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

Katia Befort, Christelle Zilliox, Dominique Filliol, ShiYi Yue, Brigitte L. Kieffer
1999 Journal of Biological Chemistry  
We have investigated whether transmembrane amino acid residues Asp 128 (domain III), Tyr 129 (domain II), and Tyr 308 (domain VII) in the mouse ␦ opioid receptor play a role in receptor activation. To do so, we have used a [ 35 S]GTP␥S (where GTP␥S 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
more » ... he wild-type receptor. In the absence of ligand, [ 35 S]GTP␥S 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 [ 35 S]GTP␥S binding in the mutated receptor preparations. The potent agonist BW373U86 stimulated [ 35 S]GTP␥S binding above basal levels with similar (D128N, Y129F, and Y129A) or markedly increased (Y308F) efficacy compared with wildtype 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 Asp 128 (III) and Tyr 308 (VII) interact with each other and that Tyr 129 (III) undergoes H bonding with His 278 (VI). Thus, Asp 128 , Tyr 129 , and Tyr 308 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.
doi:10.1074/jbc.274.26.18574 pmid:10373467 fatcat:uufeoj67qzg5hjnehvf7xhaamy