K. Befort et al., ROLE OF AROMATIC TRANSMEMBRANE RESIDUES OF THE DELTA-OPIOID RECEPTOR IN LIGAND RECOGNITION, The Journal of biological chemistry, 271(17), 1996, pp. 10161-10168
In the present study we examine the role of transmembrane aromatic res
idues of the delta-opioid receptor in ligand recognition. Three-dimens
ional computer modeling of the receptor allowed to identify an aromati
c pocket within the helices bundle which spans transmembrane domains (
Tms) III to VII and consists of tyrosine, phenylalanine, and tryptopha
n residues. Their contribution to opioid binding was assessed by singl
e amino acid replacement: Y129F and Y129A (Tm III), W173A (Tm IV), F21
8A and F222A (Tm V), W274A (Tm VI), and Y308F (Tm VII). Scatchard anal
ysis shows that mutant receptors, transfected into COS cells, are expr
essed at levels comparable with that of the wild-type receptor. Bindin
g properties of a set of representative opioids were examined. Mutatio
ns at position 129 most dramatically affected the binding of all teste
d ligands (up to 430-fold decrease of deltorphin II binding at Y129A),
with distinct implication of the hydroxyl group and the aromatic ring
, depending on the ligand under study. Affinity of most ligands was al
so reduced at Y308F mutant (up to 10-fold). Tryptophan residues seemed
implicated in the recognition of specific ligand classes, with reduce
d binding for endogenous peptides at W173A mutant (up to 40-fold) and
for nonselective alkaloids at W274A mutant (up to 65-fold). Phenylalan
ine residues in Tm V appeared poorly involved in opioid binding as com
pared with other aromatic amino acids examined. Generally, the binding
of highly selective delta ligands (TIPP psi naltrindole, and BW373U86
) was weakly modified by these mutations. Noticeably, TIPP psi binding
was enhanced at W274A receptor by 5-fold. Conclusions from our study
are: (i) aromatic amino acid residues identified by the model contribu
te to ligand recognition, with a preponderant role of Y129; (ii) these
residues, which are conserved across opioid receptor subtypes, may be
part of a general opioid binding domain; (iii) each ligand-receptor i
nteraction is unique, as demonstrated by the specific binding pattern
observed for each tested opioid compound.