Delineating the Specificity of Cannabinoid Effects by Investigating Cannabinoid Receptor-1 Trafficking and Signaling

Abstract

Pharmaceuticals target specific proteins within the body to produce desired therapeutic outcomes, and the G-protein-coupled receptor (GPCR) family represents the most common type of pharmaceutical target. Traditional models of GPCR function suggest that receptors are only active at the cell surface, however, multiple GPCRs have demonstrated activation at both surface and intracellular locations. This illuminates the possibility of GPCR spatial signaling, whereby a single GPCR can exhibit differential signaling outcomes based on its cellular localization. Therefore, GPCR trafficking could be targeted as a new pharmacological approach for modulating GPCR activity. In this thesis, I investigate cannabinoid receptor-1 (CB1) trafficking to determine how it may influence the specificity of cannabinoid-mediated effects.

CB1 is a Gi-coupled GPCR that is widely expressed throughout the central nervous system (CNS). At both excitatory and inhibitory presynaptic terminals, CB1’s Gi-coupling reduces intracellular calcium concentrations in presynaptic neurons, and this regulates synaptic release of neurotransmitters. It is suggested that CB1’s unique physiological function underlies its broad therapeutic potential for treating neurological disorders. However, the underlying trafficking mechanisms of CB1’s axonal polarization remain unknown. It is well established that CB1 trafficking exhibits a high degree of constitutive recycling, yet CB1 does not recycle after agonist stimulation. Chapter 2 identifies biosynthetic trafficking as a key regulator of CB1 surface expression. Specifically, 2AG, an endogenous cannabinoid, upregulates biosynthetic trafficking of CB1 to the cell surface. This highlights a recycling-independent mechanism by which cells are resensitized to cannabinoids after acute agonist stimulation.

Cannabinoid agonists produce many similar effects in vitro and in vivo, but there are some effects are not entirely mediated by CB1. Chapter 3 characterizes novel biological effects of WIN 55,212-2 (WIN), a synthetic cannabinoid that has been used to investigate the endocannabinoid system, extensively. This chapter demonstrates that WIN disrupts the Golgi apparatus and microtubules in cultured cells, independent of CB1. This observation signifies a need for re- interpretation of previous WIN-based studies, particularly those that have speculated the functions of CB1 and the endocannabinoid system.

Overall, the work presented in this thesis highlights GPCR trafficking as crucial element of pharmacology research. Specifically, investigating CB1 trafficking helped establish a cellular mechanism by which CB1 activity is regulated within the CNS. Additionally, investigating how cannabinoids differentially regulate CB1 trafficking provided significant insight into the use of WIN as a pharmacological tool for studying the endocannabinoid system.