Membrane receptor internalization studies follow the functional process of receptors binding to ligands or agonists. Receptor internalization, or trafficking, is a part of cell signaling. Cancer, psychoactive drug targets, virology, endocytosis, neurotransmitters and addiction are all relevant areas of study. However, membrane receptors are challenging to study given their low native expression levels.
Recombinant bioluminescent reporters and fluorescent protein labeling systems have revolutionized the study of receptor internalization. These systems can be used instead of traditional, cumbersome radioactively tagged ligand probes. Fluorescent probes are localized in cells through a process called “gating”, to be evaluated by ELISA, flow cytometry, imaging or high-throughput assays (1). Real time receptor internalization methods using time lapse confocal microscopy require the use of recombinant bioluminescent reporter proteins, protein labeling systems or antibodies. Bioluminescent reporters fluoresce regardless of receptor localization. Therefore, applications like flow cytometry or plate readers will not discriminate sub-cellular or membrane localization. To overcome this issue, SNAP-tag®, CLIP-tag™, ACP-tag and MCP-tag protein labeling systems, as well as fluorogen activating proteins (FAPs) (2), have membrane impermeant substrates. Simultaneous localization of more than one protein is possible with these substrates when targets are engineered into different systems with unique substrate requirements. These systems also have the advantage of being able turn on the signal at will, allowing time-resolved analysis of receptor internalization and protein trafficking which can be combined with FRET analysis (2).
- Evans, N. (2004) Current Protocols in Pharmacology Unit 12.6. PMID: 22294118
- Saunders, M.J. (2012) Methods, 2012 Feb 16. PMID: 22366230
- Comps-Agrar L. et al (2011) Methods Mol Biol. 756, 201-214. PMID: 21870227
SNAP-tag® is a registered trademark of New England Biolabs, Inc.
CLIP-tag™ is a trademark of New England Biolabs, Inc.