Localization of the kappa opioid receptor in lipid rafts.
Lipid rafts are microdomains of plasma membranes enriched in cholesterol and sphingolipids in the outer layer. We determined whether kappa opioid receptors (KOR) in human placenta and FLAG (DYKDDDDK)-tagged human KOR (FLAG-hKOR) expressed in Chinese hamster ovary (CHO) cells are localized in lipid rafts and whether 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 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, coinciding with high levels of caveolin-1 and cholesterol, markers of lipid rafts, which indicated that the KOR is localized in lipid rafts. Pretreatment with 2% methyl beta-cyclodextrin (MCD) reduced cholesterol content by approximately 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 the affinity of (-)-(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide (U50,488H) for the hKOR, and greatly increased U50,488H-induced [35S]guanosine 5'-O-(3-thio)triphosphate binding and p42/44 mitogen-activated protein kinase phosphorylation. Cholesterol replenishment reversed all the MCD effects. Caveolin-1 immunoprecipitated with Galphai proteins and MCD treatment reduced caveolin-1 associated with Galphai 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 affects coupling to G proteins.
Xu
Wei
W
Department of Pharmacology, Center for Substance Abuse Research, Temple University School of Medicine, 3420 N. Broad St., Philadelphia, PA 19140, USA.
Yoon
Su-In
SI
Huang
Peng
P
Wang
Yulin
Y
Chen
Chongguang
C
Chong
Parkson Lee-Gau
PL
Liu-Chen
Lee-Yuan
LY
D000818:Animals; D016466:CHO Cells; D051242:Caveolin 1 / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009842:Oligopeptides; D010455:Peptides; D010766:Phosphorylation; D010920:Placenta / Q000166:cytology / Q000378:metabolism; D021381:Protein Transport; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D017473:Receptors, Opioid, kappa / Q000378:metabolism*; D015398:Signal Transduction; D047392:beta-Cyclodextrins / Q000494:pharmacology
D051242:Caveolin 1; D008565:Membrane Proteins; D009842:Oligopeptides; D010455:Peptides; D043562:Receptors, G-Protein-Coupled; D017473:Receptors, Opioid, kappa; D047392:beta-Cyclodextrins; C106354:flotillins; C108732:methyl-beta-cyclodextrin; D002784:Cholesterol; C085706:FLAG peptide; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D019204:GTP-Binding Proteins