To extend current work, we will further document the finding that elevation of binding of 125I-EGF, which occurs on reduction of medium Ca++, does not depend on protein synthesis by including puromycin in the medium. Then, to determine whether these exposed receptors are physiologically active, we hope to develop a biochemical measure of EGF action such as glucose or ion transport. For example cells grown in glass vials will be incubated with 14C-glucose and the production of 14C-C02 measured by absorption by hydroxide of hyamine according to known methods. To extend present work demonstrating fibronectin production by keratinocytes, we will label cells with 35S methionine, extract with 1% Triton X-100, and solublize the residue with SDS, then immunoprecipitate according to Hynes after reducing the SDS concentration. The precipitates will be run on SDS gels and autoradiograms developed. Using this methodology, we will be able to examine modulation of fibronectin production by agents such as EGF and cholera toxin and we will then extend these observations to other extracellular matrix proteins including laminin and pemphigoid antigen. These studies may help determine how basement membrane proteins such as laminin, fibronectin, and pemphigoid antigen are produced only at the base of the epithelium and not at higher levels. To pursue production of anti-EGF receptor antibody, we will continue to study an antibody already produced whose specificity is uncertain. We also hope to purify receptors from A-431 cells or human placenta in nondenaturing detergents by affinity chromatography on concanavalin-A Sepharose and EGF-Sepharose using receptor phosphorylation with gamma 32P-ATP as an assay and then raise antibodies in rabbits. The antibody would be used to study EGF receptor content in cultured keratinocytes from patients with lamellar ichthyosis or psoriasis as originally proposed.
| O'Keefe, E J; Woodley, D T; Falk, R J et al. (1987) Production of fibronectin by epithelium in a skin equivalent. J Invest Dermatol 88:634-9 |
| Petersen, M J; Woodley, D T; Stricklin, G P et al. (1987) Production of procollagenase by cultured human keratinocytes. J Biol Chem 262:835-40 |
| Olashaw, N E; O'Keefe, E J; Pledger, W J (1986) Platelet-derived growth factor modulates epidermal growth factor receptors by a mechanism distinct from that of phorbol esters. Proc Natl Acad Sci U S A 83:3834-8 |
| O'Keefe, E J; Payne, R E; Russell, N (1985) Keratinocyte growth-promoting activity from human placenta. J Cell Physiol 124:439-45 |
