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Journal of Pharmacology And Experimental Therapeutics Fast Forward
First published on February 27, 2006; DOI: 10.1124/jpet.105.099507

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Received for publication December 6, 2005.
Revised February 23, 2006.
Accepted for publication February 23, 2006.

LOCALIZATION OF THE KAPPA OPIOID RECEPTOR IN LIPID RAFTS

Wei Xu 1, Su-In Yoon 2, Peng Huang 1, Yulin Wang 1, Chongguang Chen 1, Parkson Lee-Gau Chong 2, Lee-Yuan Liu-Chen 3*

1 Dept of Pharmacology, Temple University School of Medicine 2 Dept of Biochemistry, Temple Universiy School of Medicine 3 Dept of Pharmacology, Temple Universiy School of Medicine

* Address correspondence to: E-mail: lliuche{at}temple.edu

Abstract

Abstract Lipid rafts are microdomains of plasma membranes enriched in cholesterol and sphingolipids in the outer layer. We determined if {kappa} opioid receptors (KOR) in human placenta and FLAG-tagged human KOR (FLAG-hKOR) expressed in CHO cells are localized in lipid rafts and if changes in cholesterol contents affect hKOR properties and signaling. Lipid rafts were prepared from placenta membranes and CHO cells expressing FLAG-hKOR using the Na2CO3 method of Song et al. (1996) and fractionation through a sucrose density gradient. The majority of the KOR in the placenta and FLAG-hKOR in CHO cells, determined by [3H]diprenorphine binding and/or immunoblotting with an anti-FLAG antibody, was present in low density fractions, coincided with high levels of caveolin-1 and cholesterol, markers of lipid rafts, indicating that the KOR is localized in lipid rafts. Pretreatment with 2% methyl {beta}-cyclodextrin (MCD) reduced cholesterol content by ~48% and changed the cells from spindle-shaped to spherical. MCD treatment disrupted lipid rafts, shifted caveolin-1 and FLAG-hKOR to higher density fractions, increased affinity of U50,488H for the hKOR and greatly increased U50,488H-induced [35S]GTP{gamma}S binding and p42/44 MAP kinase phosphorylation. Cholesterol replenishment reversed all the MCD effects. Caveolin-1 immunoprecipitated with G{alpha}iproteins and MCD treatment reduced caveolin-1 associated with G{alpha}i proteins, which may contribute to the enhanced agonist-induced G protein activation. Caveolin-1 also immunoprecipitated with FLAG-hKOR, but MCD treatment had no effect on the association. Thus, the KOR is located in lipid rafts and its localization in the microdomains greatly impacts on coupling to G proteins.


Key words: Caveolins, Cholesterol, Gi proteins, Lipid rafts, MCD, Opioid receptor


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