Palmitoylation and membrane cholesterol stabilize μ-opioid receptor homodimerization and G protein coupling

Abstract

Background: A cholesterol-palmitoyl interaction has been reported to occur in the dimeric interface of the β₂-adrenergic receptor crystal structure. We sought to investigate whether a similar phenomenon could be observed with μ-opioid receptor (OPRM1), and if so, to assess the role of cholesterol in this class of G protein-coupled receptor (GPCR) signaling.

Results: C3.55(170) was determined to be the palmitoylation site of OPRM1. Mutation of this Cys to Ala did not affect the binding of agonists, but attenuated receptor signaling and decreased cholesterol associated with the receptor signaling complex. In addition, both attenuation of receptor palmitoylation (by mutation of C3.55[170] to Ala) and inhibition of cholesterol synthesis (by treating the cells with simvastatin, a HMG-CoA reductase inhibitor) impaired receptor signaling, possibly by decreasing receptor homodimerization and Gαi2 coupling; this was demonstrated by co-immunoprecipitation, immunofluorescence colocalization and fluorescence resonance energy transfer (FRET) analyses. A computational model of the OPRM1 homodimer structure indicated that a specific cholesterol-palmitoyl interaction can facilitate OPRM1 homodimerization at the TMH4-TMH4 interface.

Conclusions: We demonstrate that C3.55(170) is the palmitoylation site of OPRM1 and identify a cholesterol-palmitoyl interaction in the OPRM1 complex. Our findings suggest that this interaction contributes to OPRM1 signaling by facilitating receptor homodimerization and G protein coupling. This conclusion is supported by computational modeling of the OPRM1 homodimer.

Figure 1

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.

Figure 2

Palmitoylation contributes to Gαi2 coupling. (a) The colocalization between HA-tagged receptor and Gαi2 was determined in HEKOPRM1 and HEKC170A cells. Images were analyzed as described in Methods. (b) Anti-HA antibody was used to precipitate receptors in HEKOPRM1 and HEKC170A cells. Gαi2 precipitated with receptors was quantified and normalized to that in HEKOPRM1cells. (c) Co-immunoprecipitation was performed with Anti-Gαi2 antibody and the precipitated receptor was quantified. (d) CFPOPRM1 or CFPC170A was transfected into HEK cells with YFPGαi2. FRET analysis was performed. A two-tailed student t-test was used. Error bars and "*" represent the standard deviations and significant changes (p < 0.05, n > 3 for b and c; n > 10 for a and d), respectively. IP, immunoprecipitation; IB, immunoblotting.

Figure 3

Palmitoylation stabilizes homodimerization. (a) FRET analysis was performed after transfecting combinations of CFPOPRM1/CFPC170A and YFPOPRM1/YFPC170A into HEK cells. (b-c) FLAGOPRM1/FLAGC170A and HAOPRM1/HAC170A were transfected into HEK cells. The colocalization between FLAG-tagged receptor and HA-tagged receptor was determined in (b). The amounts of HA-tagged receptor precipitated with Flag-tagged receptor were normalized against the amount of HAOPRM1 precipitated with FLAGOPRM1 and summarized in (c). 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.

Figure 4

Palmitoylation stabilizes Gαi2 coupling. (a) The FRET between CFPOPRM1 and YFPGαi2 and the FRET between CFPC170A and YFPGαi2 were determined in HEKOPRM1 and HEKC170A cells. (b-c) Colocalization between FLAGOPRM1 and Gαi2 (or FLAGC170A and Gαi2) was compared in HEKOPRM1 and HEKC170A cells after transfection (b). The amounts of Gαi2 precipitated with FLAGOPRM1 and FLAGC170A in the two cell lines were compared in (c). The amount of Gαi2 precipitated with FLAGOPRM1 in HEKOPRM1 cells was used for normalization. One-way ANOVA with Dunnett's test as a post-hoc test was used. The error bars and "*" represent the standard deviations and significant changes (p < 0.05, n > 3), respectively.

Figure 5

Palmitoylation facilitates cholesterol association. (a) Cholesterol associated with receptor complex was determined in HEK, HEKOPRM1 and HEKC170A cells. The amount of cholesterol precipitated with PBS in HEK cells was used for normalization. (b-c) HEKOPRM1 and HEKC170A cells were treated with PBS (Control), 0.5 μM simvastatin (Simva), or 0.5 μM simvastatin with 20 ng/ml cholesterol (Simva+Chol) for 12 h. Membrane cholesterol contents were determined in (b) as described in Methods. Cholesterol association with receptor complex was determined in (c). One-way ANOVA with Dunnett's test (b) or two-way ANOVA with Bonferroni's test (a, c) was used. The error bars and "*" represent the standard deviations and significant changes (p < 0.05, n > 3), respectively. N/S, no significance.

Figure 6

Reducing cellular cholesterol affects homodimerization and G protein coupling. (a) HEK cells were transfected with CFPOPRM1 and YFPOPRM1 or transfected with CFPC170A and YFPC170A for 24 h. These cells were than treated with PBS (Control), 0.5 μM simvastatin (Simva), or 0.5 μM simvastatin with 20 ng/ml cholesterol (Simva+Chol) for 12 h. The FRET was analyzed to determine the amount of homodimer. (b) HEK cells were transfected with CFPOPRM1 and YFPGαi2 or transfected with CFPC170A and YFPGαi2 for 24 h. These cells were than treated as in (a) and G protein coupling was determined with FRET. One-way ANOVA with Dunnett's test was used. The error bars and "*" represent the standard deviations and significant changes (p < 0.05, n > 3), respectively.

Figure 7

Palmitoylation inhibitor impairs homodimerization and cholesterol association. (a) HEKOPRM1 or HEKC170A cells were treated with 50 μM 2-BP or vehicle for 12 h, and the cholesterol associated with receptor complex was measured. (b) HEK cells were transfected with CFPOPRM1 and YFPOPRM1 or transfected with CFPC170A and YFPC170A for 24 h. 50 μM 2-BP or vehicle was used to treat the cells for additional 12 h. FRET was then determined. Two-way ANOVA with Bonferroni's test (a) or student t-test (b) was used. Error bars and "*" represent the standard deviations and significant changes (p < 0.05, n > 3), respectively.

Figure 8

Computational modeling of the OPRM1 homodimer interface. (a) This figure illustrates the position of cholesterol relative to the palmitoyl and the OPRM1 TMH bundle. The view is from lipid looking toward TMH4 (yellow). The OPRM1 model is displayed in molecular surface view, with cholesterol (green) and palmitoyl (orange) contoured at their VDW radii. TMH3 and TMH5 are in orange and cyan, respectively. (b) This figure illustrates an extracellular view of protomers A and B forming the OPRM1 homodimer TMH4 interface. Residues that form the interface (N4.41, I4.44, C4.48, I4.51, A4.55, and P4.59) are contoured at their VDW radii and colored magenta. Also contoured at their VDW radii are the palmitoyls (orange) and cholesterols (green). In this arrangement, cholesterols associated with one protomer also interact with the opposite protomer.