Dataset Information

High-throughput identification of G protein-coupled receptor modulators through affinity mass spectrometry screening.

ABSTRACT: G protein-coupled receptors (GPCRs) represent the largest class of cell surface proteins and thus constitute an important family of therapeutic targets. Therefore, significant effort has been put towards the identification of novel ligands that can modulate the activity of a GPCR target with high efficacy and selectivity. However, due to limitations inherent to the most common techniques for GPCR ligand discovery, there is a pressing need for more efficient and effective ligand screening methods especially for the identification of potential allosteric modulators. Here we present a high-throughput, label-free and unbiased screening approach for the identification of small molecule ligands towards GPCR targets based on affinity mass spectrometry. This new approach features the usage of target-expressing cell membranes rather than purified proteins for ligand screening and allows the detection of both orthosteric and allosteric ligands targeting specific GPCRs. Screening a small compound library with this approach led to the rapid discovery of an antagonist for the 5-HT receptor and four positive allosteric modulators for GLP-1 receptor that were not previously reported.

SUBMITTER: Qin S

PROVIDER: S-EPMC5916221 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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High-throughput identification of G protein-coupled receptor modulators through affinity mass spectrometry screening.

Qin Shanshan S Meng Mengmeng M Yang Dehua D Bai Wenwen W Lu Yan Y Peng Yao Y Song Gaojie G Wu Yiran Y Zhou Qingtong Q Zhao Suwen S Huang Xiping X McCorvy John D JD Cai Xiaoqing X Dai Antao A Roth Bryan L BL Hanson Michael A MA Liu Zhi-Jie ZJ Wang Ming-Wei MW Stevens Raymond C RC Shui Wenqing W

Chemical science 20180220 12

G protein-coupled receptors (GPCRs) represent the largest class of cell surface proteins and thus constitute an important family of therapeutic targets. Therefore, significant effort has been put towards the identification of novel ligands that can modulate the activity of a GPCR target with high efficacy and selectivity. However, due to limitations inherent to the most common techniques for GPCR ligand discovery, there is a pressing need for more efficient and effective ligand screening methods ...[more]

PMID: 29732102

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Project description: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 proximity 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.

Dataset Information

High-throughput identification of G protein-coupled receptor modulators through affinity mass spectrometry screening.

Publications

High-throughput identification of G protein-coupled receptor modulators through affinity mass spectrometry screening.

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