Spatial intensity distribution analysis (SpIDA): a new tool for receptor tyrosine kinase activation and transactivation quantification

Abstract

This chapter presents a general approach for the application of spatial intensity distribution analysis (SpIDA) to pharmacodynamic quantification of receptor tyrosine kinase homodimerization in response to direct ligand activation or transactivation by G protein-coupled receptors. A custom graphical user interface developed for MATLAB is used to extract quantal brightness and receptor density information from intensity histograms calculated from single fluorescence microscopy images. This approach allows measurement of monomer/oligomer protein mixtures within subcellular compartments using conventional confocal laser scanning microscopy. Application of quantitative pharmacological analysis to data obtained using SpIDA provides a universal method for comparing studies between cell lines and receptor systems. In addition, because of its compatibility with conventional immunostaining approaches, 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.

Keywords: Confocal microscopy; Dimerization; Fluorescence distribution analysis; G protein-coupled receptors (GPCRs); Heptahelical receptors (7TM); Image analysis; Receptor transactivation; Receptor tyrosine kinase (RTK).