FUNCTIONAL INTERACTION OF LIGANDS AND RECEPTORS OF THE HEMATOPOIETIC SUPERFAMILY IN YEAST

Circulating peptide hormones and growth factors interact with cell sur face receptors to initiate specific cellular responses, These complexe s can consist of a simple association between two proteins or a more e laborate association of multiple proteins. We describe the functional expression of ligands and corresponding receptors in a microbial syste m useful for the rapid dissection of these important protein interacti ons. GH or PRL and extracellular domains of their respective receptors were functionally expressed as fusion proteins in an extended two-hyb rid protein-protein interaction system, Reversible and specific ligand -receptor interactions were demonstrated by concurrent expression of f ree ligand peptides (GH or PRL) as binding competitors. The versatilit y established by expressing three heterologous proteins allowed for th e investigation of higher order structures. Ligand-dependent GH recept or dimerization was demonstrated but PRL receptor dimerization was not observed in an analogous assay, suggesting that these related growth factors may not engage receptors in a similar manner, Additionally, si gnificant association of GH receptors was observed in the absence of l igand, suggesting that there may be substantial avidity between these receptor proteins before ligand binding, Ligand-dependent and ligand-i ndependent receptor dimerization was demonstrated by vascular endothel ial growth factor and receptor proteins in similar assays. These findi ngs indicate that extracellular protein interactions such as ligand-re ceptor association, as well as the formation of higher order protein s tructures important for the activation of hematopoietic receptors, can be rapidly investigated in this microbial expression system.