Cholesterol modulates ligand binding and G-protein coupling to serotonin(1A) receptors from bovine hippocampus.
The serotonin(1A) (5-HT(1A)) receptor is an important member of the superfamily of seven-transmembrane domain G-protein-coupled receptors. We have examined the modulatory role of cholesterol on the ligand binding activity and G-protein coupling of the bovine hippocampal 5-HT(1A) receptor by depleting cholesterol from native membranes using methyl-beta-cyclodextrin (MbetaCD). Removal of cholesterol from bovine hippocampal membranes using varying concentrations of MbetaCD results in a concentration-dependent reduction in specific binding of the agonist 8-OH-DPAT to 5-HT(1A) receptors. This is accompanied by alterations in binding affinity and sites obtained from analysis of binding data. Importantly, cholesterol depletion affected G-protein-coupling of the receptor as monitored by the GTP-gamma-S assay. The concomitant changes in membrane order were reported by changes in fluorescence polarization of membrane probes such as DPH and TMA-DPH, which are incorporated at different locations (depths) in the membrane. Replenishment of membranes with cholesterol led to recovery of ligand binding activity as well as membrane order to a considerable extent. Our results provide evidence, for the first time, that cholesterol is necessary for ligand binding and G-protein coupling of this important neurotransmitter receptor. These results could have significant implications in understanding the influence of the membrane lipid environment on the activity and signal transduction of other G-protein-coupled transmembrane receptors.
Pucadyil
Thomas J
TJ
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India.
Chattopadhyay
Amitabha
A
D017371:8-Hydroxy-2-(di-n-propylamino)tetralin / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002784:Cholesterol / Q000172:deficiency / Q000378:metabolism*; D003505:Cyclodextrins / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006624:Hippocampus / Q000378:metabolism*; D011869:Radioligand Assay; D044282:Receptor, Serotonin, 5-HT1A / Q000378:metabolism*; D058825:Serotonin 5-HT1 Receptor Agonists; D017366:Serotonin Receptor Agonists / Q000378:metabolism / Q000494:pharmacology; D014316:Tritium; D047392:beta-Cyclodextrins
D003505:Cyclodextrins; D005456:Fluorescent Dyes; D058825:Serotonin 5-HT1 Receptor Agonists; D017366:Serotonin Receptor Agonists; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D014316:Tritium; D044282:Receptor, Serotonin, 5-HT1A; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D017371:8-Hydroxy-2-(di-n-propylamino)tetralin; D002784:Cholesterol; D019204:GTP-Binding Proteins