Cannabinoid receptors distribution in mouse cortical plasma membrane compartments

Abstract

The type 1 and type 2 cannabinoid receptors (CB1 and CB2 receptors) are class A G protein-coupled receptors (GPCRs) that are activated by endogenous lipids called endocannabinoids to modulate neuronal excitability and synaptic transmission in neurons throughout the central nervous system (CNS), and inflammatory processes throughout the body. CB1 receptor is one of the most abundant GPCRs in the CNS and is involved in many physiological and pathophysiological processes, including mood, appetite, and nociception. CB2 receptor is primarily found on immunomodulatory cells of both the CNS and the peripheral immune system. In this study, we isolated lipid raft and non-lipid raft fractions of plasma membrane (PM) from mouse cortical tissue by using cold non-ionic detergent and sucrose gradient centrifugation to study the localization of CB1 receptor and CB2 receptor. Lipid raft and non-lipid raft fractions were confirmed by flotillin-1, caveolin-1 and transferrin receptor as their protein biomarkers. Both CB1 receptor and CB2 receptor were found in non-raft compartments that is inconsistent with previous findings in cultured cell lines. This study demonstrates compartmentalization of both CB1 receptor and CB2 receptor in cortical tissue and warrants further investigation of CB1 receptor and CB2 receptor compartmental distribution in various brain regions and cell types.

Keywords: Lipid rafts; Type 1 cannabinoid receptor; Type 2 cannabinoid receptor.

Fig. 1

Isolation and identification of lipid rafts from non-raft fractions by discontinuous sucrose gradient centrifugation. Tissue samples were from juvenile male C57BL/6 mouse cortex. The lipid raft compartments are fully visible as an opaque band at the interface between 5 and 30% sucrose

Fig. 2

CB receptors PM compartmental distribution in mouse cortical tissue. A Aliquots of fractions collected from top to bottom of the gradient were subjected to SDS-PAGE isolation and analyzed by western blotting with antibodies directed against CB1 receptor, CB2 receptor, flotillin-1, caveolin-1, PrPc and transferrin receptor. To investigate purity of the fractionation process, the PVDF membranes were later stripped and probed with Cytochrome C (mitochondrial marker) and SCD1 (endoplasmic reticulum marker). Mouse cortical lysate (40 µg) was loaded on each gel as positive control. B Quantification of percent of CB1 or CB2 receptor present in each fraction relative to total receptor across all fractions within sample was performed using ImageJ software. All these experiments were repeated 5 times. Data are presented as mean ± S.E.M