Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2015 Aug 15:761:235-44.
doi: 10.1016/j.ejphar.2015.05.054. Epub 2015 Jun 6.

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

Kelly M DiMattio  1 , Frederick J Ehlert  2 , Lee-Yuan Liu-Chen  3
Affiliations
Comparative Study

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

Kelly M DiMattio et al. Eur J Pharmacol. .

Abstract

Several investigators recently identified biased κ 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 [(35)S]GTPγS binding and KOP receptor internalization as measures of activation of G protein and β-arrestin pathways, respectively. The method of Ehlert and colleagues was used to quantify intrinsic relative activities at G protein activation (RAi-G) and receptor internalization (RAi-I) and the degree of functional selectivity between the two [Log RAi-G - logRAi-I, RAi-G/RAi-I and bias factor]. The parameter, RAi, 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, U69,593, and ICI-199,441. 12-Epi-salvinorin A was highly internalization-biased at the mKOP receptor, but apparently G protein-biased at hKOP receptor. U69,593 was much more internalization-biased at mKOP receptor than hKOP receptor. ICI199,441 showed internalization-biased at the mKOP receptor and G protein-biased at the hKOP receptor. Possible mechanisms for the observed species differences are discussed.

Keywords: Kappa opioid receptor; Ligand bias; Species difference.

PubMed Disclaimer

Conflict of interest statement

CONFLICT OF INTERESTS

There is no conflict of interests.

Figures

Figure 1
Figure 1. Concentration-response curves of five KOP receptor agonists at the hKOP receptor for [35S]GTPγS binding and receptor internalization in N2a-3HA-hKOP receptorcells
(A) [35S]GTPγS binding was performed on 10 µg membrane protein in the presence of 5 mM MgCl2, 150 mM NaCl and 20–25 µM GDP at 30°C for 60 min as described in the Materials and Methods. Each experiment included a concentration-response curve for U50,488H, and the data have been normalized relative to the maximal response to U50,488H at 10 µM since it gave the highest response in preliminary experiments. Each concentration-response curve represents the mean ± SEM of at least three independent experiments performed in duplicate. (B) The OCW was performed on live cells and receptor internalization was measured as compared to control. Twenty thousand cells/well were plated in 24-well plates 40 hr before the experiment was performed and the agonists were added in serum-free medium at varying concentrations. The anti-HA monoclonal antibody was added as the primary antibody, cells were fixed and then stained with two cellular stains (Sapphire700 and DRAQ5) with the 800CW goat anti-mouse antibody as described in Materials and Methods. Each value represents the mean ± SEM of at least three independent experiments performed in triplicate. The theoretical curves represent the global least squares fit of equations 1 and 2 to the data.
Figure 2
Figure 2. Concentration-response curves of five KOP receptor agonists at the mKOP receptor for [35S]GTPγS binding and receptor internalization in N2a-FLAG-mKOP receptor cells
(A) [35S]GTPγS binding was performed on 10 µg membrane protein in the presence of 5 mM MgCl2, 150 mM NaCl and 20–25 µM GDP at 30°C for 60 min as described in the Materials and Methods. Each experiment included a concentration-response curve for enadoline, and the data have been normalized to the maximal response to enadoline at 1 µM since it gave the highest response in preliminary experiments. Each concentration-response curve represents the mean ± SEM of at least three independent experiments performed in duplicate. (B) The OCW was performed on live cells and receptor internalization was measured as compared to control. Thirty thousand cells/well were plated in 24-well plates 40 hr before the experiment was performed and the agonists were added in serum-free medium at varying concentrations. The anti-FLAG M1 monoclonal antibody was added as the primary antibody, cells were fixed and then stained with two cellular stains (Sapphire700 and DRAQ5) with the 800CW goat anti-mouse antibody as described in Materials and Methods. Each value represents the mean ± SEM of at least three independent experiments performed in triplicate. The theoretical curves represent the global least squares fit of equations 1 and 2 to the data.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Beguin C, Potuzak J, Xu W, Liu-Chen LY, Streicher JM, Groer CE, Bohn LM, Carlezon WA, Jr, Cohen BM. Differential signaling properties at the kappa opioid receptor of 12-epi-salvinorin A and its analogues. Bioorg. Med. Chem. Lett. 2012;22:1023–1026. - PMC - PubMed
    1. Bruchas MR, Land BB, Aita M, Xu M, Barot SK, Li S, Chavkin C. Stress-induced p38 mitogen-activated protein kinase activation mediates kappa-opioid-dependent dysphoria. J. Neurosci. 2007;27:11614–11623. - PMC - PubMed
    1. Chen C, Wang Y, Huang P, Liu-Chen LY. Effects of C-terminal modifications of GEC1 protein and gamma-aminobutyric acid type A (GABA(A)) receptor-associated protein (GABARAP), two microtubule-associated proteins, on kappa opioid receptor expression. J. Biol. Chem. 2011;286:15106–15115. - PMC - PubMed
    1. DeWire SM, Yamashita DS, Rominger DH, Liu G, Cowan CL, Graczyk TM, Chen XT, Pitis PM, Gotchev D, Yuan C, Koblish M, Lark MW, Violin JD. A G protein-biased ligand at the mu-opioid receptor is potently analgesic with reduced gastrointestinal and respiratory dysfunction compared with morphine. J. Pharmacol. Exp. Ther. 2013;344:708–717. - PubMed
    1. Ehlert FJ. On the analysis of ligand-directed signaling at G protein-coupled receptors. Naunyn Schmiedebergs Arch. Pharmacol. 2008;377:549–577. - PubMed

Publication types

MeSH terms