Endomorphin-2: a biased agonist at the μ-opioid receptor

Abstract

Previously we correlated the efficacy for G protein activation with that for arrestin recruitment for a number of agonists at the μ-opioid receptor (MOPr) stably expressed in HEK293 cells. We suggested that the endomorphins (endomorphin-1 and -2) might be biased toward arrestin recruitment. In the present study, we investigated this phenomenon in more detail for endomorphin-2, using endogenous MOPr in rat brain as well as MOPr stably expressed in HEK293 cells. For MOPr in neurons in brainstem locus ceruleus slices, the peptide agonists [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) and endomorphin-2 activated inwardly rectifying K(+) current in a concentration-dependent manner. Analysis of these responses with the operational model of pharmacological agonism confirmed that endomorphin-2 had a much lower operational efficacy for G protein-mediated responses than did DAMGO at native MOPr in mature neurons. However, endomorphin-2 induced faster desensitization of the K(+) current than did DAMGO. In addition, in HEK293 cells stably expressing MOPr, the ability of endomorphin-2 to induce phosphorylation of Ser375 in the COOH terminus of the receptor, to induce association of arrestin with the receptor, and to induce cell surface loss of receptors was much more efficient than would be predicted from its efficacy for G protein-mediated signaling. Together, these results indicate that endomorphin-2 is an arrestin-biased agonist at MOPr and the reason for this is likely to be the ability of endomorphin-2 to induce greater phosphorylation of MOPr than would be expected from its ability to activate MOPr and to induce activation of G proteins.

Fig. 1.

Concentration-response curves for activation of the GIRK current in rat LC neurons by DAMGO, etorphine, and endomorphin-2. In individual LC neurons, concentration-response curves for DAMGO (n = 3–5) (A), etorphine (n = 3–8) (B),and endomorphin-2 (Endo-2) (n = 3–5) (C) were determined before and after treatment with the irreversible MOPr antagonist β-FNA (30 nM) for 30 min, with normalization to the maximal current induced by NA (100 μM) in the same neuron. Each concentration of agonist was applied until the response reached a steady state (∼2 min). Different concentrations were tested on each neuron to ensure that the responses to higher concentrations of agonist were not attenuated by desensitization. For graphical representation, the data were fitted to sigmoidal concentration-response curves with variable slopes.

Fig. 2.

Rate and extent of desensitization of MOPr-evoked GIRK channel currents in rat LC neurons. A, outward potassium currents were recorded from single LC neurons, clamped at −60 mV, in response to application of saturating concentrations of DAMGO (10 μM), endomorphin-2 (30 μM), morphine (30 μM), and etorphine (1 μM). Agonists, which were applied for at least 10 min, induced an outward current that was not sustained for the period of drug application (solid bars) but decreased (desensitization) to a steady state (plateau). Desensitization to etorphine was slow and needed longer to reach the plateau. The opioid receptor antagonist naloxone (Nlx) (1 μM) was perfused immediately after each agonist, to restore the basal level. B, desensitization-phase data from 3 to 10 neurons for each agonist were best fitted to a one-phase, exponential-decay model (one- and two-phase, exponential-decay models were compared with an F test for each data set). The fastest rate of desensitization observed was for endomorphin-2. Values shown are mean ± S.E.M.

Fig. 3.

Image-based quantification of agonist-induced phosphorylation of Ser375 in MOPr. HEK293 cells stably expressing HA-tagged MOPr were plated in 96-well plates and were exposed to different concentrations of MOPr agonists for 10 min. Cells were then fixed and used for immunocytochemical staining with an anti-phospho-Ser375 antibody, and image analysis was performed as described under Materials and Methods. A, representative images of the Ser375-phosphorylated MOPr immunofluorescence signal and the DAPI staining in untreated cells [phosphate-buffered saline (PBS)] and cells treated with DAMGO (10 μM), morphine (30 μM), or endomorphin-2 (30 μM) are shown. Cells in the mitotic process showed high nonspecific immunofluorescence signal levels. Scale bar, 30 μm. Results shown are representative of three to six independent experiments. B, concentration-response curves for Ser375 phosphorylation. Values are mean ± S.E.M. of three to six independent experiments. C, Western blot of phospho-Ser375-MOPr immunoprecipitated from HEK293 cells with anti-HA antibody and identified with anti-phospho-Ser375 antibody. Endomorphin-2 (E2) (30 μM) induced phosphorylation of Ser375 similar to that induced by DAMGO (D) (10 μM) and much greater than that induced by morphine (M) (30 μM). C, control; IP, immunoprecipitation; IB, immunoblotting.

Fig. 4.

Agonist-induced interaction of MOPr with arrestin-3, as measured in FRET experiments. HEK293 cells were transiently transfected with MOPr-YFP, GRK2, and arrestin-3-CFP. A, FRET trace for DAMGO (10 μM). An increase in the FRET ratio (measured as F₅₃₅/F₄₈₀) reflects the interaction between MOPr-YFP and arrestin-3-CFP. B, half-life of MOPr-YFP/arrestin-3-CFP interaction after addition of 10 μM DAMGO, 30 μM endomorphin-2, or 30 μM morphine. FRET traces were fitted to a one-phase, exponential model for calculation of t_1/2. Values are mean ± S.E.M. from at least three separate experiments in each case. **, P < 0.01, Student's t test, t_1/2 for morphine was significantly longer than that for DAMGO or endomorphin-2. C, extent of agonist-induced FRET. The maximal FRET for each agonist was expressed as a percentage of that induced with subsequent addition of 10 μM DAMGO. The value for DAMGO is greater than 100% because the second DAMGO response was always slightly less than the first (see results in A). Values are mean ± S.E.M. from at least three separate experiments in each case. **, P < 0.01, Student's t test, value for morphine was significantly lower than that for DAMGO or endomorphin-2.

Fig. 5.

Concentration- and time-dependent cell surface loss of MOPr from HEK293 cells stably expressing HA-tagged MOPr. A, cells were incubated with different concentrations of agonist for 30 min before determination of cell surface MOPr loss with enzyme-linked immunosorbent assays. Data were fitted to sigmoidal curves with variable slopes. Values are mean ± S.E.M. from four separate experiments, each performed in triplicate. B, cells were incubated with receptor-saturating concentrations of agonist (morphine, 30 μM; endomorphin-2, 30 μM; DAMGO, 10 μM; etorphine, 1 μM) for up to 30 min for determination of time-dependent cell surface MOPr loss. Values are mean ± S.E.M. from three to five separate experiments, each performed in triplicate.

Fig. 6.

Relative efficacy values for DAMGO, etorphine, endomorphin-2, and morphine for five MOPr signaling outputs. Values refer to efficacy relative to that of DAMGO, which was set to 1.00 for each output. The values for [³⁵S]GTPγS binding and arrestin-3 recruitment were taken from Table 2 of McPherson et al. (2010). The efficacy values are τ values for operational efficacy in each case except for cell surface loss, for which the values are relative efficacy (e) values obtained with the method described by Ehlert (1985). The τ value for morphine in the GIRK assay was obtained from Bailey et al. (2009). Actual values of efficacy for DAMGO in each assay are given in Table 1.

Fig. 7.

Fractional receptor occupancy-response relationships for MOPr agonists. Previously published (McPherson et al., 2010) concentration-response data for agonist-induced [³⁵S]-GTPγS binding and arrestin-3 recruitment were used to determine the occupancy-response relationships for MOPr agonists. Fractional receptor occupancy at each concentration of agonist was calculated as described under Materials and Methods. A, relationship between [³⁵S]GTPγS binding and fractional receptor occupancy for DAMGO, etorphine, morphine, and endomorphin-2. Data were fitted to a one-site binding model (hyperbola) with GraphPad Prism; the r² value for each was >0.964. Data points represent the mean response at each level of occupancy. B, relationship between arrestin-3 recruitment and fractional receptor occupancy for DAMGO, etorphine, morphine, and endomorphin-2. Data were fitted with linear regression because this yielded a better fit than a one-site binding model (hyperbola). Data points represent the mean response at each level of occupancy.

Fig. 8.

Calculation of ligand bias at MOPr. The bias factor for 16 MOPr ligands was calculated as described under Materials and Methods, using data for ligand-induced [³⁵S]GTPγS binding and arrestin-3 recruitment previously generated in HEK293 cells stably expressing MOPr (McPherson et al., 2010). Leu-enkephalin was selected as the reference unbiased ligand on the basis of its position when operational efficacy values for the two signaling outputs were plotted (see Fig. 3 of McPherson et al., 2010). For each agonist with either G protein activation or arrestin-3 recruitment, the effective signaling (σ_lig) was calculated as σ_lig = log(τ_lig/τ_ref); the bias factor (β_lig) for a particular ligand was then calculated as β_lig = (σ_lig^path1 − σ_lig^path2)/√2. A one-sample, two-tailed t test was used to determine whether the degree of bias was statistically different from 0. *, endomorphin-2 displayed a statistically significant level of bias (p < 0.05). M6G, morphine-6-glucuronide; 6-MAM, 6-monoacetylmorphine.