Systematic Affinity Purification Coupled to Mass Spectrometry Identified p62 as Part of the Cannabinoid Receptor CB2 Interactome

Abstract

The endocannabinoid system (ECS) consists particularly of cannabinoid receptors 1 and 2 (CB1 and CB2), their endogenous ligands, and enzymes that synthesize and degrade their ligands. It acts in a variety of organs and disease states ranging from cancer progression over neuropathic pain to neurodegeneration. Protein components engaged in the signaling, trafficking, and homeostasis machinery of the G-protein coupled CB2, are however largely unknown. It is therefore important to identify further interaction partners to better understand CB2 receptor functions in physiology and pathophysiology. For this purpose, we used an affinity purification and mass spectrometry-based proteomics approach of Strep-HA-CB2 receptor in HEK293 cells. After subtraction of background interactions and protein frequency library assessment we could identify 83 proteins that were classified by the identification of minimally 2 unique peptides as highly probable interactors. A functional protein association network analysis obtained an interaction network with a significant enrichment of proteins functionally involved in protein metabolic process, in endoplasmic reticulum, response to stress but also in lipid metabolism and membrane organization. The network especially contains proteins involved in biosynthesis and trafficking like calnexin, Sec61A, tubulin chains TUBA1C and TUBB2B, TMED2, and TMED10. Six proteins that were only expressed in stable CB2 expressing cells were DHC24, DHRS7, GGT7, HECD3, KIAA2013, and PLS1. To exemplify the validity of our approach, we chose a candidate having a relatively low number of edges in the network to increase the likelihood of a direct protein interaction with CB2 and focused on the scaffold/phagosomal protein p62/SQSTM1. Indeed, we independently confirmed the interaction by co-immunoprecipitation and immunocytochemical colocalization studies. 3D reconstruction of confocal images furthermore showed CB2 localization in close Frontiers in Molecular Neuroscience | www.frontiersin.org 1 September 2019 | Volume 12 | Article 224 Sharaf et al. Cannabinoid Receptor CB2 Interactomeproximity to p62 positive vesicles at the cell membrane. In summary, we provide a comprehensive repository of the CB2 interactome in HEK293 cells identified by a systematic unbiased approach, which can be used in future experiments to decipher the signaling and trafficking complex of this cannabinoid receptor. Future studies will have to analyze the exact mechanism of the p62-CB2 interaction as well as its putative role in disease pathophysiology.

Bioinformatics Methods for Mass Spectrometry-Based Proteomics Data Analysis

“…Traditional approaches tend to focus on one or a few proteins; however, with the recent developments in the sample separation and mass spectrometry technologies, it is now possible to consider a complex biological system as an integrated unit. The rapid advancements in the experimental aspects of proteomics have inspired various downstream bioinformatics analysis methods that help to discover the relationship between molecular-level protein regulatory mechanisms and phenotypic behavior, such as disease development and progression [3,4].…”

Monitoring Cannabinoid CB2 -Receptor Mediated cAMP Dynamics by FRET-Based Live Cell Imaging

“…The FRET responses of Epac1-CB2-HEK cells to 1 µM of CB2 inverse agonist AM630 showed more variability, depending on precedent CB2 agonist stimulation (Figure 6D,F,G). The mean differences in ∆R between CB2 agonist and AM630 baseline ranged between 10 6D).…”

“…As a Gαi/o -coupled GPCR, the activation of CB2 leads to the inhibition of adenylate cyclases (AC) via Gαi subunits [ 5 , 8 ], causing a decrease in 3’,5’-cyclic adenosine monophosphate (cAMP). Recently, CB2 signaling via Gαs has been demonstrated in human PBMCs [ 9 ] and binding to Gαs protein has been detected [ 10 ]. Early reports on CB2-mediated signaling from Bouaboula et al [ 8 ] already registered a high degree of constitutive receptor activity in cAMP measurements in heterologous expression systems that was later confirmed by describing the action of CB2 inverse agonists [ 11 , 12 ].…”

Impact of the Endocannabinoid System on Bone Formation and Remodeling in p62 KO Mice

“…To better understand the CB2 receptor signaling pathways, we have previously performed a screen for protein–protein interactions using tandem mass spectrometry. Here, we identified p62 (sequestosome 1, SQSTM1) as an interaction partner for the G-protein coupled CB2 receptor ( Sharaf et al, 2019 ). p62 is a signaling scaffold protein and signaling hub with multiprotein domains that mediate its interactions with various binding partners, implicating the protein in numerous signaling pathways that influence processes such as cell differentiation, survival, osteoclastogenesis, inflammation, obesity, and autophagy ( Moscat et al, 2007 ; Sanchez-Martin and Komatsu, 2018 ).…”

“…p62 is a signaling scaffold protein and signaling hub with multiprotein domains that mediate its interactions with various binding partners, implicating the protein in numerous signaling pathways that influence processes such as cell differentiation, survival, osteoclastogenesis, inflammation, obesity, and autophagy ( Moscat et al, 2007 ; Sanchez-Martin and Komatsu, 2018 ). Binding to the CB2 receptor is mediated via the ZZ-type zinc finger (ZZ) domain ( Sharaf et al, 2019 ). The ubiquitin-associated (UBA) domain of the p62 protein clusters mutations identified in patients with familial and sporadic Paget’s disease of bone (PDB) ( Morissette et al, 2006 ; Falchetti et al, 2009 ), which is characterized by focal and disorganized increases in bone turnover ( Roodman and Windle, 2005 ) and excessive bone-resorbing activity of abnormal osteoclasts ( Chamoux et al, 2009 ).…”