Abstract
Lipid rafts are cholesterol-rich plasma membrane nanodomains that facilitate the colocalisation of receptors with their associated proteins and enrich the surrounding membrane with cholesterol. This can alter the neural microenvironment and receptor signalling dynamics, potentially changing receptor conformations, with raft-associated cholesterol functioning akin to an allosteric modulator. The present study focuses on these ideas at the G protein-coupled cannabinoid CB1 receptor (CB1), a central modulator of neural processes and promising therapeutic target implicated in a variety of CNS disorders. The CB1 receptor is known to colocalise with lipid rafts in certain contexts and contains six cholesterol-binding sites which overlap with current CB1 allosteric modulators, including the well-characterised negative allosteric modulator ORG27569. The limited success of cannabinoid research in developing safe and effective CB1-targeted therapies, and the growing interest in CB1 allosteric modulators, provides rationale for a comprehensive characterisation of how cholesterol modulates CB1 receptor signalling. This research aimed to characterise the cholesterol environment of two morphologically distinct cell lines, one transfected with human CB1 and one endogenously expressing mouse CB1. It also aims to comprehensively assess how cholesterol modulates CB1 signalling kinetics in response to biologically relevant agonists and the allosteric modulator ORG27569. Cytochemical staining was used to localise and quantify cholesterol, and to determine the effects of methyl-β- cyclodextrin (MβCD), a membrane cholesterol modulator, in Human Embryonic Kidney 293 cells and Neuro-2A cells. This was coupled with kinetic BRET biosensor assays to measure cAMP accumulation, ERK1/2 phosphorylation, and β-arrestin translocation, as well as immunocytochemical analyses of receptor trafficking, both following stimulation with anandamide, 2-arachidonoylglycerol, (-)-trans-Δ9-tetrahydrocannabinol, or ORG27569. We observed cell line-specific differences in cholesterol localisation and quantity, with MβCD differentially affecting each. In signalling assays, MβCD altered HEK293 activity but not Neuro-2A activity. Cholesterol depletion modestly suppressed cAMP inhibition and enhanced β-arrestin translocation over all agonists, without impacting receptor internalisation or ORG27569-mediated signalling. These findings support hypotheses that membrane composition and lipid raft colocalisation influence CB1 signalling kinetics, and that cholesterol may act as an allosteric modulator, biasing signalling towards G⍺i/o pathways while inhibiting β-arrestin recruitment. This underscores the potential clinical relevance of lipid raft modulators or cholesterol derivatives in fine-tuning receptor activation and reducing the common adverse effects observed with current therapies.