Stabilization of μ-opioid receptor facilitates its cellular translocation and signaling

Abstract

The G protein-coupled μ-opioid receptor (μ-OR) mediates the majority of analgesia effects for morphine and other pain relievers. Despite extensive studies of its structure and activation mechanisms, the inherently low maturation efficiency of μ-OR represents a major hurdle to understanding its function. Here we computationally designed μ-OR mutants with altered stability to probe the relationship between cell-surface targeting, signal transduction, and agonist efficacy. The stabilizing mutation T315Y enhanced μ-OR trafficking to the plasma membrane and significantly promoted the morphine-mediated inhibition of downstream signaling. In contrast, the destabilizing mutation R165Y led to intracellular retention of μ-OR and reduced the response to morphine stimulation. These findings suggest that μ-OR stability is an important factor in regulating receptor signaling and provide a viable avenue to improve the efficacy of analgesics.

Keywords: GPCR; maturation; opioid receptor; protein engineering; signal transduction.

Figure 1.. Rational design of μ-OR stabilizing and destabilizing mutants.

(A) Eris scan identifies a stabilizing mutation (T315Y) and destabilizing mutation (R165Y) using μ-OR structural models (Left: inactive state, Right: active state). The mutated tyrosine residues were shown as spheres. (B) T315Y can mediate hydrogen bond network (Y128-H319-T315Y) and aromatic interactions (T315Y-W318) in the design model, while R165Y disrupts polar interactions with D124 and T279 of μ-OR.

Figure 2.. Cellular distribution of WT μ-OR and two mutants.

(A) Expression of μ-OR was visualized by immunocytochemistry (blue: Hoechst stain; red: μ-OR expression; green: EGFP expression; yellow: overlap between red and green fluorescence, showing intracellular retention of μ-OR). (B) Quantified cell surface expression levels for WT, R165Y, and T315Y indicates that increasing μ-OR stability facilitates its maturation in HEK293T cells. ** (P<0.05), *** (P<0.001). 5–7 repeats/group. (C) R165Y was located mainly intracellularly, while the majority of T315Y translocated to the plasma membrane in HEK293 cells. *** (P<0.001). 5–7 repeats/group.. (D) Quantified cell surface expression levels for WT, R165Y, and T315Y in HEK 293 cells.

Figure 3.. Measurement of μ-OR activity in HEK293 cells.

HEK293 cells transiently transfected with vectors containing μ-OR (WT, R165Y, T315Y), or empty vectors (Control). Cells were then treated with μ-OR agonist (A) morphine or (B) DAMGO at concentrations of 10⁻¹² to 10⁻⁴ M. The activities were measured with the cAMP-sensitive luciferase reporter assay. The stabilizing mutant T315Y exhibited elevated efficacy to both agonists, in comparison to the destabilizing mutant R165Y. Error bar: S.E.M., n = 3.