Intrinsic relative activities of κ opioid agonists in activating Gα proteins and internalizing receptor: Differences between human and mouse receptors

Several investigators recently identified biased kappa opioid receptor (KOP receptor) agonists. However, no comprehensive study of the functional selectivity of available KOP receptor agonists at the human and mouse KOP receptors (hKOP receptor and mKOP receptor, respectively) has been published. Here we examined the ability of over 20 KOP receptor agonists to activate G proteins and to internalize the receptor. Clonal neuro-2a mouse neuroblastoma (N2a) cells stably transfected with the hKOP receptor or mKOP receptor were used We employed agonist-induced [S-35]GTP gamma S binding and KOP receptor internalization as measures of activation of G protein and beta-arrestin pathways, respectively. The method of Wert and colleagues was used to quantify intrinsic relative activities at G protein activation (RA(i-G)) and receptor internalization (RA(i-l)) and the degree of functional selectivity between the two [Log RA(i-G) - log RA(i-l), RA(i-G)/RA(i-I) and bias factor]. The parameter, RA(i), represents a relative estimate of agonist affinity for the active receptor state that elicits a given response. The endogenous ligand dynorphin A (1-17) was designated as the balanced ligand with a bias factor of 1. Interestingly, we found that there were species differences in functional selectivity. The most striking differences were for 12-epi-salvinorin A, 1369,593, and ICI-199,441. 12Epi-salvinorin A was highly internalization biased at the mKOP receptor, but apparently G protein biased at hKOP receptor. 1369,593 was much more internalization biased at mKOP receptor than hKOP receptor. ICI199,4-41 showed internalization biased at the mKOP receptor and G protein biased at the hKOP receptor. Possible mechanisms for the observed species differences are discussed. (C) 2015 Elsevier LW. All rights reserved.