Abstract
G protein-coupled receptors (GPCRs) play vital roles in human physiology and pathophysiology. This makes the elucidation of the high-resolution blueprints of these high value membrane proteins of crucial importance for the structure-based design of novel therapeutics. However, the production and crystallization of GPCRs for structure determination comes with many challenges.
In this chapter, we provide a comprehensive protocol for expressing and purifying the thromboxane A2 receptor (TPR), an attractive therapeutic target, for use in structure studies. Guidelines for crystallizing the TPR are also included. Together, these procedures provide a template for generating crystal structures of the TPR and indeed other GPCRs in complex with pharmacologically interesting ligands.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Smyth E, Grosser T, Wang M, Yu Y, FitzGerald G (2009) Prostanoids in health and disease. J Lipid Res 50:S423–S428. https://doi.org/10.1194/jlr.r800094-jlr200
Nakahata N (2008) Thromboxane A2: physiology/pathophysiology, cellular signal transduction and pharmacology. Pharmacol Ther 118:18–35. https://doi.org/10.1016/j.pharmthera.2008.01.001
Fan H, Chen S, Yuan X, Han S, Zhang H, Xia W, Xu Y, Zhao Q, Wu B (2019) Structural basis for ligand recognition of the human thromboxane A2 receptor. Nat Chem Biol 15:27–33. https://doi.org/10.1038/s41589-018-0170-9
Luckow V, Lee S, Barry G, Olins P (1993) Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. J Virol 67:4566–4579. https://doi.org/10.1128/jvi.67.8.4566-4579.1993
Aloia A, Glatz R, McMurchie E, Leifert W (2009) GPCR expression using baculovirus-infected Sf9 cells. Methods Mol Biol 552:115–129. https://doi.org/10.1007/978-1-60327-317-6_8
Munk C, Mutt E, Isberg V, Nikolajsen L, Bibbe J, Flock T, Hanson M, Stevens R, Deupi X, Gloriam D (2019) An online resource for GPCR structure determination and analysis. Nat Methods 16:151–162. https://doi.org/10.1038/s41592-018-0302-x
Qanbar R, Bouvier M (2003) Role of palmitoylation/depalmitoylation reactions in G-protein-coupled receptor function. Pharmacol Ther 97:1–33. https://doi.org/10.1016/s0163-7258(02)00300-5
Tobin A (2008) G-protein-coupled receptor phosphorylation: where, when and by whom. Br J Pharmacol 153:S167–S176. https://doi.org/10.1038/sj.bjp.0707662
Chen Q, Miller L, Dong M (2010) Role of N-linked glycosylation in biosynthesis, trafficking, and function of the human glucagon-like peptide 1 receptor. Am J Physiol Endocrinol Metab 299:E62–E68. https://doi.org/10.1152/ajpendo.00067.2010
Caffrey M (2015) A comprehensive review of the lipid cubic phase or in meso method for crystallizing membrane and soluble proteins and complexes. Acta Crystallogr F Struct Biol Commun 71:3–18. https://doi.org/10.1107/s2053230x14026843
Caffrey M, Cherezov V (2009) Crystallizing membrane proteins using lipidic mesophases. Nat Protoc 4:706–731. https://doi.org/10.1038/nprot.2009.31
Caffrey M, Porter C (2010) Crystallizing membrane proteins for structure determination using lipidic mesophases. J Vis Exp. https://doi.org/10.3791/1712
Li D, Boland C, Walsh K, Caffrey M (2012) Use of a robot for high-throughput crystallization of membrane proteins in lipidic mesophases. J Vis Exp. https://doi.org/10.3791/4000
Misquitta Y, Caffrey M (2003) Detergents destabilize the cubic phase of monoolein: implications for membrane protein crystallization. Biophys J 85:3084–3096. https://doi.org/10.1016/s0006-3495(03)74727-4
Gasteiger E (2003) ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res 31:3784–3788. https://doi.org/10.1093/nar/gkg563
Caffrey M (2011) Crystallizing membrane proteins for structure–function studies using lipidic mesophases. Biochem Soc Trans 39:725–732. https://doi.org/10.1042/bst0390725
Acknowledgments
We thank D. Weichert for training and guidance in GPCR expression, purification, and crystallization, B. Wu and H. Fan for providing the plasmid for the TPR construct, and past and present members of the Membrane Structural and Functional Biology group. This work was supported in part by Science Foundation Ireland award 16/IA/4435 and an Irish Research Council Postgraduate Scholarship GOIPG/2019/3074.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Krawinski, P., Caffrey, M. (2022). Thromboxane A2 G Protein-Coupled Receptor Production and Crystallization for Structure Studies. In: Mus-Veteau, I. (eds) Heterologous Expression of Membrane Proteins. Methods in Molecular Biology, vol 2507. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2368-8_13
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2368-8_13
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2367-1
Online ISBN: 978-1-0716-2368-8
eBook Packages: Springer Protocols