Electron Paramagnetic Resonance Gives Evidence for the Presence of Type 1 Gonadotropin-Releasing Hormone Receptor (GnRH-R) in Subdomains of Lipid Rafts.

Tilen Koklič,Valentina Kubale,Milka Vrecl,Marjeta Šentjurc,Ismael Rodríguez-Espigares,Ramon Guixà-González,Jana Selent,Alenka Hrovat,Ana Plemenitaš,Damaris Navio,Robert Frangež,Matjaž Uršič

doi:10.3390/molecules26040973

Abstract

This study investigated the effect of type 1 gonadotropin releasing hormone receptor (GnRH-R) localization within lipid rafts on the properties of plasma membrane (PM) nanodomain structure. Confocal microscopy revealed colocalization of PM-localized GnRH-R with GM1-enriched raft-like PM subdomains. Electron paramagnetic resonance spectroscopy (EPR) of a membrane-partitioned spin probe was then used to study PM fluidity of immortalized pituitary gonadotrope cell line αT3-1 and HEK-293 cells stably expressing GnRH-R and compared it with their corresponding controls (αT4 and HEK-293 cells). Computer-assisted interpretation of EPR spectra revealed three modes of spin probe movement reflecting the properties of three types of PM nanodomains. Domains with an intermediate order parameter (domain 2) were the most affected by the presence of the GnRH-Rs, which increased PM ordering (order parameter (S)) and rotational mobility of PM lipids (decreased rotational correlation time (τc)). Depletion of cholesterol by methyl-β-cyclodextrin (methyl-β-CD) inhibited agonist-induced GnRH-R internalization and intracellular Ca2+ activity and resulted in an overall reduction in PM order; an observation further supported by molecular dynamics (MD) simulations of model membrane systems. This study provides evidence that GnRH-R PM localization may be related to a subdomain of lipid rafts that has lower PM ordering, suggesting lateral heterogeneity within lipid raft domains.

Highlights

The discovery of lipid rafts and other domains of the plasma membrane (PM) has led to a re-evaluation of the classical Singer-Nicolson fluidic mosaic model, which described cell membranes as a more or less homogeneous phospholipid bilayer [1]
R internalization and [Ca2+]i, we investigated the effect of receptor-lipid raft association on PM properties by Electron paramagnetic resonance spectroscopy (EPR) spectroscopy using MeFASL(10,3) as a spin probe
Partitioning/clustering of membrane proteins in specific PM nanodomains can lead to reorganization of nanodomain composition and/or their physical properties

Summary

Introduction

The discovery of lipid rafts and other domains of the plasma membrane (PM) has led to a re-evaluation of the classical Singer-Nicolson fluidic mosaic model, which described cell membranes as a more or less homogeneous phospholipid bilayer [1]. There is a growing consensus that the potential for lateral segregation of PM is based on the preferential association among sphingolipids, sterols, and specific proteins, which underlies the raft concept of PM subcompartmentalization (reviewed in [2]). Lipid rafts ( called lipid nanodomains) in the PM outer leaflet have been defined as small (10–200 nm), heterogeneous, and highly dynamic domains enriched with glycosphingolipids and cholesterol that compartmentalize cellular processes [3,4]. The tight packing of the saturated acyl chains of sphingolipids and the presence of cholesterol appear to result in a domain that is thicker, more ordered, and less fluid than the surrounding phosphatidylcholine-enriched PM environment. Protein interactions with scaffolding raft-resident proteins—e.g., caveolin—transmembrane domain (TM) length, oligomerization, and preferential interaction of protein segments at the membrane interface with certain lipid components could be responsible for lipid raft association (reviewed in [5,7,8,9])

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Results

Conclusion