We are studying the effect of a toxin on cell membrane lipids which binds to receptors RPTP-alpha & beta but their localization in lipid rafts of cell membrane is not well characterized, so, how to study this?
Lipid rafts are microdomains of plasma membranes enriched in cholesterol and sphingolipids in the outer layer. 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.