The epidermal growth factor receptor (EGF-R) on human epidermal carcinoma cells, A431, is associated with the Triton X-100 insoluble cytoskeleton. The cytoskeletally associated receptor retains both its ability to bind epidermal growth factor (EGF) and its tyrosine kinase activity. This proposal has as one of its principal goals to establish the significance of the structural association of the EGF-R in internalization and processing of the hormone-receptor complex by the cell. Treatment of intact A431 cells with EGF, followed by detergent lysis and incubation of the resulting cytoskeletons with gamma-32P-ATP in situ revealed EGF-stimulated autophosphorylation of the EGF-R and high molecular weight cytoskeletal proteins on tyrosine residues. These proteins will be examined for the presence of phosphotyrosyl residues following in vivo labeling with 32Pi and treatment with EGF. EGF treatment of cells at low temperatures and binding of EGF directly to cytoskeletons failed to stimulate protein phosphorylation. This proposal will examine the role of structural association of the EGF-R in regulation of the EGF-R tyrosine kinase activity. Tyrosine kinase activity will be measured both by phosphorylation of the small peptide substrate angiotensin II and by the autophsphorylation of the EGF-R, which may be constrained due to its association with the cytoskeleton. In addition, studies are proposed to determine if other cellular events are involved in regulation of the EGF-R kinase activity. The nature of the protein-protein interactions responsible for the association of the EGF-R with the cytoskeleton will be explored with chemical cross linking reagents to identify proteins interacting with the EGF-R.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM034908-01A1
Application #
3286767
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1986-07-01
Project End
1989-06-30
Budget Start
1986-07-01
Budget End
1987-06-30
Support Year
1
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Medical University of South Carolina
Department
Type
Schools of Medicine
DUNS #
183710748
City
Charleston
State
SC
Country
United States
Zip Code
29425
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