Selectivity of μ-opioid receptor determined by interfacial residues near third extracellular loop

Abstract

We hypothesized that the selectivity profile of the rat μ-opioid receptor for opioid receptor-selective ligands is determined by the nature of the amino acid residues at highly divergent sites in the ligand-binding pocket. To determine which characteristics of these residues contribute to opioid receptor ligand selectivity, we made various mutant receptors that replaced the Lys³⁰³ and Trp³¹⁸ residues near the extracellular interface of transmembrane domains VI and VII, respectively. Ligand binding determinations using transiently transfected monkey kidney epithelial (COS-1) cells show that Lys³⁰³ mutations cause little change in the receptor binding profile, whereas the Trp³¹⁸ mutant receptors have considerably lower affinity for μ-opioid receptor-selective ligands and greatly increased affinity for δ-opioid receptor-selective ligands. The nature of these mutations show that this effect is not due to sterics or charge alone. [³⁵S]guanosine-5′-O-(3-thio)-triphosphate ([³⁵S]GTPγS) activity assays show that these residues may influence functional, as well as binding selection. We conclude that a primary role for Trp³¹⁸ is to form a basis for ligand selectivity.

Introduction

Due to their sequence homology and to the availability of a wide range of ligands, the G-protein-coupled opioid receptors offer an excellent opportunity for studying the molecular basis of ligand selectivity and activity. Little work has been done in determining the contribution of divergent amino acid residues within the transmembrane domains of opioid receptors in determining selectivity or function. Few sites in the transmembrane domains of these receptors demonstrate convincing divergence in amino acid chemical functionality Meng et al., 1993, Thompson et al., 1993, Kieffer et al., 1992, Bunzow et al., 1994. One such site is the Lys³⁰³ in the rat μ-opioid receptor. This residue corresponds to Trp²⁸⁴ in the rat δ-opioid receptor, Glu²⁹⁷ in the rat κ-opioid receptor, and Gln²⁸⁶ in the rat orphan-opioid receptor. Among this set of amino acids, there is considerable diversity in chemical functionality (i.e., positively charged, aromatic hydrophobic, negatively charged, and polar neutral residues).

This current analysis was undertaken in an effort to determine the functional implications of amino acid divergences of this type. One possibility is that the nature of the amino acid does not matter at these sites, and that any amino acid will suffice. Alternatively, these amino acids may form the basis of opioid receptor ligand selectivity. If the latter argument is true, then one should be able to substitute the amino acid of one receptor type for the other and show that it leads to altered, or even reversed opioid receptor ligand selectivity. For example, if one were to replace Trp³¹⁸ in the μ-opioid receptor with leucine (the homologous δ-opioid receptor residue) to yield the W318L-μ receptor mutant, and subsequently show that this mutant binds δ-opioid receptor-selective ligands with higher affinity, and μ-opioid receptor-selective ligands with lower affinity, then that would be clear evidence in support of the argument that the residue serves an important role in discriminating between ligands of different opioid receptor selectivities.

Site-directed mutagenesis studies which yield a “gain-of-function” of this type have been reported previously by this laboratory — the orphan-opioid receptor was converted into a receptor which recognized opioid receptor-selective antagonists (Meng et al., 1996). In the present work, two divergent sites (Lys³⁰³ and Trp³¹⁸ in the μ-opioid receptor) were chosen in an effort to determine their importance in producing opioid receptor selectivity. Mutations at these sites were made and assayed for their ability to bind opioid receptor-selective ligands as determined by radioligand competition, and for their ability to transduce message, as determined by agonist-induced [³⁵S]guanosine-5′-O-(3-thio)-triphosphate ([³⁵S]GTPγS) binding.

Few mutational analyses have been performed on these two sites or their homologues in the other opioid receptors. Published reports include a κ-opioid receptor mutant (E297K), that was shown to have decreased affinity for the κ-opioid receptor-selective ligand norbinaltorphimine, yet unchanged affinity for the less opioid receptor-selective ligands diprenorphine and naloxone (Hjorth et al., 1995). The wild-type κ-opioid receptor was also shown to be convertible to one which bound with high affinity and was activated by the μ-opioid receptor-selective agonist peptide [d-Ala², N-MePhe⁴,Gly-ol]enkephalin (DAMGO) by changing only four residues, two of which being homologous to the ones reported in this work (Seki et al., 1998). More attention has been paid to Trp²⁸⁴ in the δ-opioid receptor Varga et al., 1996, Valiquette et al., 1996, although these latter two studies produced somewhat disparate results, possibly due to the difference in the amino acid to which the target residue was mutated. Recently, it was reported that a W318A mutation of the μ-opioid receptor yields a receptor in which specific binding was nearly undetectable (Xu et al., 1999). However, only a very limited number of ligands was tested, and the gain-of-function towards non-μ-opioid receptor-selective ligands was not explored.

Thus, although there is scattered evidence that these sites are important for determining the pharmacological profiles of opioid receptor-selective ligands, these sets of data have yet to reveal a full picture of how the opioid receptor amino acid residues influence ligand characteristics such as affinity, selectivity, and potency. The μ-opioid receptor has a complex set of determinants for these ligand characteristics Onogi et al., 1995, Seki et al., 1998. When one is making mutant opioid receptors, one should evaluate carefully the nature of the amino acid residue of the wild-type and consider the nature of the amino acid to which the residue will be mutated. In the present study, Lys³⁰³ was mutated to glutamine, glutamic acid, and tryptophan. This chemically diverse set of amino acids at this position were chosen to help reveal the chemical requirements which determine μ-opioid receptor-selective ligand affinity, activity, and selectivity. These mutated residues represent the amino acids present at the homologous positions in the other (non-μ) opioid receptors. Similarly, Trp³¹⁸ was mutated to leucine and lysine, both to elucidate the chemical requirements at this site for high affinity ligand binding, and to determine whether this residue acts as a selectivity switch. For both sets of μ-opioid receptor mutants (K303 and W318), the homologous δ-opioid receptor residue is represented. If these mutant receptors bind a class of molecules which are traditionally considered as selective for the δ-opioid receptor, then there is compelling reason to conclude that the mutated residues are important in determining selectivity.