μ Opioid receptor: role for the amino terminus as a determinant of ligand binding affinity

doi:10.1016/S0169-328X(99)00332-0

Molecular Brain Research

Volume 76, Issue 1, 10 March 2000, Pages 64-72

https://doi.org/10.1016/S0169-328X(99)00332-0 Get rights and content

Abstract

The importance of the amino-terminal domain of the μ opioid receptor (MOR) as a component of the high affinity ligand-binding pocket was evaluated. A deletion mutant lacking 64 amino acids from the amino-terminus of MOR (ΔN64) was constructed and expressed in HEK 293 cells. The affinities of bremazocine and cyclazocine were similar for the truncated and full-length MORs. Affinities of the μ receptor antagonist, naloxone, and the μ receptor agonist, morphine, were decreased 3.5-fold and 6-fold, respectively, for the truncated receptor relative to the wild-type MOR. Similarly, the affinities of the opioid peptide agonists, DAMGO (Tyr-d-Ala-Gly-MePhe-Gly-ol), β-endorphin, and DADL (Tyr-d-Ala-Gly-Phe-d-Leu), for the ΔN64 receptor were decreased from 3- to 8-fold as a result of the deletion. In contrast, the affinities of the alkaloid agonists, methadone and fentanyl, and the peptide agonists, endomorphin 1 and endomorphin 2, for the truncated receptor relative to MOR were reduced dramatically by 20- to 60-fold. MOR is glycosylated when expressed in HEK 293 cells; however, analysis of N-glycosidase F-treated membranes indicated that N-glycan chains within the amino-terminal domain of MOR do not contribute significantly to ligand affinities. These results indicate that amino acid residues within the amino-terminal domain of MOR play a crucial role in the composition of the binding pocket for a select group of agonists.

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 G_i and G_o 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 [ $^{3} H$ ]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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Research reportμ Opioid receptor: role for the amino terminus as a determinant of ligand binding affinity

Abstract

Introduction

Section snippets

Construction of the ΔN64 μ opioid receptor

Transfection and radioligand binding assays

Stable expression of μ and ΔN64 opioid receptors in HEK 293 cells

Discussion

Acknowledgements

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Biochem. Biophys. Res. Commun.

The conserved aspartate residue in the third putative transmembrane domain of the δ-opioid receptor is not the anionic counterpart for cationic opiate binding but is a constituent of the receptor binding site

Mol. Pharmacol.

Mutagenesis of a single amino acid in the rat μ-opioid receptor discriminates ligand binding

J. Neurochem.

Research report
μ Opioid receptor: role for the amino terminus as a determinant of ligand binding affinity