsevier B.V., All rights reserved.
PY - 1992/6/25
Y1 - 1992/6/25
N2 - Cross-linking of β-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 δ-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 δ-opioid affinity, but not by μ-opioid, κ-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 band (Kd= 6 nM) that is similar to that reported for [D-Ala2,D-Leu5]enkephalin binding to NG108-15 membranes; this affinity decreased ∼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.
AB - Cross-linking of β-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 δ-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 δ-opioid affinity, but not by μ-opioid, κ-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 band (Kd= 6 nM) that is similar to that reported for [D-Ala2,D-Leu5]enkephalin binding to NG108-15 membranes; this affinity decreased ∼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.
UR - http://www.scopus.com/inward/record.url?scp=0026612529&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0026612529&partnerID=8YFLogxK
M3 - Article
C2 - 1320002
AN - SCOPUS:0026612529
SN - 0021-9258
VL - 267
SP - 12722
EP - 12727
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 18
ER -