Research reportμ Opioid receptor: role for the amino terminus as a determinant of ligand binding affinity
Introduction
Opioid drugs are powerful analgesic agents that are the drugs of choice for the treatment of pain. The pharmacological effects of opioid drugs and the physiological effects of endogenous opioid peptides are initiated through the binding and activation of membrane-bound opioid receptors that are prevalent in the central and peripheral nervous systems. Opioid receptors have been classified into three different types, μ, δ and κ, on the basis of extensive pharmacological and behavioral studies 23, 35, 40. The existence of the proposed opioid receptor types has been confirmed by molecular cloning 6, 9, 10, 18, 30, 45, 48, 54. The amino acid sequences of putative transmembrane spanning segments and the three intracellular loops are highly conserved among opioid receptor types, whereas the extracellular amino termini, second and third extracellular loops, and the intracellular carboxyl termini are divergent. Overall, the opioid receptors exhibit approximately 60% identity in their amino acid sequences. The opioid receptor types display characteristic ligand selectivity profiles 13, 37and neuroanatomical distribution patterns [26]. The μ opioid receptor (MOR) is of particular clinical and social importance since the more potent analgesic drugs, such as morphine, heroin, fentanyl and methadone, elicit their beneficial pharmacological effects as well as their addictive liability through activation of the μ receptor 24, 27, 41.
Opioid receptors belong to the family of guanine nucleotide binding protein (G protein)-coupled receptors 42, 51. G-protein-coupled receptors share several features: they contain seven transmembrane domains, ligands approach and engage the receptor from the extracellular space, and receptor activation results in coupling to heterotrimeric G proteins on the intracellular face of membrane. The amino-termini of nearly all G-protein-coupled receptors contain consensus amino acid sequences for asparagine-linked glycosylation, Asn-X-Ser/Thr, where X is any amino acid [21]. The opioid receptors have consensus N-linked glycosylation sites in the amino-terminal domains: two in the δ and κ receptors, and five in the μ receptor [40]. The diversity of cellular responses to G-protein-coupled receptor activation arises from differential coupling of receptors to various G proteins, which regulate the activity of distinct intracellular effector systems. Several studies have confirmed that opioid receptors interact with multiple members of the pertussis toxin-sensitive Gi and Go protein families, to regulate adenylyl cyclase, Ca2+ and K+ channels 5, 22, 35, 36.
In the course of cloning the rat MOR, Wang et al. [48]isolated a partial cDNA, that when transcribed and translated, yielded a receptor lacking the N-terminal 64 amino acids. The truncated MOR (ΔN64) was capable of binding tritiated DAMGO and naloxone, and activation of the receptor led to inhibition of adenylyl cyclase activity [43]. Our group has also reported previously that the ΔN64 receptor bound []bremazocine with an affinity that was very similar to its affinity for the full-length MOR [39]. The aim of this study was to characterize further the contribution of the μ receptor amino terminus toward high affinity interactions with an expanded set of peptide and alkaloid ligands. Based on this study, the amino-terminal domain of the MOR appears to be an important constituent of the high affinity binding pocket for a select group of μ receptor agonists.
Section snippets
Construction of the ΔN64 μ opioid receptor
ΔN64, which contains a deletion of 64 amino acids from the amino terminus of the wild-type rat MOR, was generated using PCR, and ligated into the pCR 3.1 mammalian expression vector (Invitrogen, Carlsbad, CA) as described previously [39]. In the ΔN64 construct, the AUG codon for Met65 of the full-length MOR serves as the translational initiator codon for the remainder of the receptor open reading frame.
Transfection and radioligand binding assays
HEK 293 cells (ATCC CRL, 1573) were transfected with expression plasmids encoding the
Stable expression of μ and ΔN64 opioid receptors in HEK 293 cells
The ΔN64 receptor has a deletion of 64 amino acids at the amino terminus, and lacks all five of the asparagine-linked glycosylation sites at positions 9, 31, 38, 46, and 53, as well as cysteine residues present at positions 13, 22, 43 and 57 (Fig. 1). Based on an empirical rule for predicting the orientation of eukaryotic membrane-spanning proteins [14], it is assumed that the truncated ΔN64 receptor has the same transmembrane orientation as the full-length μ receptor, that is, with the
Discussion
The major outcomes and conclusions drawn from this study are as follows. (1) The amino terminus of the μ receptor plays a key role as a determinant of the high affinity binding site for a subset of agonists, including methadone, fentanyl, endomorphin 1, and endomorphin 2. For these ligands, there was a 20- to 60-fold decrease in affinity for the truncated ΔN64 receptor relative to the full-length μ receptor. (2) MOR is glycosylated when expressed in HEK 293 cells. N-glycan chains within the
Acknowledgements
The authors thank Drs. Lei Yu and Mark von Zastrow for opioid receptor cDNAs. Technical assistance by Mr. Norihiro Chinen is gratefully acknowledged. This research was supported by grant DA 09113 from the National Institute on Drug Abuse (to R.D.H.).
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