No abstract

It is unclear how receptor/ligand families that are evolutionarily closely related achieve functional separation. To address this question, we focus here on the newly discovered Orphanin FQ, a peptide homologous to the opioid peptide Dynorphin, and its receptor, the Orphanin FQ receptor, which is highly homologous to the opioid receptors. In spite of this high degree of homology in terms of both ligands and receptors, there is little direct cross-talk between the Orphanin FQ system and the endogenous opioid system. Thus, the opioid peptides show either relatively low affinity or no affinity toward the Orphanin FQ receptor; conversely, Orphanin FQ has no affinity toward any of the opioid receptors. We sought to investigate the molecular basis of such discrimination by attempting to reverse it and endowing the Orphanin FQ receptor with the ability to bind opioids. We report that by mutating as few as four amino acids, we can produce a receptor that recognizes pro-Dynorphin products with very high affinity and yet still binds Orphanin FQ as well as the wild-type receptor. This suggests that the Orphanin FQ receptor has developed features that specifically exclude the opioids and that these features are distinct from those required for the high affinity binding of its own endogenous ligand.The three major types of opioid receptors, , ␦, and , have been cloned and shown to belong to the seven transmembrane domain, G protein-coupled family (1, 2). In addition, several laboratories have cloned a protein highly homologous to these opioid receptors but that, nevertheless, does not bind with high affinity any known opioid peptides or alkaloids (3). The endogenous ligand for this opioid-like orphan receptor has recently been isolated by two independent groups (3, 4). Interestingly, it is a 17-amino acid peptide with a significant degree of sequence homology to DynA-(1-17). This novel peptide has been termed Nociceptin by Meunier et al. (3) to denote its ability to increase pain responsiveness and Orphanin FQ by Civelli and coworkers (4). We shall refer here to the ligand as Orphanin FQ and to its receptor as the Orphanin FQ receptor.It is clear from ongoing anatomical studies that Orphanin FQ and its receptor represent a novel and distinct peptidergic system with a unique anatomical distribution within the central nervous system 1 and the gastrointestinal system 2 . Behavioral results have already demonstrated a novel profile for Orphanin FQ in pain and place preference tests and in patterns of tolerance development (5, 6). Orphanin FQ has at its amino terminus the sequence Phe-Gly-Gly-Phe, which only differs from the common opioid core (Tyr-Gly-Gly-Phe) by a single OH group. Yet, since the N-terminal tyrosine is critical for the binding of all opioid peptides (7), this change is sufficient to preclude Orphanin FQ from binding to any of the opioid receptors. Conversely, reciprocal events must have taken place to preclude the opioid peptides from being recognized by the Orphanin FQ receptor. Thus, this system offers a most...

Structural elements of the rat 1.t-opioid receptor important in ligand receptor binding and selectivity were examined using a site-directed mutagenesis approach. Five single amino acid mutations were made, three that altered conserved residues in the~8, and K receptors (Asn 15°to Ala, His297 to Ala, and Tyr326 to Phe) and two designed to test for~i/8 selectivity (lie198 to Val and Va1202 to lie). Mutation of His297 in transmembrane domain 6 (TM6) resulted in no detectable binding with [3H]DAMGO (3H-iabeied c-A1a2,N-Me-Phe4,Giy-o15-enkephalin), [3H]bremazocine, or [3H]ethyiketocyciazocine. Mutation of Asn15°in TM3 produces a three-to 20-fold increase in affinity for the opioid agonists morphine, DAMGO, fentanyl,~3-endorphin 1_31, JOM-13, deltorphin Ii, dynorphin1_13, and U50,488, with no change in the binding of antagonists such as naloxone, naitrexone, naitrindole, and nor-binaltorphamine. in contrast, the Tyr 326 mutation in TM7 resulted in a decreased affinity for a wide spectrum of~u, 8, and K agonists and antagonists. Altering Va1202 to lie in TM4 produced no change on hgand affinity, but lie198 to Val resulted in a four-to fivefold decreased affinity for the~i agonists morphine and DAMGO, with no change in the binding affinities of K and 8 igands.

Within the large family of G-protein-coupled receptors, a picture is emerging which contrasts the binding of small ligands and the binding of peptides to the seven-helix configuration of the proteins. Because of its unique richness in both peptide and non-peptide ligands, the opioid receptor family offers several advantages for achieving a better understanding of similarities and differences in ligand/receptor interactions across different classes of agonists and antagonists. Since multiple, naturally occurring, ligands interact with the multiple receptors with varying degrees of selectivity, this family is also an excellent model for examining the structural basis of selectivity. Thus, the molecular basis of binding affinity and selectivity of the kappa and the delta opioid receptors was investigated by the construction of four kappa/delta chimeric receptors. The pharmacological profiles of these chimeras as well as those of the wild type kappa and delta receptors were determined by their binding with several different categories of opioid ligands. A linear model was used to deduce the relative contribution of each corresponding pairs of kappa-delta receptor segments to the binding of a given ligand. The results show that the kappa and delta receptors bind the same opioid core differently and achieve their selectivity through different mechanisms. In addition, the interaction of a peptide ligand with a receptor appears to be different from that of a small ligand. Furthermore, these results point to a particularly important role of the second extracellular loop and the top half of transmembrane domain 4 in the binding of prodynorphin products. Together, the results suggest that these peptide receptors can be bound and activated via multiple binding pockets as a function of their own topography and the nature of the interacting ligand.

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.