Role for G protein-coupled receptor kinase in agonist-specific regulation of mu-opioid receptor responsiveness

Abstract

The G protein-coupled mu-opioid receptor (mu OR) mediates the physiological effects of endogenous opioid peptides as well as the structurally distinct opioid alkaloids morphine and etorphine. An intriguing feature of mu OR signaling is the differential receptor trafficking and desensitization properties following activation by distinct agonists, which have been proposed as possible mechanisms related to opioid tolerance. Here we report that the ability of distinct opioid agonists to differentially regulate mu OR internalization and desensitization is related to their ability to promote G protein-coupled receptor kinase (GRK)-dependent phosphorylation of the mu OR. Although both etorphine and morphine effectively activate the mu OR, only etorphine elicits robust mu OR phosphorylation followed by plasma membrane translocation of beta-arrestin and dynamin-dependent receptor internalization. In contrast, corresponding to its inability to cause mu OR internalization, morphine is unable to either elicit mu OR phosphorylation or stimulate beta-arrestin translocation. However, upon the overexpression of GRK2, morphine gains the capacity to induce mu OR phosphorylation, accompanied by the rescue of beta-arrestin translocation and receptor sequestration. Moreover, overexpression of GRK2 also leads to an attenuation of morphine-mediated inhibition of adenylyl cyclase. These findings point to the existence of marked differences in the ability of different opioid agonists to promote mu OR phosphorylation by GRK. These differences may provide the molecular basis underlying the different analgesic properties of opioid agonists and contribute to the distinct ability of various opioids to induce drug tolerance.

Figure 1

Agonist-mediated internalization of the μ-opioid receptor. (A) Effect of wild-type and mutant GRK2, β-arrestin1, or dynamin I on etorphine-promoted internalization of the μOR. (B) Effect of wild-type and mutant GRK2 and/or β-arrestin1 on morphine-promoted internalization of the μOR. HEK 293 cells were transiently transfected with plasmids containing cDNAs for HA epitope-tagged μOR together with empty vector (Control) or various other DNA constructs as indicated. Data represent mean ± SE of three to five independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 vs. control μOR sequestration.

Figure 2

Differential phosphorylation of the μ-opioid receptor in response to etorphine and morphine in the absence or presence of overexpressing GRK2. HA epitope-tagged μORs were transiently expressed in HEK 293 cells together with or without cotransfected GRK2. The cells were then treated with serum-free medium (−) or medium containing agonists etorphine (E) or morphine (M) as described. (A) Autoradiograph from a representative experiment showing the whole cell phosphorylation of the μOR in HEK 293 cells in the absence and presence of overexpressing GRK2 in response to etorphine (E) and morphine (M). (B) Mean ± SE of three independent experiments quantified by PhosphorImager analysis. Data were normalized to the etorphine-induced μOR phosphorylation in the absence of GRK2. Morphine-induced μOR phosphorylation in the absence of GRK2 is significantly different from etorphine-induced μOR phosphorylation without GRK2 (P < 0.001), as well as morphine-induced μOR phosphorylation in the presence of GRK2 (P < 0.05).

Figure 3

Differential βarr2/GFP translocation in response to μOR activation by etorphine (A) or morphine (B) in the absence and presence of overexpressing GRK2 or GRK-K220M. HEK 293 cells were transiently transfected to express βarr2/GFP and μOR together with or without (Control) GRK2 or GRK2-K220M. Experiments were done on a heated microscope stage set at 30°C. Shown are representative confocal microscopic images of βarr2/GFP fluorescence obtained before (− Etorphine, − Morphine) and 10 min following the addition of etorphine (+ Etorphine) or morphine (+ Morphine) to the medium. Experiments were performed independently on three to five different occasions and each time four to six cells from independent stimulation by each agonist were recorded.

Figure 4

Effect of overexpressing GRK2 on μOR-mediated inhibition of adenylyl cyclase activity by morphine. HEK 293 cells were transiently transfected to express μOR and adenylyl cyclase type V in the absence (•, control) or presence (○) of GRK2. Whole cell adenylyl cyclase activity was measured as described. (A) Dose-dependent inhibition of adenylyl cyclase activity by morphine. (B) Time course of morphine-induced inhibition of adenylyl cyclase activity. The results were normalized to the forskolin-stimulated cellular cyclase response. Data shown represent means ± SE of three independent experiments analyzed by graphpad prism software. **, P < 0.01 and ***, P < 0.001 vs. matched adenylyl cyclase activity in the absence of GRK2.