Interaction of opiate receptor binding sites and guanine nucleotide regulatory sites: selective protection from N-ethylmaleimide.

Guanine nucleotides decrease the binding of opiate agonists to opiate receptors by increasing dissociation rates. To explore the relationship between receptor binding and GTP regulation, rat brain membranes were incubated with N-ethylmaleimide (NEM) and assayed for GTP-induced decrease of 3H-agonist binding. NEM decreased both binding of [3H]dihydromorphine and the effect of GTP on binding. The GTP effect on D-[3H]Ala2-Met5-enkephalinamide (D-Ala-enk) binding was less sensitive to NEM than the binding itself. Incubation of membranes with 0.2 microM D-Ala-enk before addition of NEM significantly protected D-[3H]Ala-enk binding, but did not protect the GTP effect, actually increasing the effect of NEM and eliminating the GTP effect. These "unregulated" receptors were still inhibited by NaCl, but were not stimulated by divalent cations. Although opiate protection alone could not protect the GTP regulation, preincubation of membranes with GTP or guanyl-yl-5'-imidodiphosphate fully protected the GTP effect from NEM and also partially protected the binding site. Preincubation of membranes either with NaCl alone or with NaCl plus D-Ala-enk protected both the GTP effect and the binding site. These results suggest that although the sites for GTP regulation and opiate receptor binding are physically distinct, interactions between sites occur upon the binding of either opiate or guanine nucleotide ligands to their respective sites, and that sodium may play a crucial role in the coupling of these interactions.