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Abstract
Insolubility in non-ionic detergents such as Triton X-100 at low temperature is a widely used biochemical criterion for characterization of membrane domains. In view of the emerging role of membrane organization in the function of G-protein coupled receptors, we have examined detergent insolubility of the 5-HT1A receptor in CHO cells using a novel GFP fluorescence approach developed by us. Using this approach, we have explored the membrane organization of the serotonin1A receptor tagged to enhanced yellow fluorescent protein (5-HT1AR-EYFP) stably expressed in CHO-K1 cells under conditions of varying detergent concentration, reduced membrane cholesterol and agonist stimulation. Our results show that a small yet significant fraction of the 5-HT1A receptor exhibits detergent insolubility, which increases upon depletion of membrane cholesterol. Stimulation of 5-HT1AR-EYFP by its endogenous ligand, serotonin, did not cause a significant change in the detergent insolubility of the receptor. Taken together, our results on detergent insolubility of 5-HT1AR-EYFP provide new insights into the membrane organization of the 5-HT1A receptor and could be relevant in the analysis of membrane organization of other G-protein coupled receptors.
| Acronyms | ||
| 5-hydroxytryptamine | = | 5-HT |
| 5-hydroxytryptamine-1A receptor | = | 5-HT1A receptor |
| 5-hydroxytryptamine-1A receptor tagged to EYFP | = | 5-HT1AR-EYFP |
| β1-adrenergic receptor | = | β1AR |
| β2-adrenergic receptor | = | β2AR |
| adenylate cyclase | = | AC |
| bicinchoninic acid | = | BCA |
| Chinese hamster ovary cells | = | CHO cells |
| detergent resistant membrane | = | DRM |
| dialkylindocarbocyanine | = | DiI |
| 1,1′-dihexadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate | = | DiIC16 |
| enhanced yellow fluorescent protein | = | EYFP |
| 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate | = | FAST DiI |
| green fluorescent protein | = | GFP |
| G-protein coupled receptor | = | GPCR |
| glycosylphosphoinositol | = | GPI |
| methyl-β-cyclodextrin | = | MβCD |
| Acronyms | ||
| 5-hydroxytryptamine | = | 5-HT |
| 5-hydroxytryptamine-1A receptor | = | 5-HT1A receptor |
| 5-hydroxytryptamine-1A receptor tagged to EYFP | = | 5-HT1AR-EYFP |
| β1-adrenergic receptor | = | β1AR |
| β2-adrenergic receptor | = | β2AR |
| adenylate cyclase | = | AC |
| bicinchoninic acid | = | BCA |
| Chinese hamster ovary cells | = | CHO cells |
| detergent resistant membrane | = | DRM |
| dialkylindocarbocyanine | = | DiI |
| 1,1′-dihexadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate | = | DiIC16 |
| enhanced yellow fluorescent protein | = | EYFP |
| 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate | = | FAST DiI |
| green fluorescent protein | = | GFP |
| G-protein coupled receptor | = | GPCR |
| glycosylphosphoinositol | = | GPI |
| methyl-β-cyclodextrin | = | MβCD |