Characterization of β-125I-endorphin cross-linked proteins in NG108-15 cell membranes. A 25-kilodalton protein with properties of δ-opioid-binding site

J. L. Ko, N. M. Lee, Horace H Loh

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6 Scopus citations

Abstract

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 (K(d) = 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.

Original languageEnglish (US)
Pages (from-to)12722-12727
Number of pages6
JournalJournal of Biological Chemistry
Volume267
Issue number18
StatePublished - Jan 1 1992

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