Biology Reference
In-Depth Information
1.3.2.2 Determining the Monomeric QB for Antibody Labeling using Detection
of a Reference Monomeric Protein............................................................................ 9
1.3.2.3 Determining the Monomeric QB by using Pharmacological Agents that
Block Oligomerization and Induce Monomeric Conformation ...................................11
1.3.3 Image Acquisitions ........................................................................... 11
1.3.4 Analysis with the SpIDA Program Graphical User Interface................... 12
1.3.4.1 Description of Histogram Parameters ..........................................13
1.3.4.2 SpIDA GUI Procedures ...............................................................13
1.3.5 Determination of Analog Detector Signal Broadening ........................... 15
1.3.5.1 Analog Detector Calibration Procedure ........................................16
1.3.6 Data Interpretation and Pharmacological Analysis ............................... 16
1.4 Discussion......................................................................................................... 17
Acknowledgments ..................................................................................................... 18
References ............................................................................................................... 18
Abstract
This chapter presents a general approach for the application of spatial intensity dis-
tribution 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.
INTRODUCTION
Receptor protein kinases (RTKs) constitute a large superfamily of membrane
proteins implicated in several biological processes including cell proliferation,
differentiation, motility, and survival (
Yarden & Ullrich, 1988
). The RTK protein
family includes 58 different single-transmembrane-domain glycoproteins that can
be subdivided in 20 subfamilies, which include most of the receptors for growth
