Opioids such as codeine and morphine, are the active pharmaceutical ingredients of many drugs such as pain relievers (analgesics), cough suppressants, sedatives, etc. Opioids are derivatives of opium and bind to the μ-opioid receptor (μOR). The coupling of the μOR to Gi, the inhibitory subunit of the G-protein, blocks the activation of adenylyl cyclase and results in the analgesic effect. The opioid receptors belong to the G protein-coupled receptors (GPCRs) superfamily and specifically to the rhodopsin-like family A. These receptors consist of two closely related members, namely, δOR and κOR (Manglik et al. 2012, p. 322). The GPCRs have been long known to mediate major cellular signal transduction processes and the corresponding physiological responses in the mammalian body, which makes them potential targets for novel therapeutic drugs (Katritch et al. 2013, p. 531).
The elucidation of the general structure of GPCRs was a major breakthrough in understanding the biochemistry of the receptors, their ligand-binding mechanisms and the variance in their efficacy to the various ligands. GPCRs are characterized by the presence of seven transmembrane (TM) α helices that are connected by three intracellular (ICL) and three extracellular loops (ECL) (Rosenbaum et al. 2009, p. 356). One of the major findings was the difference in ligand efficacy that was produced by the binding of a full agonist, a partial agonist, an inverse agonist and a neutral agonist to the ligand-binding site in the receptors. The spectrum of interaction suggested that the signal transduction of GPCRs is a very complex process (Rosenbaum et al. 2009, p. 357). Another major discovery was the difference in the state of the bound receptor that was stabilized by the different agonists. Scientists have discovered that the efficacy of the signal transduction depends on the type of ligand that binds to the receptor and the state of the receptor that the ligand stabilizes (Rosenbaum et al. 2009, p. 357).
The process of crystallization of the GPCRs in their intact, native state posed quite a few challenges for the researchers. Firstly, the native proteins were produced in meager quantities that did not facilitate extraction and purification. This challenge was overcome by using Sf9 insect cells, Hi5 insect cells and COS-1 mammalian cells, which could overproduce the GPCRs for purification. Secondly, the GPCRs were thermally unstable and susceptible to proteolysis. This problem was overcome by using stabilizing ligands such as T4 lysozyme (T4L), introducing mutations such as truncation at the C-terminal, using high salt concentration solvents and purification of the GPCRs using lipids (Rosenbaum et al. 2009, p. 359).
The fact that μORs are very similar in structure and function to other GPCRs such as rhodopsin and β-adrenergic receptors (β1AR and β2AR), the structure elucidation has paved the way for the development of new drugs that have maximum efficacy, low side effects and low dependency issues. Another motivating factor is the development of tolerance to the prescribed doses of opioids in patients. Such desensitization of the μOR results in endocytosis of the receptor from the cell membrane in an effort to re-sensitize the receptor by regulating the concentration of the receptors (Lee and Ho 2013, p. 690).
2. Structure of μOR
The opioid receptor subfamily has been researched upon widely and the structures of all the four closely related ORs, namely, κOR, δOR, μOR and nociception OR (NOR) have been elucidated. The NOR has 60% sequence identity to the other ORs but has very different ligand specificity (Katritch et al. 2013, p. 534). The crystal lattice of μOR displays seven TM helices and an eighth loose helix. These helices are connected by three ECLs and three ICLs. The ECL2 exhibits a β-hairpin, which is exhibited by all three members of this subfamily (Granier et al. 2012, p. 401). The receptor is a dimeric structure, which appears as alternating layers of aqueous and lipid phases. The intradimeric interactions are observed among 28 residues between TM5 and TM6, each of which exist in a bundle of four helices (Manglik et al. 2012, p. 325). Adjacent dimers interact via interdimeric contact between TM1, TM2 and the eighth helix. A disulfide bridge between C1403.25 and C217 connects TM3 to ECL2. The side chain of K233 is the site of covalent binding of the morphinan ligand β-FNA, which is an antagonistic ligand (Manglik et al. 2012, p. 322).
The intracellular side of the μOR exhibits structural similarities with the rhodopsin’s TM3, TM5 and TM6 ligand (Manglik et al. 2012, p. 322). When a ligand binds to the GPCRs on the cell surface, the intracellular side undergoes a rotational change in conformation, called as rotamer conformational transition. It is also called as a rotamer toggle switch, which creates an environment that is conducive for G-protein binding. This switch is a highly conserved region that consists of the residues aspartate/glutamate, arginine and tyrosine (D/ERY) (Rosenbaum et al. 2009, p. 362). Some GPCRs such as rhodopsin, form a stabilizing ionic lock, where the arginine binds to the aspartate to form a salt bridge, which in turn interacts with the TM3 and TM4 in the cytoplasmic side. This ionic lock is absent in μOR and many other GPCRs (Katritch et al. 2013, p. 535). However, the formation of the salt bridge is observed in μOR between R165 and D164. Mutation of the threonine 279 residue to lysine has been found to become constitutively active (producing a physiological response even in the absence of a bound ligand) ligand (Manglik et al. 2012, p. 323).
The ligand-binding sites of GPCRs are usually located inside the helical structures. However, in μOR, the binding site for β-funaltrexamine (β-FNA), which results in irreversible binding, is located quite superficially near the extracellular surface. The location of the ligand-binding site so close to the surface is the reason behind the very rapid dissociation of all opioid drugs. This nature of the binding site is also helpful in reversing the effect of the drugs in case of overdose (Manglik et al. 2012, p. 323). Of the fourteen residues that interact with β-FNA, nine residues are conserved in other members of ORs as well. The affinity towards morphinan ligands such as naltrindole is noted in δOR, where the interaction is non-covalent instead of covalent as seen in μOR (Granier et al. 2012, p. 401).
Granier, S., Manglik, A., Kruse, A. C., Kobilka, T. S., Thian, F. S., Weis, W. I., & Kobilka, B. K. (2012). Structure of the δ-opioid receptor bound to naltrindole. Nature. Vol. 485, no. 7398, pp. 400-404.
Katritch, V., Cherezov, V., & Stevens, R. C. (2013). Structure-Function of the G-protein-Coupled Receptor Superfamily. Annual review of pharmacology and toxicology. Vol. 53, pp. 531-556.
Lee, C. W. S., & Ho, I. K. (2013). Pharmacological Profiles of Oligomerized μ-Opioid Receptors. Cells. Vo. 2, no. 4, pp. 689-714.
Manglik, A., Kruse, A. C., Kobilka, T. S., Thian, F. S., Mathiesen, J. M., Sunahara, R. K., & Granier, S. (2012). Crystal structure of the [micro]-opioid receptor bound to a morphinan antagonist. Nature. Vol. 485, no. 7398, pp. 321-326.
Rosenbaum, D. M., Rasmussen, S. G., & Kobilka, B. K. (2009). The structure and function of G-protein-coupled receptors. Nature. Vol. 459, no. 7245, pp. 356-363.