Constitutive activation of the δ opioid receptor by mutations in transmembrane domains III and VII

We have investigated whether transmembrane amino acid residues Asp(128) (domain III), Tyr(129) (domain II), and Tyr(308) (domain VII) in the mouse delta opioid receptor play a role in receptor activation. To do so, we have used a [S-35]GTP gamma S (where GTP gamma 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 the wild-type receptor. In the absence of ligand, [S-35]GTP gamma 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 cu-aminobutyric acid) efficiently reduced basal [S-35]GTP gamma S binding in the mutated receptor preparations. The potent agonist BW373U86 stimulated [S-35]GTP gamma 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 delta 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.