Abstract
Interactions between extracellular domains (ECDs) are crucial for many physiological processes in the cell, most importantly perception of its environment. However, studying these often-transient interactions can be challenging. Here we describe a method that allows for in vitro detection of extracellular domain interactions through an oligomerization-based cell surface interaction (CSI) assay. In a CSI, bait- and prey-tagged proteins are produced and secreted by Drosophila S2 cells to ensure proper folding and post-translational modifications. Subsequently, Bait (FC fragment) and Prey (pentamer domain and alkaline phosphatase) tags allow the detection of interactions in protein A-coated 96 wells plates through a colorimetric readout. Due to the easy detection of interactions this approach can be used for high-throughput screening and mapping of extracellular interaction networks.
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References
Belkhadir Y, Yang L, Hetzel J, Dangl JL, Chory J (2014) The growth defense pivot: crisis management in plants mediated by LRR-RK surface receptors. Trends Biochem Sci 39(10):447–456. https://doi.org/10.1016/j.tibs.2014.06.006
Song W, Han Z, Wang J, Lin G, Chai J (2017) Structural insights into ligand recognition and activation of plant receptor kinases. Curr Opin Struct Biol 43:18–27. https://doi.org/10.1016/j.sbi.2016.09.012
Hohmann U, Lau K, Hothorn M (2017) The structural basis of ligand perception and signal activation by receptor kinases. Annu Rev Plant Biol 68(1):109–137. https://doi.org/10.1146/annurev-arplant-042916-040957
Özkan E, Carrillo Robert A, Eastman Catharine L, Weiszmann R, Waghray D, Johnson Karl G, Zinn K, Celniker Susan E, Garcia KC (2013) An extracellular Interactome of immunoglobulin and LRR proteins reveals receptor-ligand networks. Cell 154(1):228–239. https://doi.org/10.1016/j.cell.2013.06.006
Smakowska-Luzan E, Mott GA, Parys K, Stegmann M, Howton TC, Layeghifard M, Neuhold J, Lehner A, Kong J, Grünwald K, Weinberger N, Satbhai SB, Mayer D, Busch W, Madalinski M, Stolt-Bergner P, Provart NJ, Mukhtar MS, Zipfel C, Desveaux D, Guttman DS, Belkhadir Y (2018) An extracellular network of Arabidopsis leucine-rich repeat receptor kinases. Nature 553(7688):342–346. https://doi.org/10.1038/nature25184
Mott GA, Smakowska-Luzan E, Pasha A, Parys K, Howton TC, Neuhold J, Lehner A, Grünwald K, Stolt-Bergner P, Provart NJ, Mukhtar MS, Desveaux D, Guttman DS, Belkhadir Y (2019) Map of physical interactions between extracellular domains of Arabidopsis leucine-rich repeat receptor kinases. Scientific Data 6(1):190025. https://doi.org/10.1038/sdata.2019.25
Acknowledgments
This work was supported by The NWO Talent Programme Vidi grant VI.Vidi.193.074 to E.S.L.
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Stouthamer, J., Martin-Ramirez, S., Smakowska-Luzan, E. (2023). An Interactome Assay for Detecting Interactions between Extracellular Domains of Receptor Kinases. In: Mukhtar, S. (eds) Protein-Protein Interactions. Methods in Molecular Biology, vol 2690. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3327-4_18
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DOI: https://doi.org/10.1007/978-1-0716-3327-4_18
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