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Titolo:
The conserved cysteine 7.38 residue is differentially accessible in the binding-site crevices of the mu, delta, and kappa opioid receptors
Autore:
Xu, W; Chen, CG; Huang, P; Li, J; de Riel, JK; Javitch, JA; Liu-Chen, LY;
Indirizzi:
Temple Univ, Sch Med, Dept Pharmacol, Philadelphia, PA 19140 USA Temple Univ Philadelphia PA USA 19140 armacol, Philadelphia, PA 19140 USA Temple Univ, Sch Med, Ctr Subst Abuse Res, Philadelphia, PA 19140 USA Temple Univ Philadelphia PA USA 19140 use Res, Philadelphia, PA 19140 USA Temple Univ, Sch Med, Fels Inst Mol Biol & Canc Res, Philadelphia, PA 19140 USA Temple Univ Philadelphia PA USA 19140 anc Res, Philadelphia, PA 19140 USA Columbia Univ Coll Phys & Surg, Ctr Mol Recognit, New York, NY 10029 USA Columbia Univ Coll Phys & Surg New York NY USA 10029 w York, NY 10029 USA
Titolo Testata:
BIOCHEMISTRY
fascicolo: 45, volume: 39, anno: 2000,
pagine: 13904 - 13915
SICI:
0006-2960(20001114)39:45<13904:TCC7RI>2.0.ZU;2-H
Fonte:
ISI
Lingua:
ENG
Soggetto:
MEMBRANE-SPANNING SEGMENT; PROTEIN-COUPLED RECEPTORS; DOPAMINE D2 RECEPTOR; FUNCTIONAL EXPRESSION; N-ETHYLMALEIMIDE; RAT-BRAIN; SULFHYDRYL-REAGENTS; MOLECULAR-CLONING; OPIATE RECEPTORS; INACTIVATION;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
64
Recensione:
Indirizzi per estratti:
Indirizzo: Liu-Chen, LY Temple Univ, Sch Med, Dept Pharmacol, Philadelphia, PA 19140 USA Temple Univ Philadelphia PA USA 19140 adelphia, PA 19140 USA
Citazione:
W. Xu et al., "The conserved cysteine 7.38 residue is differentially accessible in the binding-site crevices of the mu, delta, and kappa opioid receptors", BIOCHEM, 39(45), 2000, pp. 13904-13915

Abstract

Binding pockets of the opioid receptors are presumably formed among the transmembrane domains (TMDs) and are accessible from the extracellular medium. In this study, we determined the sensitivity of binding of [H-3]diprenorphine, an antagonist, to mu, delta, and kappa opioid receptors to charged methanethiosulfonate (MTS) derivatives and identified the cysteine residues within the TMDs that confer-red the sensitivity. Incubation of the mu opioidreceptor expressed in HEK293 cells with MTS ethylammonium (MTSEA), MTS ethyltrimethylammonium (MTSET), or MTS ethylsulfonate (MTSES) inhibited [H-3]diprenorphine binding with the potency order of MTSEA > MTSET > MTSES. Pretreatment of mu, delta, and kappa opioid receptors with MTSEA dose-dependently inhibited [H-3]dipnnorphine binding with MTSEA a sensitivity in the orderof kappa > mu much greater than delta. The effects of MTSEA occurred rapidly, reaching the maximal inhibition in 10 min. (-)-Naloxone, but not (+)-naloxone, prevented the MTSEA effect, demonstrating that the reaction occurs within or in the vicinity of the binding pockets. Each cysteine residue in the TMDs of the three receptors was mutated singly, and the effects of MTSEA treatment were examined. The mutants had similar affinities for [H-3]diprenorphine, and C7.38(321)S, C7.38(303)S, and C7.38(315)S mutations renderedmu, delta, and kappa opioid receptors less sensitive to the effect of MTSEA, respectively. These results indicate that the conserved Cys7.38 is differentially accessible in the binding-site crevice of these receptors. The second extracellular loop of the kappa receptor, which contains several acidic residues, appears to play a role, albeit small, in its higher sensitivityto MTSEA, whereas the negative charge of Glu6.58(297) did not. To the bestof our knowledge, this is the first report to show that a conserved residue among highly homologous G protein-coupled receptors is differentially accessible in the binding-site crevice. In addition, this represents the firstsuccessful generation of MTSEA-insensitive mutants of mu, delta, and kappaopioid receptors, which will allow determination of residues accessible inthe binding-site crevices of these receptors by the substituted cysteine accessibility method.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 19/01/20 alle ore 09:31:32