Abstract
G protein-coupled receptors represent one of the largest families of cell-surface receptors. They play important roles in various physiological systems in response to a range of ligands with diverse physicochemical properties. Ligand stimulation triggers G protein-coupled receptor (GPCR) endocytosis, which controls cellular desensitization and resensitization processes by regulating the number of GPCRs on the plasma membrane. Notably, it has recently been shown that endosome-localized GPCRs contribute significantly to both G protein-dependent and G protein-independent signaling in the acute and subacute phase. Studying individual GPCR behaviors during endocytosis is thus becoming increasingly more important in GPCR research. Here we describe a simple and versatile protocol for monitoring GPCR endocytosis utilizing a pulse-chase covalent labeling technique based on the HaloTag technology.
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
References
Vassilatis DK, Hohmann JG, Zeng H, Li F, Ranchalis JE, Mortrud MT, Brown A, Rodriguez SS, Weller JR, Wright AC, Bergmann JE, Gaitanaris GA (2003) The G protein-coupled receptor repertoires of human and mouse. Proc Natl Acad Sci U S A 100(8):4903–4908. doi:10.1073/pnas.0230374100
Rajagopal S, Rajagopal K, Lefkowitz RJ (2010) Teaching old receptors new tricks: biasing seven-transmembrane receptors. Nat Rev Drug Discov 9(5):373–386. doi:10.1038/nrd3024
Irannejad R, Tomshine JC, Tomshine JR, Chevalier M, Mahoney JP, Steyaert J, Rasmussen SGF, Sunahara RK, El-Samad H, Huang B, von Zastrow M (2013) Conformational biosensors reveal GPCR signalling from endosomes. Nature 495(7442):534–538. doi:10.1038/nature12000
Eichel K, Jullie D, von Zastrow M (2016) beta-Arrestin drives MAP kinase signalling from clathrin-coated structures after GPCR dissociation. Nat Cell Biol 18(3):303–310. doi:10.1038/ncb3307
Ranjan R, Gupta P, Shukla AK (2016) GPCR Signaling: beta-arrestins Kiss and Remember. Curr Biol 26(7):R285–R288. doi:10.1016/j.cub.2016.02.056
Nuber S, Zabel U, Lorenz K, Nuber A, Milligan G, Tobin AB, Lohse MJ, Hoffmann C (2016) beta-Arrestin biosensors reveal a rapid, receptor-dependent activation/deactivation cycle. Nature 531(7596):661–664. doi:10.1038/nature17198
Los GV, Wood K (2007) The HaloTag: a novel technology for cell imaging and protein analysis. Methods Mol Biol 356:195–208
Los GV, Encell LP, McDougall MG, Hartzell DD, Karassina N, Zimprich C, Wood MG, Learish R, Ohana RF, Urh M, Simpson D, Mendez J, Zimmerman K, Otto P, Vidugiris G, Zhu J, Darzins A, Klaubert DH, Bulleit RF, Wood KV (2008) HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem Biol 3(6):373–382. doi:10.1021/cb800025k
Kumagai H, Ikeda Y, Motozawa Y, Fujishiro M, Okamura T, Fujio K, Okazaki H, Nomura S, Takeda N, Harada M, Toko H, Takimoto E, Akazawa H, Morita H, Suzuki J, Yamazaki T, Yamamoto K, Komuro I, Yanagisawa M (2015) Quantitative measurement of GPCR endocytosis via pulse-chase covalent labeling. PLoS One 10(5), e0129394. doi:10.1371/journal.pone.0129394
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Kumagai, H., Ikeda, Y. (2016). Pulse-Chase Covalent Labeling Technique for Monitoring GPCR Endocytosis. In: Shukla, A. (eds) Chemical and Synthetic Approaches in Membrane Biology. Springer Protocols Handbooks. Humana Press, New York, NY. https://doi.org/10.1007/8623_2016_2
Download citation
DOI: https://doi.org/10.1007/8623_2016_2
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6835-0
Online ISBN: 978-1-4939-6836-7
eBook Packages: Springer Protocols