Morphine-activated opioid receptors elude desensitization by beta-arrestin

Abstract

mu opioid receptors are targets of native opioid peptides and addictive analgesic drugs. A major clinical liability of opiate drugs is their ability to cause physiological tolerance. Individual opiates, such as morphine and etorphine, differ both in their ability to promote physiological tolerance and in their effects on receptor regulation by endocytosis. Here, we demonstrate that arrestins play a fundamental role in mediating this agonist-selective regulation and that morphine-activated mu receptors fail to undergo arrestin-dependent uncoupling from cognate G proteins. Thus, highly addictive opiate drugs elude a fundamental mode of physiological regulation that is mediated by agonist-specific interaction of opioid receptors with arrestins.

Figure 1

Mu opioid receptors were endocytosed in a Dyn-dependent manner. HEK293 cells expressing FLAG-epitope-tagged μ opioid receptors (μOR) were stained as described in Materials and Methods. (A) Receptors remained predominantly in the plasma membrane in the absence of agonist stimulation. (B) Cells were treated with 5 μM etorphine for 30 min at 37°C and stained. In the presence of etorphine, receptors were endocytosed as indicated by redistribution of antibody-labeled receptors from the plasma membrane to numerous endocytic vesicles visualized throughout the cytoplasm. (C) Cells were treated with 5 μM morphine for 30 min at 37°C and stained. In the presence of morphine, receptors remained in the plasma membrane. (D and E) HEK293 cells expressing the FLAG-tagged μ opioid receptor were transfected transiently with an HA-tagged dominant negative Dyn, K44E. Cells were then treated with 5 μM etorphine and stained for both receptor in red (D) and Dyn in green (E). Cells expressing K44E Dyn failed to endocytose the receptor (D and E, open arrows), whereas adjacent cells not expressing K44E Dyn did endocytose receptor (D and E, closed arrows). (F and G) Cells transiently transfected with HA-tagged wild-type Dyn also were treated and stained. HA-tagged wild-type Dyn did not affect receptor endocytosis, as cells expressing (F and G, open arrows) and not expressing (F and G, closed arrows) Dyn-endocytosed receptors. (H) Slides stained as above were coded, and receptor-containing vesicles were counted from cells expressing both wild-type and mutant Dyn as well as cells not expressing any additional Dyn.

Figure 2

Overexpression of β-arrestin could facilitate μ opioid receptor internalization in the presence of morphine. (A) HEK293 cells stably expressing FLAG-epitope-tagged μ opioid receptors were transiently transfected with a plasmid overexpressing β-arrestin and stained for both receptor in red (μOR) and arrestin in green. In the absence of agonist, β-arrestin overexpression failed to stimulate endocytosis of μ opioid receptors (Aa and Ab). In the presence of 5 μM etorphine, receptors were endocytosed in both cells overexpressing (Ac and Ad, closed arrows) and those expressing only endogenous arrestins (Ac and Ad, open arrows). Cells expressing endogenous arrestins and treated with 5 μM morphine (Ae and Af, open arrows) failed to endocytose the receptor. However, receptors were endocytosed in the presence of morphine in cells overexpressing β-arrestin (Ae and Af, closed arrows). (B) HEK293 cells stably expressing FLAG-epitope-tagged μ opioid receptors and stably overexpressing an EE-tagged version of β-arrestin were used to quantitate receptor internalization. Surface-biotinylated μ opioid receptors (lanes 1 and 6) were cleaved in the presence glutathione (lanes 2 and 7). In the absence of agonist, little endocytosis of receptors was observed in cells expressing endogenous levels of β-arrestin (lane 3) or in cells overexpressing β-arrestin (lane 8). Etorphine-induced endocytosis of μ opioid receptors was observed readily in both cell lines (lanes 4 and 9). Although morphine caused no detectable stimulation of receptor endocytosis in cells expressing β-arrestin at endogenous levels (cf. lanes 3 and 5), a substantial amount of morphine-induced endocytosis of receptors was observed in cells overexpressing β-arrestin (cf. lanes 8 and 10). (C) Quantitation of these effects demonstrated that etorphine-induced (ET) endocytosis observed at steady state (30 min) was <2-fold enhanced in cells overexpressing β-arrestin (bars 3 and 4). Overexpression of β-arrestin enhanced morphine (MS)-induced endocytosis of μ opioid receptors by >40-fold (bars 5 and 6), far in excess of the effect of β-arrestin on either agonist-independent or etorphine-induced endocytosis.

Figure 3

Morphine stimulated GTP exchange but failed to promote uncoupling of receptor from G protein and failed to desensitize adenylyl cyclase activity. (A) Cells expressing μ opioid receptor were pretreated for either 5 or 30 min with DAMGO (DG) or morphine (MS) or left untreated and residual agonist washed from cells. Membranes were prepared from these cells and the ability of the μ receptors in these membranes to activate GTP exchange on G proteins in vitro was measured. Receptors from cells that were not pretreated with agonist stimulated GTP exchange efficiently with both morphine and DAMGO (open bars). Receptors pretreated with DAMGO for either 5 or 30 min were very inefficient at promoting GTP exchange upon DAMGO stimulation (black bars), indicating that the μ opioid receptors in these cells had become uncoupled from their G proteins during the DAMGO pretreatment. Receptors pretreated with morphine were still as effective at stimulating GTP exchange upon DAMGO stimulation (shaded bars) indicating that pretreatment with morphine failed to uncouple the receptors from their G proteins. (B) Cells expressing both μ opioid receptor and EE-tagged β-arrestin were pretreated with morphine or left untreated and membranes prepared as above. Receptors from cells overexpressing arrestin (hatched bars) pretreated with morphine were significantly impaired in their ability to stimulate GTP exchange, differing markedly from cells expressing endogenous levels of arrestin (solid bars), demonstrating that overexpression of arrestin facilitated functional uncoupling of morphine-bound opioid receptors. These assays were done in triplicate three times with comparable results. (C) Cells expressing μ opioid receptor were pretreated for 5 min with DAMGO (DG) or morphine (MS) or left untreated, and residual agonist washed from cells. Membranes were prepared from these cells, and the ability of the μ receptors in these membranes to inhibit forskolin-stimulated adenylyl cyclase activity was measured. Receptors from cells that were not pretreated with agonist inhibited adenylyl cyclase efficiently with both morphine and DAMGO stimulation (white bars). Receptors from cells pretreated with DAMGO for 5 min were significantly impaired in their ability to inhibit adenylyl cyclase activity upon DAMGO stimulation (black bar). Receptors from cells pretreated with morphine were still as effective at inhibiting adenylyl cyclase upon DAMGO (shaded bar). (D) Cells expressing μ opioid receptor and overexpressing β-arrestin-EE were pretreated for 5 min with morphine (MS) or left untreated, and residual agonist washed from cells. Membranes were prepared and the ability of the μ receptors to inhibit forskolin-stimulated adenylyl cyclase activity was measured. Receptors from cells that were not pretreated with agonist inhibited adenylyl cyclase as efficiently as cells expressing endogenous arrestins. Cells overexpressing arrestin that were pretreated with morphine were significantly less efficient at inhibiting adenylyl cyclase than cells expressing endogenous levels of arrestins (cf. hatched and unhatched shaded bars). These assays were repeated twice in triplicate with similar results.

Figure 4

Overexpression of GRK2 facilitated morphine-induced receptor internalization. Cells stably transfected with FLAG-epitope-tagged μ opioid receptor were transiently transfected with GRK2. Cells were treated with morphine (C and D) or left untreated (A and B) and stained as for receptor (A and C) and GRK2 (B and D). In the absence of agonist, GRK2 overexpression failed to stimulate receptor endocytosis (A and B, open arrows). However, in the presence of morphine, overexpression of GRK2 facilitated receptor internalization (C and D, open arrows), even though adjacent cells expressing endogenous GRKs failed to endocytose receptor (C and D, closed arrows ).