The opiate receptor: a single 110 kDa recognition molecule appears to be conserved in Tetrahymena, leech, and rat.
We compared the molecular nature of the rat brain opiate receptor with that of the invertebrate leech, Haemopis marmorata, and the protozoan, Tetrahymena, in order to examine the issue of apparent receptor heterogeneity with respect to biochemical structure. A binding study with rat brain membrane verified that [125I]beta-endorphin [( 125I]beta E), a broad specificity ligand, is displaced by the antagonist (-)-naloxone, but not the inactive stereoisomer (+)-naloxone; agonists considered prototypes for mu, delta, and kappa opiate receptors all displayed stereospecific binding displacement. For SDS-PAGE analysis of the opiate receptor [125I]beta-endorphin was covalently affixed to its recognition molecule with the cross-linking reagent DSS. Primary reaction products occur at 110, 58/55, and 29 kDa. Cross-linking products of all 3 molecular weights are effectively reversed by opiate ligands, regardless of their mu, delta, or kappa specificities. Peptide mapping studies in SDS gels, using limited proteolysis, showed that the 110 kDa band can be digested into 58 and 29 kDa fragments and the 58 kDa band into a 29 kDa fragment. Additional smaller molecular weight fragments were generated from the 110, 58/55, and 29 kDa bands which shared their molecular weights. Two possible explanations for the extensive sequence homology between the three major cross-linking products are: (1) the 110 kDa species is the opiate receptor, and the 58 and 29 kDa species are proteolytic fragments; and (2) one of the lower molecular weight species is the opiate receptor, and adjacent receptors are aggregated into the 110 kDa complex through cross-linking.(ABSTRACT TRUNCATED AT 250 WORDS)
Zipser
B
B
Clinical Neuroscience Branch, National Institute of Mental Health, Bethesda, MD 20892.
Ruff
M R
MR
O'Neill
J B
JB
Smith
C C
CC
Higgins
W J
WJ
Pert
C B
CB
D000818:Animals; D001667:Binding, Competitive; D003432:Cross-Linking Reagents; D004723:Endorphins / Q000378:metabolism*; D007865:Leeches / Q000378:metabolism*; D008297:Male; D008970:Molecular Weight; D009270:Naloxone / Q000494:pharmacology; D051381:Rats / Q000378:metabolism*; D011919:Rats, Inbred Strains; D011957:Receptors, Opioid / Q000032:analysis / Q000502:physiology*; D013045:Species Specificity; D013768:Tetrahymena / Q000378:metabolism*
D003432:Cross-Linking Reagents; D004723:Endorphins; D011957:Receptors, Opioid; D009270:Naloxone