Peptide receptors can be categorized as those which are sequence-specific for their ligands and those which will bind sterically accessible peptides without respect to sequence. In the former case, ligand-receptor interactions presumably involve amino acyl side chains of the ligand, while in the latter case, the backbone of the ligand must provide most of the sites of interaction. We are investigating membrane-associated peptide receptors of both of these classes. In the first class, we are studying the epidermal growth factor (EGF) receptor and EGF-stimulable protein kinase. We have affinity labeled the kinase in A431 cells and have provided substantive evidence that the EGF receptor and the EGF-stimulable protein kinase are parts of the same polypeptide chain. We have hypothesized that for the EGF system, a primary transmembrane signaling event is the allosteric activation of the kinase triggered by the binding of EGF to the receptor. We are planning to continue our study of the EGF receptor/protein kinase by determining the stoichiometry of kinase sites to receptor sites. We also plan to fluorescent label the kinase so as to be able to detect by fluorescence spectroscopy a conformational change in that site on binding of EGF to the receptor. Further, we plan to study by cross-linking the intermolecular interactions of the clustered EGF receptor/protein kinase with other molecules in the membrane and the interactions of the receptor/kinase with elements of the cytoskeleton. In the second class, we are investigating chemotaxis/transport receptors in Escherichia coli. We have obtained preliminary evidence that E. coli are chemotactically attracted toward peptides. In addition, we have photoaffinity labeled peptide binding proteins in the E. coli envelope with a reagent which we have shown to be a potent photoaffinity inhibitor of peptide transport. We plan to test the hypothesis that the E. coli chemotaxis/transport systems are coupled in that they share initial receptors for peptides. We plan to identify by photoaffinity labeling and to purify using affinity chromatography these initial receptors. We then plan to investigate the interactions of these initial receptors with other components of the chemotaxis/transport systems.
