Cys170 is the palmitoylation site of OPRM1. (a) Palmitoylation assays were performed in HEK and HEKOPRM1 cells. The amounts of palmitoylated receptor were normalized against that in HEKOPRM1 (Lane 2). 50 μM 2-BP was used to treat HEKOPRM1 for 12 h (Lane 3). Individual steps (treatment with NEM, hydroxylamine, or btn-BMCC) were omitted to validate the assay (Lanes 4-6). (b) The palmitoylation assay was performed in HEK, HEKOPRM1, HEKOPRM1 treated with 50 μM 2-BP for 12 h, HEKC170A, HEKC346A, and HEKC351A cells. The amounts of palmitoylated receptor were normalized against that in HEKOPRM1 (Lane 2). (c-e) Membrane receptor levels were determined with binding assay (c), FACS (d), and immunoblotting (e). (f) HEKOPRM1 cells were treated with PBS (C), 1 μM morphine (M) and 10 nM fentanyl (F) for 5 min. Receptor palmitoylation was then determined. (g) HEKOPRM1, HEKOPRM1 treated with 50 μM 2-BP for 12 h, and HEKC170A cells were treated with 1 μM morphine for 5 min. Phosphorylated ERK and total-ERK were determined by immunoblotting. One-way ANOVA with Dunnett's test as a post-hoc test was used for analysis. The error bars and "*" represent the standard deviations and significant changes (p < 0.05, n > 3), respectively.
Zheng et al. BMC Cell Biology 2012 13:6 doi:10.1186/1471-2121-13-6