Quantitative proteomics reveals CLR interactome in primary human cells

Manolis, Dimitrios; Hasan, Shirin; Maraveyas, Anthony; O'Brien, Darragh P.; Kessler, Benedikt M.; Kramer, Holger; Nikitenko, Leonid L.

Abstract

The G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) mediates essential functions in several cell types and is implicated in cardiovascular pathologies, skin diseases, migraine and cancer. To date, the network of proteins interacting with CLR (“CLR interactome”) in primary cells, where this GPCR is expressed at endogenous (physiologically relevant) levels, remains unknown. To address this knowledge gap, we established a novel integrative methodological workflow/approach for conducting a comprehensive/proteome-wide analysis of Homo sapiens CLR interactome. We used primary human dermal lymphatic endothelial cells and combined immunoprecipitation (IP) utilising anti-human CLR antibody with label-free quantitative nano-liquid chromatography-tandem mass spectrometry (nano LC-MS/MS) and quantitative in situ proximity ligation assay (PLA). By using this workflow, we identified 37 proteins interacting with endogenously expressed CLR amongst 4,902 detected members of the cellular proteome (by quantitative nano LC-MS/MS) and revealed direct interactions of two kinases and two transporters with this GPCR (by in situ PLA). All identified interactors have not been previously reported as members of CLR interactome. Our approach and findings uncover the hitherto unrecognized compositional complexity of the interactome of endogenously expressed CLR and contribute to fundamental understanding of the biology of this GPCR. Collectively, our study provides a first-of-its-kind integrative methodological approach and datasets as valuable resources and robust platform/springboard for advancing the discovery and comprehensive characterization of physiologically relevant CLR interactome at a proteome-wide level in a range of cell types and diseases in future studies.

Citation

Manolis, D., Hasan, S., Maraveyas, A., O'Brien, D. P., Kessler, B. M., Kramer, H., & Nikitenko, L. L. (2024). Quantitative proteomics reveals CLR interactome in primary human cells. Journal of Biological Chemistry, 300(6), Article 107399. https://doi.org/10.1016/j.jbc.2024.107399

• Journal Article Type: Article
• Acceptance Date: May 14, 2024
• Online Publication Date: Jun 17, 2024
• Publication Date: 2024-06
• Deposit Date: Jun 18, 2024
• Publicly Available Date: Jun 19, 2024
• Journal: Journal of Biological Chemistry
• Print ISSN: 0021-9258
• Publisher: American Society for Biochemistry and Molecular Biology
• Peer Reviewed: Peer Reviewed
• Volume: 300
• Issue: 6
• Article Number: 107399
• DOI: https://doi.org/10.1016/j.jbc.2024.107399
• Keywords: Calcitonin receptor–like receptor; GPCR; endothelial cell; endogenous; interactome; proteome
• Public URL: https://hull-repository.worktribe.com/output/4710269