Involvement of Trp-284, Val-296, and Val-297 of the Human δ-Opioid Receptor in Binding of δ-Selective Ligands

Manon Valiquette,Huy K Vu,Shi Yi Yue,Claes Wahlestedt,Philippe Walker

doi:10.1074/jbc.271.31.18789

Manon Valiquette, Huy K Vu + Show 3 more

Open Access

https://doi.org/10.1074/jbc.271.31.18789

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Abstract

Given the high homology in amino acid sequence between the delta-opioid receptor and the two other types (mu and kappa), distinct residues in this receptor may confer its selectivity to some ligands. In order to identify molecular determinants in the human delta receptor responsible for the selectivity of delta-selective ligands, two different delta/mu chimeras were constructed. In the first one, the delta sequence from the top of transmembrane 5 to the C terminus was replaced by the equivalent mu sequence, and in the second one, 13 consecutive residues in the third extracellular loop region of the delta receptor were replaced by the mu counterpart. These two chimeras retained the ability to bind the nonselective bremazocine but completely lost the ability to bind different delta-selective ligands. These results suggested that the region of the third extracellular loop of the delta receptor is crucial for the type selectivity. Furthermore, an alanine scan was performed by site-directed mutagenesis of 20 amino acids located in or proximal to the third extracellular loop. Among all the point mutations, only mutations of Trp-284, Val-296, or Val-297 significantly decreased the binding of delta-selective ligands tested. Moreover, combined mutation of Trp-284, Val-296, and Val-297 considerably decreased the affinities of the receptor for delta-selective ligands compared with the single point mutations. These findings suggest that Trp-284, Val-296, and Val-297 are crucial residues involved in the delta receptor type selectivity.

Highlights

Similarity in the transmembrane domains and intracellular loops, distinct residues in these receptors may confer their selectivity to some ligands
A stretch of 13 consecutive amino acids in the region of the third extracellular loop of the ␦ receptor was replaced for the equivalent sequence of the ␮ receptor (Fig. 3, A and B)
We used chimeric constructs between ␦and ␮-opioid receptors as an initial approach to locate critical regions involved in the selectivity displayed by the ␦ receptor. ␦1–214/␮215–381 and ␦/␮288 –300 chimeric receptors allowed us to show that the region of the third extracellular loop is essential to the binding of tested ␦-selective ligands

Summary

EXPERIMENTAL PROCEDURES

PcDNA3 vector was purchased from Invitrogen. 293S cells were obtained from Cold Spring Harbor. [3H]Bremazocine was purchased from DuPont NEN. ␦1–214/ ␮215–381 chimera was created using two distinct BglII restriction sites conserved in the ␦ and ␮ cDNA containing vector. Wild-type and mutant ␦-opioid receptor constructs were transfected for transient expression into 293S cells by a modified calciumphosphate precipitation method described in Ref. 20. Binding reactions were performed at 25 °C for 1 h in a buffer containing 50 mM Tris, pH 7.4, 3 mM MgCl2, 1 mg/ml bovine serum albumin. Duplicate contained Ϸ5 nM [3H]bremazocine and 1 pM to 10 ␮M SNC-80, DPDPE, or deltorphin II or 0.01 pM to 100 nM naltrindole. The seven initial helices were built using the identified sequences and were submitted to a conformational search using a mixed Molecular Dynamics and Monte-Carlo procedure. Sequence homology, and final structure refinement were performed using Quanta/CHARMM (Biosym/MSI) software

RESULTS

DISCUSSION

DELT II