Protection of opiate receptors in NG108-15 against modification by N- ethylmaleimide

Abstract

Two different -SH groups associated with the opiate receptors of the mouse neuroblastoma X rat glioma hybrid NG108-15 have been identified. Modification of these by N-ethylmaleimide (NEM) (presumed to be via alkylation) or by para-chloromercuribenzoic acid (presumed to be via formation of mercury adducts) decreases the binding of both opiate agonists and antagonists to these receptors. Agonist binding is more sensitive than antagonist binding to modification by NEM. Losses in antagonist binding are accounted for totally by decreases in the number of binding sites; there are no corresponding losses in antagonist affinity. Losses of antagonist binding exhibit a pseudo-first order rate constant; the modification of only one such group completely destroys the binding site. Both agonists and antagonists protect against modification of this group by NEM. Sodium and lithium, but not GTP, also protect this group, indicating that the action of these monovalent cations is directly on the receptor moiety. Losses in agonist binding stem not only from decreases in receptor number but also from selective losses in affinity. This -SH group appears to be different from the one at the binding site as sodium, GTP, and antagonist ligands do not protect against losses in agonist affinity. Agonist high affinity also is lost in a pseudo-first order fashion indicating that an alteration of only one -SH group per receptor complex is sufficient to produce this effect. The possible roles of two sulfhydryls in opiate receptor function are discussed.

Different effects of N-ethylmaleimide on M1 and M2 muscarine receptors in rat brain.

“…clase (17)(18)(19)(20). In sharp contrast, MalNEt has been found to ie results are in accord with our prior evidence that Malstabilize ternary complexes of agonist-p8-adrenergic recepdoes not modify the rates of association or dissociation tors-nucleotide-binding protein, and to slow the dissociation e antagonist [3H]QNB to or from either Ml or M2 recepof 8-adrenergic agonists (26,27).…”

“…Parallel effects of p[NH]ppG and MalNEt also have been noted for certain a-adrenoreceptors, dopamine receptors, opiate receptors, and other receptors (17)(18)(19)(20), all of which are believed to act via the attenuation of adenylate cyclase (11).…”

Transfer of functional opiate receptors from membranes to recipient cells by polyethylene glycol‐induced fusion

“…1, NEM treatment of NG108-15 membranes almost completely abolishes adenylate cyclase activity and, under the conditions used, has little effect on opiate binding to receptors. As shown by others [12,13], NaCI protects opiate-binding sites from inactivation with NEM and in our experiments was found to be necessary to ensure selective impairment of adenylate cyclase only. Inclusion of opiate ligands (either agonists or antagonists) gave only a slight further protection of binding sites and was not generally deemed necessary in our experiments.…”

Studies on Inhibition of μ and δ Opioid Receptor Binding by Dithiothreitol and N-Ethylmaleimide

“…The results suggested that the agonists became tethered to the receptor via a mixed disulfide linkage that was in the vicinity of the receptor binding site. Other studies provided evidence that NEM affected opioid agonist binding by at least two mechanisms, direct inhibition (as mentioned above) and indirect inhibition due to uncoupling of receptors from G proteins (14,15).…”

“…The results suggested that the agonists became tethered to the receptor via a mixed disulfide linkage that was in the vicinity of the receptor binding site. Other studies provided evidence that NEM affected opioid agonist binding by at least two mechanisms, direct inhibition (as mentioned above) and indirect inhibition due to uncoupling of receptors from G proteins (14,15).Several other, but not all, G protein-coupled receptors are also sensitive to sulfhydryl reagents. Susceptible receptors include the thyrotropin-releasing hormone (16), D1 and D2 dopamine (17, 18), substance P (19), ␣1 and ␣2 adrenoreceptor (20), platelet-activating factor (21), leukotriene B 4 (22), vasopressin (23), follicle-stimulating hormone (24), and cannabinoid receptors (25).…”