Electron Paramagnetic Resonance Gives Evidence for the Presence of Type 1 Gonadotropin-Releasing Hormone Receptor (GnRH-R) in Subdomains of Lipid Rafts
Tilen Koklič,
Alenka Hrovat,
Ramon Guixà-González,
Ismael Rodríguez-Espigares,
Damaris Navio,
Robert Frangež,
Matjaž Uršič,
Valentina Kubale,
Ana Plemenitaš,
Jana Selent,
Marjeta Šentjurc,
Milka Vrecl
Affiliations
Tilen Koklič
Laboratory of Biophysics, Department of Condensed Matter Physics, Jožef Stefan Institute, 1000 Ljubljana, Slovenia
Alenka Hrovat
Veterinary Faculty, Institute of Preclinical Sciences, University of Ljubljana, Gerbičeva 60, 1000 Ljubljana, Slovenia
Ramon Guixà-González
Research Programme on Biomedical Informatics (GRIB), Department of Experimental and Health Sciences, Hospital del Mar Medical Research Institute (IMIM), Pompeu Fabra University (UPF), 08003 Barcelona, Spain
Ismael Rodríguez-Espigares
Research Programme on Biomedical Informatics (GRIB), Department of Experimental and Health Sciences, Hospital del Mar Medical Research Institute (IMIM), Pompeu Fabra University (UPF), 08003 Barcelona, Spain
Damaris Navio
Research Programme on Biomedical Informatics (GRIB), Department of Experimental and Health Sciences, Hospital del Mar Medical Research Institute (IMIM), Pompeu Fabra University (UPF), 08003 Barcelona, Spain
Robert Frangež
Veterinary Faculty, Institute of Preclinical Sciences, University of Ljubljana, Gerbičeva 60, 1000 Ljubljana, Slovenia
Matjaž Uršič
Veterinary Faculty, Institute of Preclinical Sciences, University of Ljubljana, Gerbičeva 60, 1000 Ljubljana, Slovenia
Valentina Kubale
Veterinary Faculty, Institute of Preclinical Sciences, University of Ljubljana, Gerbičeva 60, 1000 Ljubljana, Slovenia
Ana Plemenitaš
Faculty of Medicine, Institute of Biochemistry and Molecular Genetics, University of Ljubljana, 1000 Ljubljana, Slovenia
Jana Selent
Research Programme on Biomedical Informatics (GRIB), Department of Experimental and Health Sciences, Hospital del Mar Medical Research Institute (IMIM), Pompeu Fabra University (UPF), 08003 Barcelona, Spain
Marjeta Šentjurc
Laboratory of Biophysics, Department of Condensed Matter Physics, Jožef Stefan Institute, 1000 Ljubljana, Slovenia
Milka Vrecl
Veterinary Faculty, Institute of Preclinical Sciences, University of Ljubljana, Gerbičeva 60, 1000 Ljubljana, Slovenia
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.
