This project combines resources of two established laboratories in membrane protein biophysics and biochemistry to study details of the intermolecular interactions of the epidermal growth factor (EGF) receptor with its ligands and with other receptors of the ErbB family of receptor kinases. The ErbB family of receptors, and, in particular, the EGF receptor, are among the most widely studied polypeptide hormone receptors because of their importance in normal development, wound healing, and, when deregulated by mutation or over-expression, neoplasia. Nonetheless, fundamental questions concerning the mechanisms of signal transduction triggered by the binding of the EGF family of ligands to the ErbB family of receptors remain unresolved. The long term goal of this project is to develop an in-depth understanding of the earliest events in the signal transduction pathway that occur when EGF family ligands bind to their respective receptors.
The specific aims for this budget period will exploit well characterized fluorescent ligands, new cell lines that have been shown to withstand turbulent stoppedflow rapid mixing and that express selected ErbB family receptors in the absence of endogenous ErbB receptors, as well as a custom stopped-flow fluorometer specifically designed and optimized for these studies. Studies will address hypotheses a) that expression of other ErbB family members affects the proportion of EGF receptors in high and low affinity states, b) that ligands recognized by ErbB3 or ErbB4 can modulate the binding of EGF to its receptor when the receptors are present in the same cell, c) that the dynamic on and off rates for receptor binding are important parameters for determining the mitogenic activity ofpeptide hormones, and d) that heterogeniety in glycosylation can affect receptor affinity for EGF. Studies will also test competing hypotheses concerning the interactions between the EGF receptor and ErbB2: that EGF receptor-ErbB2 dimers form directly when EGF binds to the receptor, or that EGF receptors dimerize on binding EGF and subsequently recruit ErbB2.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM055056-06
Application #
6763045
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Basavappa, Ravi
Project Start
1997-01-01
Project End
2007-06-30
Budget Start
2004-07-01
Budget End
2005-06-30
Support Year
6
Fiscal Year
2004
Total Cost
$295,583
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Physiology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Stein, Richard A; Staros, James V (2006) Insights into the evolution of the ErbB receptor family and their ligands from sequence analysis. BMC Evol Biol 6:79
Whitson, Kristin B; Whitson, Stefanie R; Red-Brewer, Monica L et al. (2005) Functional effects of glycosylation at Asn-579 of the epidermal growth factor receptor. Biochemistry 44:14920-31
Whitson, Kristin B; Beechem, Joseph M; Beth, Albert H et al. (2004) Preparation and characterization of Alexa Fluor 594-labeled epidermal growth factor for fluorescence resonance energy transfer studies: application to the epidermal growth factor receptor. Anal Biochem 324:227-36
Zhen, Yuejun; Caprioli, Richard M; Staros, James V (2003) Characterization of glycosylation sites of the epidermal growth factor receptor. Biochemistry 42:5478-92
Wilkinson, John C; Beechem, Joseph M; Staros, James V (2002) A stopped-flow fluorescence anisotropy method for measuring hormone binding and dissociation kinetics with cell-surface receptors in living cells. J Recept Signal Transduct Res 22:357-71
Wilkinson, John C; Staros, James V (2002) Effect of ErbB2 coexpression on the kinetic interactions of epidermal growth factor with its receptor in intact cells. Biochemistry 41:8-14
Wilkinson, J C; Stein, R A; Guyer, C A et al. (2001) Real-time kinetics of ligand/cell surface receptor interactions in living cells: binding of epidermal growth factor to the epidermal growth factor receptor. Biochemistry 40:10230-42
Stein, R A; Wilkinson, J C; Guyer, C A et al. (2001) An analytical approach to the measurement of equilibrium binding constants: application to EGF binding to EGF receptors in intact cells measured by flow cytometry. Biochemistry 40:6142-54