Structural and dynamic parameters of PM are strongly influenced by cholesterol. Besides that, cholesterol is an important component of membrane domains which are involved in the regulation of distribution and function of membrane proteins including GPCR. We study the role of membrane domains in GPCR signaling by means of biochemical methods. In parallel, we analyze the structure and function of cell membrane by methods of fluorescence spectroscopy (steady-state and time-resolved fluorescence polarization measurements).

The effect of the disruption of plasma membrane integrity on GPCR mobility in living cells is studied using the advanced methods of fluorescence microscopy, e.g., Fluorescence Recovery After Photobleaching (FRAP) or Raster Image Correlation Spectroscopy (RICS), as illustrates Fig. 1 - see the publication Brejchová et al. (2015) focusing on the effect of cholesterol depletion on the mobility and function of thyrotropin-releasing hormone receptor (TRH-R).

Using fluorescence microscopy (Confocal Laser Scanning Microscopy, CLSM), we also studied the effect of cholesterol depletion on the internalization of δ-opioid receptor (δ-OR). Results of this study were recently published (Brejchová et al. 2016) and are illustrated by Fig. 2. (For further information please see also publications Brejchová et al., 2011; Ostašov et al., 2013; Roubalová et al., 2015 and PhD Thesis of J. Brejchová, 2014.)

Fig. 1. The effect of cholesterol depletion on the mobility and function of TRH-R

Left panel: Distribution of TRH-R in HEK293 cells stably expressing fusion protein of TRH-R with green fluorescent protein (TRH-R-eGFP) before (control) and after cholesterol depletion induced by incubation of cells with β-cyclodextrin. Middle panel: Values of the apparent diffusion coefficient (D_app) of TRH-R-eGFP in living control (CTR) and cholesterol-depleted (β-CDX) cells which were determined by FRAP. Right panel: Result of the analysis of the effect of cholesterol depletion on the ability of TRH-R to activate cognate G-proteins as determined by high-affinity [³⁵S]GTPγS binding assay.

Fig. 2. The effect of cholesterol depletion on agonist-induced internalization of δ-OR - HEK293 cells transiently expressing FLAG-δ-OR

Left panel: Representative micrographs displaying the distribution of FLAG-δ-OR in control cells (control) and cells treated with β-cyclodextrin (β-CDX), agonist (DADLE), and both substances together (DADLE + β-CDX). Right panel: Values of fraction of internalized receptors determined by micrographs quantification performed with ImageJ software.