Cross-linking of [125I]beta-endorphin to mu-opioid receptors during development.
Radioiodinated human beta-endorphin was cross-linked to opioid receptors from rat brain membranes using the bifunctional reagents bis-[2-(succinimidooxycarbonyloxy)ethyl] sulfone (BSCOES) and disuccinimidyl suberate (DSS). Major radiolabeled bands migrated with Mr values of 65,000, 55,000 and 33,000, however the presence of the 55 kDa band was variable. The 65 kDa band was characterized as the mu-receptor: the binding of [125I]beta-endorphin to this band was displaced by mu-selective ligands and blocked by alkylation of the receptor by mu-specific, but not delta-specific alkylating agents. The cross-linked receptor underwent alterations in mol. wt. during development. Early in development, embryonic day 18 and postnatal day 1, the [125I]beta-endorphin-labeled material migrated as a single band of mol. wt. 55 kDa. By day 21 postnatally the higher mol. wt. band of 65 kDa was present, as was material of 53, 47 and 43 kDa. Although the protein labeled early in development migrated with a mol. wt. of 55 kDa similar to the delta-receptor isolated from NG108-15 neuroblastoma-glioma cells, competition studies suggest this protein is not the delta-receptor. The 65 kDa band, tentatively identified as the mu-receptor, was present in adults but not detected in neonates, despite competition binding data indicating the presence of mu-sites. The results suggest that the 55 kDa band found in the 1-day-old neonate may be an immature form of the mu-opioid receptor that undergoes posttranslational modification, perhaps glycosylation, during development.
McLean
S
S
Laboratory of Neurophysiology, NIMH, Bethesda, MD 20892.
Rothman
R B
RB
Chuang
D M
DM
Rice
K C
KC
Spain
J W
JW
Coscia
C J
CJ
Roth
B L
BL
D000478:Alkylation; D000818:Animals; D001345:Autoradiography; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005283:Fentanyl / Q000031:analogs & derivatives / Q000494:pharmacology; D017879:Isothiocyanates; D008297:Male; D008861:Microsomes / Q000378:metabolism; D051381:Rats; D011957:Receptors, Opioid / Q000378:metabolism*; D017450:Receptors, Opioid, mu; D013388:Succinimides / Q000494:pharmacology; D013570:Synaptic Membranes / Q000378:metabolism; D013861:Thiocyanates / Q000494:pharmacology; D001615:beta-Endorphin / Q000378:metabolism*
D003432:Cross-Linking Reagents; D017879:Isothiocyanates; D011957:Receptors, Opioid; D017450:Receptors, Opioid, mu; D013388:Succinimides; D013861:Thiocyanates; C011683:bis(2-(succinimidooxycarbonyloxy)ethyl)sulfone; D001615:beta-Endorphin; C037091:fentanyl isothiocyanate; C042097:2-(4-ethoxybenzyl)-1-diethylaminoethyl-5-isothiocyanatobenzimidazole; C035760:bis(sulfosuccinimidyl)suberate; D005283:Fentanyl