Chemical biology tools for investigating heteromeric complexes of cannabinoid receptors

点击翻译按钮获取中文摘要

Project Summary The Gi/o coupled cannabinoid receptors 1 and 2 (CB1 and CB2) co-express in central and peripheral tissues. They individually play fundamental roles in membrane plasticity, vesicle secretion, cell migration, and inflammation. While CB1 and CB2 receptors share only 40% structural homology, both are fully activated by 2- arachidonoylglycerol (2-AG) and associate with the same G-proteins. Surprisingly, opposite signaling outcomes have been reported after selective activation of CB1 or CB2 in both central and peripheral organs. The two receptors are individually known to interact with several protein complexes beyond the established role of heterotrimeric G-proteins, namely through heteromeric complexes with other G protein-coupled receptors or receptor tyrosine kinases. Co-expressed CB1 and CB2 receptors are suggested to be juxtaposed in a way that one's activation regulates the other's activity. As 2-AG activates both receptors, I hypothesize that the endocannabinoid coordinates heteromerization mechanisms of CB1 and CB2 with other proteins. This project aims at combining protein engineering and proteomics to investigate the prevalence, the biological functions and the composition of cannabinoid receptor heteromers in living cells. CB1, CB2 and their respective interactomes need to be jointly investigated to distinguish individual from synergistic effects on cellular activity. Investigating the biological function of CB1 and CB2 heteromers in living cells ultimately requires novel tools to activate, label and purify cannabinoid receptors and their interacting proteins. Additionally, a new strategy to manipulate the formation of these protein complexes in a spatially and temporally resolved manner is needed. The speed and complexity of heteromerization mechanisms are challenging to capture with traditional techniques. I will develop new molecular tools for the study of cannabinoid receptor heteromers including imaging tools and those to characterize and manipulate the CB1 and CB2 interactomes at the molecular level. The techniques involved in this project include cannabinoid receptor visualization and photo-crosslinking, reversible manipulation of 2-AG levels, and manipulation of heteromer formation by light. This K99/R00 award will broaden my education through critical training in proteomics and cutting-edge protein engineering techniques. Aim 1. To investigate subcellular locations and composition of heteromeric complexes of cannabinoid receptors. Aim 2. To investigate the biological function of endogenous receptor heteromer formation by light.