title: Quantification of receptor tyrosine kinase activation and transactivation by G-protein-coupled receptors using spatial intensity distribution analysis (SpIDA).
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.
authors: Department of Psychiatry and Neuroscience, Faculty of Medicine, Laval University, Québec, QC, Canada.
authors: Annie
authors: A
authors: Barbeau
authors: Antoine G
authors: AG
authors: Godin
authors: Jody L
authors: JL
authors: Swift
authors: Yves
authors: Y
authors: De Koninck
authors: Paul W
authors: PW
authors: Wiseman
authors: Jean-Martin
authors: JM
authors: Beaulieu
mesh_terms: D000818:Animals; D002460:Cell Line; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods* / Q000706:statistics & numerical data; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D016012:Poisson Distribution; D055503:Protein Multimerization; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D015533:Transcriptional Activation
chemical_list: D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D020794:Receptor Protein-Tyrosine Kinases