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

Methods Enzymol. 2013;522:109-31. doi: 10.1016/B978-0-12-407865-9.00007-8.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Image Processing, Computer-Assisted / methods*
  • Image Processing, Computer-Assisted / statistics & numerical data
  • Microscopy, Confocal / methods*
  • Microscopy, Fluorescence / methods*
  • Poisson Distribution
  • Protein Multimerization
  • Receptor Protein-Tyrosine Kinases / genetics
  • Receptor Protein-Tyrosine Kinases / metabolism*
  • Receptors, G-Protein-Coupled / genetics
  • Receptors, G-Protein-Coupled / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Signal Transduction
  • Transcriptional Activation*

Substances

  • Receptors, G-Protein-Coupled
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins
  • Receptor Protein-Tyrosine Kinases