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BMC Cell Biol. 2012; 13: 6.
Published online Mar 19, 2012. doi:  10.1186/1471-2121-13-6
PMCID: PMC3317874
Palmitoylation and membrane cholesterol stabilize μ-opioid receptor homodimerization and G protein coupling
Hui Zheng,corresponding author1,2 Elizabeth A Pearsall,3 Dow P Hurst,3 Yuhan Zhang,1 Ji Chu,1 Yali Zhou,1 Patricia H Reggio,3 Horace H Loh,1 and Ping-Yee Law1
1Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455
2Stem Cell and Cancer Biology Group, Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
3Department of Chemistry and Biochemistry, Center for Drug Discovery, University of North Carolina, Greensboro, North Carolina 27402
corresponding authorCorresponding author.
Hui Zheng: zhenhui2055/at/hotmail.com; Elizabeth A Pearsall: biochemliz/at/gmail.com; Dow P Hurst: dphurst/at/uncg.edu; Yuhan Zhang: emarald_cici/at/hotmail.com; Ji Chu: chuxji/at/hotmail.com; Yali Zhou: zyl2326/at/hotmail.com; Patricia H Reggio: PHREGGIO/at/uncg.edu; Horace H Loh: lohxx001/at/umn.edu; Ping-Yee Law: lawxx001/at/umn.edu
Received September 19, 2011; Accepted March 19, 2012.
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.
Keywords: Palmitoylation, Cholesterol, Homodimerization, G protein coupling
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