A Chimeric Study of the Molecular Basis of Affinity and Selectivity of the κ and the δ Opioid Receptors.

Abstract

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.

On the role of extracellular loops of opioid receptors in conferring ligand selectivity

“…In a study of 5/x chimera, it was found that a chimeric mutant possessing the C-terminal portion (from helix 5 to the C-terminus) of the receptor could bind U50,488 with affinities very similar to that of the x wild type. Other ~/x chimera showed decreased binding for U50,488 relative to the wild type x receptor [15].…”

“…Recently, a more comprehensive study of the molecular basis of affinity and selectivity between ~ and x receptors has appeared which examines the binding of various selective and non-selective, peptide and non-peptide, agonist and antagonist opioid ligands to 6, K, and 5/K chimeric receptors [15]. Some of the most interesting results from this study regard the selectivity of enkephalin and dynorphin to ~ and x receptors.…”

“…To explain these results, it has been suggested that the ~ reeep-tor contains a high affinity binding pocket for the 'opioid core' (defined as Tyr-Gly-Gly-Phe) in the C-terminal domain capable of binding both enkephalins and dynorphin. This pocket is then said to be relatively weak in the x receptor and the binding of dynorphin to x is dependent on the coulombic interactions between the basic residues of the peptide and the anionic residues found in EL-2 of the x receptor [15].…”

“…First, although dynorphin possesses highest affinity for x receptors, it has been shown to have K~ values in the nanomolar range for both ~ and p [14,15]. This demonstrates that interactions between the Cterminal end of dynorphin and the anionic residues found on EL-2 of x are not required for opioid binding.…”

“…In order to isolate regions of the opioid receptor subtypes which play a role in selectivity, various studies involving chimeric mutants which allow for the exchange of various extracellular loops among subtypes have been reported [3,[10][11][12][13][14][15][16]. Chimeric mutants between all possible combinations (6/K, ~/fl, and x/~t) have been generated and in several studies, potential interactions between selective ligands and extracellular loop regions have been suggested [3,13,16].…”