The binding of epidermal growth factor (EGF), a Mr = 6045 polypeptide hormone, to its plasma membrane receptor initiates a broad array of cellular responses, ultimately culminating in mitosis. The EGF receptor is a transmembrane glycoprotein of Mr = 170,000 with no apparent subunit structure. When EGF binds to the receptor, a tyrosyl residue specific protein kinase is stimulated. The EGF binding site and the tyrosyl kinase site have been shown to reside in the same polypeptide chain. The EGF receptor/kinase is, therefore, viewed as a transmembrane allosteric enzyme, with the active (kinase) site accessible from the cytoplasm and the effector (EGF binding) site accessible from the extracytoplasmic milieu. The mechanism for coupling the binding of EGF to kinase activation is unknown. The long-term goal of this proposal is to develop an in-depth understanding of the structure of the EGF receptor/kinase as it relates to this signal transduction process. The proposal focuses on three functionally important structural domains of the EGF receptor/kinase, the active site, the transmembrane domain, and the EGF binding site. The structure of the active site of the kinase domain will be investigated through the use of a set of ATP-analog affinity labels which are expected to label different parts of the ATP binding site. Sites of labeling will be identified by tryptic digestion of the labeled receptor/kinase, peptide mapping, and sequencing of the labeled peptides. Site-directed antibodies will be used to probe other parts of the active site. Site-directed antibodies will also be used to investigate the topology of the EGF receptor/kinase. These studies will test the hypothesis that the polypeptide chain of the receptor/kinase crosses the lipid bilayer multiple times. The sites of glycosylation will be determined by sequencing glycosylated peptides from a tryptic digest of the receptor/kinase. Possible oligomeric forms of the receptor/kinase will be probed by cross- linking. Residues near the EGF binding site will be identified by cross-linking EGF to the receptor and sequencing the cross-linked peptide from a digest. Residues in the EGF binding site will be identified by photoaffinity labeling with an analog of an EGF fragment and sequencing the labeled site. Once these initial positions have been identified, further delineation of the EGF binding site will be carried out using site-directed antibodies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK025489-10
Application #
3227429
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1980-06-01
Project End
1991-11-30
Budget Start
1989-12-01
Budget End
1990-11-30
Support Year
10
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
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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
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Stein, R A; Staros, J V (2000) Evolutionary analysis of the ErbB receptor and ligand families. J Mol Evol 50:397-412
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Tong, K; Guyer, C A; Staros, J V (1996) Steric constraints in the recognition of peptide substrates for the epidermal growth factor receptor kinase. Int J Pept Protein Res 47:219-26
Stein, R A; Staros, J V (1996) Thermal inactivation of the protein tyrosine kinase of the epidermal growth factor receptor. Biochemistry 35:2878-84
Coker, K J; Staros, J V; Guyer, C A (1994) A kinase-negative epidermal growth factor receptor that retains the capacity to stimulate DNA synthesis. Proc Natl Acad Sci U S A 91:6967-71

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