The F238L Point Mutation in the Cannabinoid Type 1 Receptor Enhances Basal Endocytosis via Lipid Rafts.

Melanie Wickert,Ruth A Ross,Keri L Hildick,Beat Lutz,Miriam Schneider,Ruth Jelinek,Gemma L Baillie,Krisztina Monory,Alejandro Aparisi Rey,Jeremy M Henley

doi:10.3389/fnmol.2018.00230

Abstract

Defining functional domains and amino acid residues in G protein coupled receptors (GPCRs) represent an important way to improve rational drug design for this major class of drug targets. The cannabinoid type 1 (CB1) receptor is one of the most abundant GPCRs in the central nervous system and is involved in many physiological and pathophysiological processes. Interestingly, cannabinoid type 1 receptor with a phenylalanine 238 to leucine mutation (CB1F238L) has been already linked to a number of both in vitro and in vivo alterations. While CB1F238L causes significantly reduced presynaptic neurotransmitter release at the cellular level, behaviorally this mutation induces increased risk taking, social play behavior and reward sensitivity in rats. However, the molecular mechanisms underlying these changes are not fully understood. In this study, we tested whether the F238L mutation affects trafficking and axonal/presynaptic polarization of the CB1 receptor in vitro. Steady state or ligand modulated surface expression and lipid raft association was analyzed in human embryonic kidney 293 (HEK293) cells stably expressing either wild-type cannabinoid type 1 receptor (CB1wt) or CB1F238L receptor. Axonal/presynaptic polarization of the CB1F238L receptor was assessed in transfected primary hippocampal neurons. We show that in vitro the CB1F238L receptor displays increased association with lipid rafts, which coincides with increased lipid raft mediated constitutive endocytosis, leading to a reduction in steady state surface expression of the CB1F238L receptor. Furthermore, the CB1F238L receptor showed increased axonal polarization in primary hippocampal neurons. These data demonstrate that endocytosis of the CB1 receptor is an important mediator of axonal/presynaptic polarization and that phenylalanine 238 plays a key role in CB1 receptor trafficking and axonal polarization.

Highlights

The cannabinoid type 1 (CB1) receptor is an attractive target for therapeutic drugs because it plays an important role in the regulation of numerous physiological processes, including neural development (Maccarrone et al, 2014), the regulation of synaptic processes (Soltesz et al, 2015) and of various behaviors, e.g., learning and memory (Lutz, 2009; Ruehle et al, 2012), food intake (Silvestri and Di Marzo, 2013), and addiction (Moreira et al, 2015)
We demonstrate that the mutant receptor shows increased basal internalization in human embryonic kidney 293 (HEK293) cells, which depends on caveolae/lipid raft mediated endocytosis
The authors state that the lower molecular bands are deglycosylated forms of the CB1 receptor located in the intracellular biosynthetic or degradation pathway, which protects them from trypsin cleavage (Grimsey et al, 2010)

Summary

Introduction

The cannabinoid type 1 (CB1) receptor is an attractive target for therapeutic drugs because it plays an important role in the regulation of numerous physiological processes, including neural development (Maccarrone et al, 2014), the regulation of synaptic processes (Soltesz et al, 2015) and of various behaviors, e.g., learning and memory (Lutz, 2009; Ruehle et al, 2012), food intake (Silvestri and Di Marzo, 2013), and addiction (Moreira et al, 2015) It is associated with many psychiatric disorders and neuropathological conditions (Pacher et al, 2006). Mutations in the second extracellular loop and helix 8 (Ahn et al, 2009, 2010), as well as truncation of the N-terminus (Andersson et al, 2003) have been shown to affect CB1 receptor forward trafficking

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Results

Conclusion