G protein-coupled receptors (GPCRs), the largest class of therapeutic targets, are profoundly influenced by their surrounding lipid environment. This lecture explores our recent findings on how membrane cholesterol and various steroidal compounds—including neurosteroids and steroid hormones—allosterically modulate the function of muscarinic and opioid receptors.
Using molecular dynamics simulations and site-directed mutagenesis, we identified a novel, non-canonical binding site at the intracellular half of transmembrane helix 6. We demonstrate that cholesterol, steroid hormones like progesterone and corticosterone, and synthetic neuroactive steroids compete for this common site to differentially modulate receptor activity and ligand binding. Furthermore, to overcome the limitations of traditional radioligand assays in studying lipid-GPCR interactions, we developed novel steroid-based fluorescence-quenching probes tagged with Tide Quencher 1 (TQ1). These probes enable real-time kinetic studies and mutational mapping of cholesterol-binding sites. Our research underscores the physiological significance of these interactions in conditions like stress or pregnancy and highlights the potential for developing tissue-specific, sterol-based pharmacotherapies.
Biography: Jan Jakubík, PhD, is Head of the Laboratory of Neurochemistry at IPHYS. He is a neuropharmacologist specialising in GPCR signalling, cholinergic neurotransmission, receptor theory, and allosteric modulation, with emphasis on biased agonism and lipid regulation of receptor function. He earned his PhD from Charles University and completed research fellowships at the NIH, University of Minnesota, and University of Tokyo. He has authored over 75 publications, which have led to key advances in muscarinic receptor pharmacology, like conceptualisation of signalling bias at the G-protein level, discoveries of allosteric agonists, positive allosteric modulators of acetylcholine and long-acting antagonists, and elucidation of cholesterol-dependent receptor modulation.
Contact at IPHYS: Jan Jakubík, jan.jakubik@fgu.cas.cz
