The conserved aspartate residue in the third putative transmembrane domain of the delta-opioid receptor is not the anionic counterpart for cationic opiate binding but is a constituent of the receptor binding site.
Opioids are cationic compounds that mediate their biological action through three highly homologous receptors (mu, delta, and kappa) known to belong to the G protein-coupled receptor (GPR) family. The third putative transmembrane domain of opioid receptors contains a conserved aspartate residue that is typically found in biogenic amine binding GPRs and is generally believed to form an ion pair with the cationic neurotransmitters. Using site-directed mutagenesis, we investigated the possibility of an identical role for this residue (Asp128) in the mouse delta-opioid receptor. Removal of the carboxylate group via an aspartate-to-alanine mutation did not modify binding affinity of a representative set of opioid compounds, including bremazocine, diprenorphine, naloxone, Tyr-D-Thr-Gly-Phe-Leu-Thr, [D-Ala2,D-Leu5]enkephalin, cyclic[D-penicillamine2,D-penicillamine5]enkephalin, deltorphin II, (+/-)-4-[(a-R*)-a-[(2S*,5R*)-4-allyl-2,5-di-methyl-1- piperazinyl]-3-hydroxybenzyl]-N,N-diethylbenzamide, and naltrindole. It nevertheless decreased receptor expression level and affected the binding of three agonists ([D-Ala2,D-Leu5]enkephalin, Tyr-D-Thr-Gly-Phe-Leu-Thr, and (+/-)-4-[(a-R*)-a-[(2S*,5R*)-4-allyl-2,5-di- methyl-1-piperazinyl]-3-hydroxybenzyl]-N,N-diethylbenzamide) when the receptor was under Na(+)-induced low affinity state. On the other hand, the aspartate-to-asparagine mutation strongly impaired the binding of all of the above ligands and highlighted differential modes of interaction for alkaloids and peptides. Finally, removal of the homologous carboxylate group in the mouse mu receptor had distinct effects because it dramatically reduced the binding potency of some, but not all, tested ligands. Taken together, these results demonstrate that (i) the direct ligand/receptor interaction previously demonstrated for the beta-adrenergic receptor does not take place in the delta receptor, (ii) Asp128 nevertheless contributes to stabilization of the spatial conformation of the binding pocket, and (iii) these conclusions cannot be extended to the closely related mu receptor.
Befort
K
K
Ecole Supérieure de Biotechnologie, Parc d'Innovation, Illkirch-Graffenstaden, France.
Tabbara
L
L
Bausch
S
S
Chavkin
C
C
Evans
C
C
Kieffer
B
B
D000595:Amino Acid Sequence; D000818:Animals; D000838:Anions; D001224:Aspartic Acid; D001483:Base Sequence; D001665:Binding Sites; D002412:Cations; D002417:Cattle; D002460:Cell Line; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D004285:Dogs; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009294:Narcotics / Q000378:metabolism / Q000494:pharmacology; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D017433:Protein Structure, Secondary; D051381:Rats; D017465:Receptors, Opioid, delta / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014162:Transfection
D000838:Anions; D002412:Cations; D009294:Narcotics; D009838:Oligodeoxyribonucleotides; D017465:Receptors, Opioid, delta; D011994:Recombinant Proteins; D001224:Aspartic Acid