The mechanism of growth factor and receptor interaction is the objective of this project. We utilize the murine CSF-l receptor as a prototypic growth factor receptor. As the initial step, the receptor has been cloned, sequenced and subsequently introduced into tissue culture cells via retroviral gene transfer techniques to demonstrate its functionality. The specific question being addressed is whether ligand (growth factor) activation of the receptor at an enzyme (tyrosine kinase) occurs via an intramolecular or intermolecular mechanism. In one case we modify the receptor (at the cDNA level) such that the extracellular ligand binding domain is structurally separated from the rest of the receptor although still covalently attached. In this manner we hope to disrupt the transmission of any intramolecular conformational signal. In the second case, we construct a hybrid glycophorin A - CSF-l receptor that can be aggregated by an antiglycophorin antibody in order to study enzyme activation via an intermolecular process. We are also in the process of raising polyclonal antibodies to synthetic peptides representing different parts of the receptor for use as conformational probes. Finally, to enable biochemical and biophysical studies to be performed on growth factor receptors, we are overexpressing the extracellular domain of the CSF-l receptor in mammalian and insect cell systems.
