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J Biol Chem, Vol. 274, Issue 26, 18574-18581, June 25, 1999

Constitutive Activation of the  Opioid Receptor by Mutations in Transmembrane Domains III and VII

Katia Befort, Christelle Zilliox, Dominique Filliol, ShiYi Yue^§, and Brigitte L. Kieffer

From the  Ecole Supérieure de Biotechnologie, Parc d'Innovation, Boulevard Sébastien Brandt, F-67400 Illkirch-Graffenstaden, France and the ^§ Departments of Chemistry and Molecular Biology, Astra Research Center Montreal, Montreal, Quebec H4S 1Z9, Canada

We have investigated whether transmembrane amino acid residues Asp¹²⁸ (domain III), Tyr¹²⁹ (domain II), and Tyr³⁰⁸ (domain VII) in the mouse  opioid receptor play a role in receptor activation. To do so, we have used a [³⁵S]GTPS (where GTPS is guanosine 5'-3-O-(thio)triphosphate) binding assay to quantify the activation of recombinant receptors transiently expressed in COS cells and compared functional responses of D128N, D128A, Y129F, Y129A, and Y308F point-mutated receptors to that of the wild-type receptor. In the absence of ligand, [³⁵S]GTPS binding was increased for every mutant receptor under study (1.6-2.6-fold), suggesting that all mutations are able to enhance constitutive activity at the receptor. In support of this finding, the inverse agonist N,N-diallyl-Tyr-Aib-Aib-Phe-Leu (where Aib represents -aminobutyric acid) efficiently reduced basal [³⁵S]GTPS binding in the mutated receptor preparations. The potent agonist BW373U86 stimulated [³⁵S]GTPS binding above basal levels with similar (D128N, Y129F, and Y129A) or markedly increased (Y308F) efficacy compared with wild-type receptor. BW373U86 potency was maintained or increased. In conclusion, our results demonstrate that the mutations under study increase functional activity of the receptor. Three-dimensional modeling suggests that Asp¹²⁸ (III) and Tyr³⁰⁸ (VII) interact with each other and that Tyr¹²⁹ (III) undergoes H bonding with His²⁷⁸ (VI). Thus, Asp¹²⁸, Tyr¹²⁹, and Tyr³⁰⁸ may be involved in a network of interhelical bonds, which contributes to maintain the  receptor under an inactive conformation. We suggest that the mutations weaken helix-helix interactions and generate a receptor state that favors the active conformation and/or interacts with heterotrimeric G proteins more effectively.

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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

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