Quantification of receptor tyrosine kinase activation and transactivation by G-protein-coupled receptors using spatial intensity distribution analysis (SpIDA).

TitleQuantification of receptor tyrosine kinase activation and transactivation by G-protein-coupled receptors using spatial intensity distribution analysis (SpIDA).
Publication TypeJournal Article
Year of Publication2013
AuteursBarbeau, A, Godin, AG, Swift, JL, De Koninck, Y, Wiseman, PW, Beaulieu, J-M
JournalMethods Enzymol
Volume522
Pagination109-31
Date Published2013
ISSN1557-7988
KeywordsAnimals, Cell Line, Green Fluorescent Proteins, Humans, Image Processing, Computer-Assisted, Microscopy, Confocal, Microscopy, Fluorescence, Poisson Distribution, Protein Multimerization, Receptor Protein-Tyrosine Kinases, Receptors, G-Protein-Coupled, Recombinant Fusion Proteins, Signal Transduction, Transcriptional Activation
Abstract

This chapter presents a general approach for the application of spatial intensity distribution analysis (SpIDA) to the pharmacodynamic quantification of receptor tyrosine kinase homodimerization in response to direct ligand activation or transactivation by G-protein-coupled receptors. Intensity histograms are generated from single fluorescence microscopy images. These histograms are then fit with Poissonian distributions to obtain density maps and quantal brightness values of the labeled proteins underlying the images. This approach allows resolving monomer/oligomer protein mixtures within subcellular compartments using conventional confocal laser scanning microscopy. The application of quantitative pharmacological analysis to data obtained using SpIDA provides a universal method for comparing studies between cell lines and receptor systems. In contrast to methods based on resonance energy transfer, SpIDA is suitable not only for use in recombinant systems but also for the characterization of mechanisms involving endogenous proteins. Therefore, SpIDA enables these biological processes to be monitored directly in their native cellular environment.

DOI10.1016/B978-0-12-407865-9.00007-8
Alternate JournalMeth. Enzymol.
PubMed ID23374183