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Abstract
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.
Highlights
Cross-linking of &‘2SI-endorphin toNG108-15 cell either that form adirect covalent bond with the receptor membranes labeled bandws ith molecular masses o5f5, or thatcan be linked covalently to thereceptor
When membranes were cross-linked with @-129-endorphinin the presence of increasing concentrations of unlabeled DADLE (2 nM to 1 PM), there was a decrease in cross-linking to the 25-kDa band (Fig. 2)
This decrease closely paralleled the decrease in equilibrium binding o[f‘HHIdiprenorphine to NG108-15 cell membranes in the presence of 2 nM to 1 pM DADLE, with a linear correlationcoefficiencyof 0.97 (Fig. 3) and anICso of 12 f 4 nM ( n = 4)
Summary
Cross-linking of &‘2SI-endorphin toNG108-15 cell either that form adirect covalent bond with the receptor membranes labeled bandws ith molecular masses o5f5, (affinity label) or thatcan be linked covalently to thereceptor. Chronic agonist treatment of NGlO8-15 cells reduced cross-linking to the 25-kDa band, but not to others, in a mannerparalleltodown-regulationof opioid receptors. Diprenorphine binding(0)or the intensityof the 25-kDa cross-linked band (0)against various concentrations of DADLE.
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