Interrelationships have been established in the response of cells to EGF, FGF, PDGF and insulin. All these hormones interact at the level of receptor down regulation. Further kinetic studies are proposed, leading to an approach to identify and isolate common, down regulation process-limiting components. Preliminary studies indicate that the process of internalization of the EGF receptor is subject to cell density in 3T3 cells. A thorough and systematic study is proposed to investigate this phenomenon, and to compare the effects of cell density on the inhibition of EGF receptor down regulation, EGF receptor display and EGF-induced stimulation of DNA synthesis. Upon exposure of 3T3 cells to EGF at 22 degrees and extended incubation, EGF adds covalently to its receptor; a characterization of this reaction is proposed. Using the direct labeling reaction, the EGF receptor has been identified in normal human fibroblasts and in the A431 human tumor line; it appears that the same species is present in both. Fingerprinting studies are proposed to test for subtle differences. In A431 lines the receptor accounts for a large share of the integral membrane proteins. A small scale isolation procedure is described; a large scale purification is proposed. The purified receptor protein will be purified and biochemically characterized, and antibodies will be raised to receptor protein. In the event that the immune serum cross reacts significantly with other membrane proteins, the cross reacting proteins will be identified. Procedures are described which may allow the assignment of specific receptor function to certain of these proteins. The antiserum will also be tested to see if it can elicit an EGF-like response on normal human cells, possibly by causing patching and/or endocytosis of receptor.
NIH ExPorter
NIH ExPorter