Constitutive activation of the delta opioid receptor by mutations in transmembrane domains III and VII.
We have investigated whether transmembrane amino acid residues Asp128 (domain III), Tyr129 (domain III) [corrected], and Tyr308 (domain VII) in the mouse delta opioid receptor play a role in receptor activation. To do so, we have used a [35S]GTPgammaS (where GTPgammaS 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]GTPgammaS 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 alpha-aminobutyric acid) efficiently reduced basal [35S]GTPgammaS binding in the mutated receptor preparations. The potent agonist BW373U86 stimulated [35S]GTPgammaS 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 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.
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Ecole Supérieure de Biotechnologie, Parc d'Innovation, Boulevard Sébastien Brandt, F-67400 Illkirch-Graffenstaden, France.
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D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D016308:Enkephalin, Leucine-2-Alanine / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009271:Naltrexone / Q000031:analogs & derivatives / Q000378:metabolism; D009292:Narcotic Antagonists / Q000378:metabolism; D017433:Protein Structure, Secondary; D017465:Receptors, Opioid, delta / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
D008024:Ligands; D009292:Narcotic Antagonists; D017465:Receptors, Opioid, delta; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009271:Naltrexone; D016308:Enkephalin, Leucine-2-Alanine; C055382:naltrindole