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Research ArticleArticle

Role of G Protein–Coupled Receptor Kinases 2 and 3 in μ-Opioid Receptor Desensitization and Internalization

Janet D. Lowe, Helen S. Sanderson, Alexandra E. Cooke, Mehrnoosh Ostovar, Elena Tsisanova, Sarah L. Withey, Charles Chavkin, Stephen M. Husbands, Eamonn Kelly, Graeme Henderson and Chris P. Bailey
Molecular Pharmacology August 2015, 88 (2) 347-356; DOI: https://doi.org/10.1124/mol.115.098293
Janet D. Lowe
School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (J.D.L., H.S.S., A.E.C., E.T., S.L.W., E.K., G.H.); Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington (C.C.); and Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom (M.O., S.M.H., C.P.B.)
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Helen S. Sanderson
School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (J.D.L., H.S.S., A.E.C., E.T., S.L.W., E.K., G.H.); Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington (C.C.); and Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom (M.O., S.M.H., C.P.B.)
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Alexandra E. Cooke
School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (J.D.L., H.S.S., A.E.C., E.T., S.L.W., E.K., G.H.); Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington (C.C.); and Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom (M.O., S.M.H., C.P.B.)
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Mehrnoosh Ostovar
School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (J.D.L., H.S.S., A.E.C., E.T., S.L.W., E.K., G.H.); Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington (C.C.); and Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom (M.O., S.M.H., C.P.B.)
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Elena Tsisanova
School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (J.D.L., H.S.S., A.E.C., E.T., S.L.W., E.K., G.H.); Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington (C.C.); and Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom (M.O., S.M.H., C.P.B.)
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Sarah L. Withey
School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (J.D.L., H.S.S., A.E.C., E.T., S.L.W., E.K., G.H.); Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington (C.C.); and Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom (M.O., S.M.H., C.P.B.)
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Charles Chavkin
School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (J.D.L., H.S.S., A.E.C., E.T., S.L.W., E.K., G.H.); Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington (C.C.); and Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom (M.O., S.M.H., C.P.B.)
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Stephen M. Husbands
School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (J.D.L., H.S.S., A.E.C., E.T., S.L.W., E.K., G.H.); Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington (C.C.); and Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom (M.O., S.M.H., C.P.B.)
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Eamonn Kelly
School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (J.D.L., H.S.S., A.E.C., E.T., S.L.W., E.K., G.H.); Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington (C.C.); and Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom (M.O., S.M.H., C.P.B.)
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Graeme Henderson
School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (J.D.L., H.S.S., A.E.C., E.T., S.L.W., E.K., G.H.); Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington (C.C.); and Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom (M.O., S.M.H., C.P.B.)
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Chris P. Bailey
School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom (J.D.L., H.S.S., A.E.C., E.T., S.L.W., E.K., G.H.); Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington (C.C.); and Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom (M.O., S.M.H., C.P.B.)
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    Fig. 1.

    Inhibition of MOPr desensitization by Cmpd101 in rat LC neurons. Traces in (A), (D), (G), and (J) show outward potassium currents recorded from rat LC neurons in response to receptor-saturating concentrations of Met-Enk (30 μM), DAMGO (10 μM), endomorphin-2 (10 μM), and morphine (morph; 30 μM). The Met-Enk, DAMGO, and endomorphin-2 responses desensitized rapidly over the 10 minutes of agonist application. The desensitization induced by morphine was less than that produced by the other agonists and was measured after 15 minutes of morphine application. Agonist responses returned to baseline after washout (Met-Enk) or when naloxone (nalox; 1 μM) was applied. The middle column of traces, (B), (E), (H), and (K), show currents induced by each agonist in slices exposed to Cmpd101 (30 μM) for at least 15 minutes before and during the application of the opioid agonists. Traces in (C), (F), (I), and (L) show pooled data for the percentage desensitization after 10 minutes of Met-Enk, DAMGO, or endomorphin-2 application and 15 minutes of morphine application from experiments such as those illustrated in the two columns of experimental traces. Cmpd101 significantly inhibited the desensitization of all four agonists. Met-Enk: n = 5 for all experiments; DAMGO: n = 4 for all experiments; endomorphin-2: n = 6 for all experiments; morphine: n = 6 for all experiments. *P < 0.05, analysis of variance compared with the appropriate control. All scale bars, 50 pA, 2 minutes. GF, GF109203X.

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    Fig. 2.

    Effect of GRK3 KO and Cmpd101 on Met-Enk–induced MOPr desensitization in mouse LC neurons. (A) Representative potassium currents in response to a receptor-saturating concentration of Met-Enk (30 μM) recorded from mouse LC neurons in slices taken from either WT (top) or GRK3 KO (bottom) mouse brains. (B) Pooled data from experiments as illustrated in (A). The desensitization induced by Met-Enk over a 10-minute application was not different in GRK3 KO mice compared with WT littermate controls (GRK3 WT, n = 6; GRK3 KO, n = 6; t test, P > 0.05). (C) Representative potassium currents in response to Met-Enk (30 μM) recorded from LC neurons from slices taken from C57BL/6J mice that were either untreated (top) or pretreated with Cmpd101 (30 μM) for at least 15 minutes prior to and during the application of Met-Enk (bottom). (D) Pooled data for the percentage desensitization over the 10 minutes of opioid agonist application from experiments such as those illustrated in (C). Cmpd101 (3 and 30 μM) significantly inhibited Met-Enk–induced desensitization measured after 10 minutes of agonist application. n = 5 for each; *P < 0.05, analysis of variance compared with control. All scale bars, 15 pA, 2 minutes.

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    Fig. 3.

    Cmpd101 reduced the depression of the maximum response to morphine produced by DAMGO-induced desensitization. (A and B) Representative potassium current traces showing the amplitude of the maximum response to morphine (morph) compared with that of NA (100 μM) in the absence of (A) or after induction of desensitization induced by application of (B) DAMGO (10 μM for 12 minutes). The opioid antagonist naloxone (nalox, 1 μM) was added after morphine to bring the response back to baseline prior to application of NA. (C) Representative current trace from an experiment in which slices were exposed to Cmpd101 (30 μM) for at least 15 minutes before and during the application of the opioid agonists. (D) Pooled data from experiments as illustrated in (A–C). DAMGO-induced desensitization inhibited the maximum response to morphine. Cmpd101 concentration-dependently reversed the DAMGO desensitization–induced decrease in the morphine response. n = 4 for all experiments; *P < 0.05, analysis of variance. All scale bars, 60 pA, 2 minutes.

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    Fig. 4.

    Inhibition of DAMGO-induced MOPr phosphorylation and arrestin recruitment by Cmpd101. (A) HEK 293 cells stably expressing HA-tagged rat MOPr were pretreated with Cmpd101 for 30 minutes prior to stimulation with DAMGO (10 μM for 5 minutes). Agonist-induced phosphorylation was assessed by Western blot analysis using an antibody targeting phospho-Ser375 (pS375). Anti-HA and anti-tubulin antibodies confirmed equal loading of the gels. (B) Western blots as illustrated in (A) were quantified by densitometry and expressed as a percentage of the maximal phosphorylation in response to DAMGO (10 μM) in each experiment. Cmpd101 (30 μM) abolished DAMGO-induced MOPr phosphorylation at Ser375 (n = 3; ***P < 0.001, analysis of variance (ANOVA) compared with control + DAMGO). No phosphorylation was seen under control conditions or with Cmpd101 alone (n = 3). (C) DAMGO-induced arrestin-3 translocation to the receptor was measured using the DiscoveRx PathHunter assay. DAMGO (10 μM) application produced a robust recruitment of arrestin-3 to the receptor that was significantly inhibited in cells that were pretreated with Cmpd101 (30 μM) for 30 minutes (n = 3; *P < 0.05, ANOVA). (D) Internalization of HA-MOPrs expressed in HEK 293 cells assessed by ELISA using an anti-HA antibody to label surface receptors. DAMGO (10 μM) induced a time-dependent loss of surface receptors that was significantly inhibited by Cmpd101 (n = 3–4; *P < 0.05, ANOVA compared with control). (E) Confocal images of HA-MOPrs following incubation with anti-HA antibody and fluorescein-tagged secondary antibody (green), counterstained with Hoechst 33258 nucleic acid stain (blue) following incubation with DAMGO (10 μM) and/or Cmpd101 (30 μM). Images are from one experiment repeated 3 times. Scale bar, 10 μM.

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    Fig. 5.

    Effect of Cmpd101 on the phosphorylation of ERK1/2 and Elk-1. (A) ERK1/2 activity in HEK 293 cells expressing HA-MOPrs was assessed by Western blot using an antibody targeting phospho-ERK1/2. DAMGO (10 μM) application for 5 minutes produced a robust phosphorylation of ERK1/2 in both control cells and cells that had been pretreated with Cmpd101 for 30 minutes. Antibodies against total ERK1/2 and tubulin confirmed equal loading of the gels. (B) Western blots as illustrated in (A) were quantified by densitometry and expressed as a percentage of the maximal ERK1/2 phosphorylation in response to DAMGO (10 μM) in each experiment. Cmpd101 did not affect DAMGO-induced ERK1/2 phosphorylation (P > 0.05, analysis of variance), but at the higher 30 μM concentration, Cmpd101 produced a modest but significant activation of ERK1/2 on its own (P < 0.05, one-sample t test versus control; n = 4 for each). (C) Phosphorylation of the ERK1/2 substrate Elk-1 in response to DAMGO was assessed by Western blot using an antibody targeting pSer383 (of Elk-1). Treatment of HA-MOPr cells with DAMGO (10 μM) for 5 minutes produced phosphorylation of Elk-1, which was unaffected by pretreatment with Cmpd101 (30 μM) for 30 minutes. In contrast, agonist-dependent phosphorylation of both Elk-1 and ERK1/2 was significantly reduced following pretreatment of 30 minutes with the MEK1 inhibitor PD98059 (10 μM). Antibodies against total ERK1/2 and tubulin confirmed equal loading of the gels. (D) Western blots of pElk-1 as shown in (C) were quantified by densitometry and expressed as a percentage of the maximal Elk-1 phosphorylation in response to DAMGO (10 μM) in each experiment. Cmpd101 did not reduce DAMGO-induced Elk-1 phosphorylation at a concentration of 30 μM (n = 3). (E) The ERK1/2 inhibitor PD98059 (10 μM) reduced both DAMGO-induced pElk-1 and pERK1/2 activation to basal levels. Data as in (C) were quantified by densitometry. *P < 0.05, one-sample t test versus control; n = 3.

Additional Files

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    Files in this Data Supplement:

    • Supplemental Data -

      Supplementary Data Table 1 - Inhibition of protein kinases by Compound 101

      Supplemental Figure 1 - Supplementary Data Table 1. Inhibition of protein kinases by Compound 101

      Supplemental Figure 2 - DAMGO-induced MOPr desensitization was unaffected by inhibition of off-target kinases inhibited by Compound 101

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Molecular Pharmacology: 88 (2)
Molecular Pharmacology
Vol. 88, Issue 2
1 Aug 2015
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Research ArticleArticle

Role of GRKs in MOPr Desensitization

Janet D. Lowe, Helen S. Sanderson, Alexandra E. Cooke, Mehrnoosh Ostovar, Elena Tsisanova, Sarah L. Withey, Charles Chavkin, Stephen M. Husbands, Eamonn Kelly, Graeme Henderson and Chris P. Bailey
Molecular Pharmacology August 1, 2015, 88 (2) 347-356; DOI: https://doi.org/10.1124/mol.115.098293

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Research ArticleArticle

Role of GRKs in MOPr Desensitization

Janet D. Lowe, Helen S. Sanderson, Alexandra E. Cooke, Mehrnoosh Ostovar, Elena Tsisanova, Sarah L. Withey, Charles Chavkin, Stephen M. Husbands, Eamonn Kelly, Graeme Henderson and Chris P. Bailey
Molecular Pharmacology August 1, 2015, 88 (2) 347-356; DOI: https://doi.org/10.1124/mol.115.098293
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