Receptor tyrosine kinases (RTKs), which include the insulin and epidermal growth factor (EGF) receptor families, mediate cell growth and differentiation in many animal tissues. RTKs consist of an extracellular ligand binding region, a single transmembrane spanning region, and a cytoplasmic kinase. Inappropriate activation of RTKs results in abnormal cell growth, and dysfunctional RTKs have been implicated in the genesis and severity of many human cancers and become the target of many anticancer drugs: For example, overexpression of the EGF receptor homolog HER2 is found in 20-30% of human breast cancers and correlates with more aggressive tumors and a poorer prognosis, and an anti-HER2 monoclonal antibody, Herceptin, has proven an effective treatment for these cancers. My laboratory has recently developed an approach to producing high levels of soluble cysteine-rich glycoproteins, which includes the extracellular regions of most RTKs, in forms suitable for X-ray crystallographic analysis. This work has led to atomic resolution structures of extracellular regions of the EGF receptor, HER2, HER2 complexed with the Herceptin Fab, and the EGF receptor homolog HER3. These structures have provided much insight into how ligand binding generates signals and inspired new approaches to anticancer drug design. Little insight has been gained, however, into how the signal produced by ligand binding is transduced across the cell membrane. Atomic resolution structures of intact receptors are needed to solve this problem. A major roadblock to determination of these structures is the expression and purification of sufficient amounts of intact receptors in crystallizable form.
Our first aim i s to adapt the expression methods that we have developed for soluble cysteine-rich glycoproteins to the expression and purification of intact receptors. These methods will be generally applicable to structural studies of all cell surface proteins.
Our second aim i s to apply these methods to overexpress members of the EGF and insulin receptor families.
Our third aim i s to purify and characterize these receptors.
Our final aim i s to produce diffraction-quality crystals of these receptors both alone and complexed with ligand. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM071591-03
Application #
7118777
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Chin, Jean
Project Start
2004-09-01
Project End
2007-08-31
Budget Start
2006-09-01
Budget End
2007-08-31
Support Year
3
Fiscal Year
2006
Total Cost
$159,658
Indirect Cost
Name
Johns Hopkins University
Department
Physiology
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Qiu, Chen; Tarrant, Mary K; Boronina, Tatiana et al. (2009) In vitro enzymatic characterization of near full length EGFR in activated and inhibited states. Biochemistry 48:6624-32