The objective of this research is to explore the biochemical events that lead from nuclear quiescence to mitogenesis using epidermal growth factor (EGF) as the activating agent. We have characterized the EGF-receptor from both mouse and human as a cell-surface polypeptide of Mr 170,000-180,000. We have antireceptor antibodies directed against the human receptor. Also we have demonstrated spontaneous and selective transfer of exogenous EGF-receptors from donor membranes to receptor-negative variant cells. These techniques and others (receptor fragmentation, photochemical crosslinking) will be exploited to investigate the receptor molecule domains that are involved in crucial interactions with other proteins in the membrane at the beginning of the mitogenic pathway. We will also use an insulin-receptor system as a model to study receptor development and expression in vitro. Regarding the intracellular events that are induced by EGF, we have described an EGF-induced cytoplasmic protein that can stimulate DNA synthesis in a cell-free nuclear assay. This in vitro assay will be used to study the protein factors that regulate entry into the DNA synthetic phase. A method for measuring loss of commitment to DNA replication has been developed by us, and our studies suggest that commitment is a whole-cell state that can decay in a single step. We plan to use 2-dimensional gel electrophoresis to identify proteins whose appearance or decay can be correlated with the cell's commitment or loss of commitment to DNA synthesis. In summary, our goal is to study molecular events at the receptor end of the mitogenic pathway, among intermediates, and at the final commitment stage.
Specific aims are directed at each of these stages.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
9R01CA043787-07
Application #
3186133
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1979-08-01
Project End
1991-03-31
Budget Start
1986-04-01
Budget End
1987-03-31
Support Year
7
Fiscal Year
1986
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Kesavan, P; Mukhopadhayay, S; Murphy, S et al. (1991) Thyroid hormone decreases the expression of epidermal growth factor receptor. J Biol Chem 266:10282-6
Das, M; Kesavan, P; Khire, J (1990) Control of expression and maturation of epidermal growth factor receptor. J Cell Biochem 44:61-8
Das, M; Khire, J; Kesavan, P (1990) Maturation and externalization of EGF-receptor: a cell-surface located oncoprotein. Indian J Biochem Biophys 27:438-42
Kesavan, P; Das, P; Kern, J et al. (1990) Regulation of stability and synthesis of EGF-receptor mRNAs encoding for intact and truncated receptor forms. Oncogene 5:483-8
Basu, A; Raghunath, M; Bishayee, S et al. (1989) Inhibition of tyrosine kinase activity of the epidermal growth factor (EGF) receptor by a truncated receptor form that binds to EGF: role for interreceptor interaction in kinase regulation. Mol Cell Biol 9:671-7
Bishayee, S; Majumdar, S; Khire, J et al. (1989) Ligand-induced dimerization of the platelet-derived growth factor receptor. Monomer-dimer interconversion occurs independent of receptor phosphorylation. J Biol Chem 264:11699-705
Das, M; Chauhan, S S; Mishra, V S et al. (1989) Aberrant postendocytotic fate of a 34-kDa molecular mass growth factor from human trophoblasts. Cancer Res 49:2761-5
DePalo, L; Das, M (1988) Epidermal growth factor-induced stimulation of epidermal growth factor-receptor synthesis in human cytotrophoblasts and A431 carcinoma cells. Cancer Res 48:1105-9
Roy-Choudhury, S; Mishra, V S; Low, M G et al. (1988) A phospholipid is the membrane-anchoring domain of a protein growth factor of molecular mass 34 kDa in placental trophoblasts. Proc Natl Acad Sci U S A 85:2014-8
Chianese, D; Roy-Choudhury, S; Murthy, U et al. (1988) Cell- and tissue-specific expression of a 34,000-molecular-weight peptide growth factor in humans. Hum Pathol 19:190-4

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