Cholesterol modulates ligand binding and G-protein coupling to serotonin(1A) receptors from bovine hippocampus

Biochim Biophys Acta. 2004 May 27;1663(1-2):188-200. doi: 10.1016/j.bbamem.2004.03.010.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 8-Hydroxy-2-(di-n-propylamino)tetralin / metabolism
  • 8-Hydroxy-2-(di-n-propylamino)tetralin / pharmacology
  • Animals
  • Cattle
  • Cell Membrane / chemistry
  • Cell Membrane / metabolism
  • Cholesterol / deficiency
  • Cholesterol / metabolism*
  • Cyclodextrins / pharmacology
  • Dose-Response Relationship, Drug
  • Fluorescence Polarization
  • Fluorescent Dyes / chemistry
  • Fluorescent Dyes / metabolism
  • GTP-Binding Proteins / metabolism*
  • Guanosine 5'-O-(3-Thiotriphosphate) / metabolism
  • Hippocampus / metabolism*
  • Radioligand Assay
  • Receptor, Serotonin, 5-HT1A / metabolism*
  • Serotonin 5-HT1 Receptor Agonists
  • Serotonin Receptor Agonists / metabolism
  • Serotonin Receptor Agonists / pharmacology
  • Tritium
  • beta-Cyclodextrins*

Substances

  • Cyclodextrins
  • Fluorescent Dyes
  • Serotonin 5-HT1 Receptor Agonists
  • Serotonin Receptor Agonists
  • beta-Cyclodextrins
  • methyl-beta-cyclodextrin
  • Tritium
  • Receptor, Serotonin, 5-HT1A
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • 8-Hydroxy-2-(di-n-propylamino)tetralin
  • Cholesterol
  • GTP-Binding Proteins