Creating a Functional Opioid Alkaloid Binding Site in the Orphanin FQ Receptor through Site-Directed Mutagenesis

Abstract

Although much has been learned about the mechanisms of ligand selectivity between different opioid receptor subtypes, little is known about the common opioid binding pocket shared by all opioid receptors. The recently discovered orphanin system offers a good opportunity to study the mechanisms involved in the binding of opioid versus nonopioid ligands. In the current study, we adopt a "gain of function" approach aimed at shifting the binding profile of the orphanin FQ receptor toward that of the opioid receptors. After two rounds of mutagenesis, several orphanin FQ receptor mutants can be labeled with the opiate alkaloid [3H]naltrindole and show greatly increased affinities toward the opiate antagonists naltrexone, nor-binaltrophine HCl, and (-)-bremazocine. These orphanin FQ receptor mutants also display stereospecificity similar to that of opioid receptors. Furthermore, the orphanin FQ receptor mutant that has the best affinities toward the opioid alkaloids shows, in the presence of GTP and high salt concentration, an affinity-shift profile similar to that of the delta receptor. Most strikingly, the same mutant exhibits naltrindole-sensitive etorphine-stimulated [35S]guanosine-5'-O-(3-thio)triphosphate binding, whereas the effect of etorphine on GTP binding cannot be inhibited by naltrindole in the wild-type receptor. Our results indicate that 1) several residues in the orphanin FQ receptor are critical to its selectivity against the opiate alkaloids, particularly antagonists; and 2) mutating these residues to those of the opioid receptor at the corresponding position preserves the agonist/antagonist nature of opiate alkaloids as they interact with the mutant receptor. It is reasonable to hypothesize that the corresponding residues in the opioid receptors may form a functional common binding pocket for opiate alkaloids. These findings may be helpful to medicinal chemists in designing ligands for the orphanin FQ receptor based on the structure of the opiate alkaloids.

Cloning, pharmacological characterization and tissue distribution of an ORL1 opioid receptor from an amphibian, the rough-skinned newt Taricha granulosa

“…These amino acid substitutions created a mutant receptor capable of binding both oFQ and prodynorphin products with sub-nanomolar affinity. The addition of a fifth mutation to the rat ORL1 receptor construct (Ala 213 -Lys in TMV) caused its affinity towards selective opioid antagonists to increase two or three orders of magnitude (Meng et al 1998). Interestingly, the nORL receptor matches the classical opioid receptors at three of the aforementioned residues targeted for mutation (Ile 303 in TMVII, Ile 277 and Ile 279 in TMVI; nORL amino acid numbering).…”

“…Also, the newt receptor shares identity with the classic opioid receptors at three residues (Ile 277 , Ile 279 , and Ile 303 ; newt numbering) shown to be important in the recognition of ligands that are not conserved in the mammalian ORL1 receptor (Meng et al 1996a(Meng et al , 1998.…”

“…Mutating four residues of the rORL1 to the conserved classical opioid receptor counterparts (Val-Gln-Val 276-278 to Ile-His-Ile in TMVI and Thr 302 to Ile in TMVII) enables the receptor to bind prodynorphin peptides with subnanomolar affinity (Meng et al 1996a); the nORL matches the classical opioid receptors at three of the four residues (black highlights). The addition of a fifth mutation to the rORL1 (Ala to Lys in TMV) increases its affinity towards selective opioid antagonists by two or three orders of magnitude (Meng et al 1998); the nORL has a unique residue at this position (Gly 214 ; black highlight). Percentage amino acid identities to the nORL are listed at the end of the alignment.…”

Pharmacological characterization of a nociceptin receptor from zebrafish (Danio rerio)

“…2; Mouledous et al 2000). Site-directed mutagenesis studies have yielded receptors with both NOP and KOR characteristics (Meng et al 1996, Mollereau et al 1996, Meng et al 1998. Interestingly, in drNOP and other non-mammalian NOPs, some of the amino acids, which are conserved in other NOPs, are replaced by prototypical k residues which are involved in k-ligand recognition (Table 1).…”

Orphan G-protein-coupled Receptors

“…Although ORLl displayed high identity to the opioid receptors (greater than 50% ovenll) it was unable to bind opioid ligands, however, single amino acid replacement experiments demonsfrated that ORLl could be endowed with the ability to bind opioid ligands (Mollereau et al, 1996;Meng et al. 1997;Meng et al, 1998 uses to exclude the binding of opioid ligands, and in addition would help reveal information about the opioid binding pocket in the opioid receptors. Although a limited number of ligands were assayed here, it does suggest that the residues mutated in GPR8 might not contribute to a generic opioid binding pocket, and also that GPR8 is incapable of forming an opioid binding pocket, however, other interpretations might explain the negative data For example.…”