ANALYSIS OF A PEPTIDE HORMONE-RECEPTOR INTERACTION IN THE YEAST 2-HYBRID SYSTEM

Interaction between a peptide hormone and extracellular domains of its receptor is a crucial step for initiation of hormone action, We have developed a modification of the yeast two-hybrid system to study this interaction and have used it to characterize the interaction of insuli n-like growth factor 1 (IGF-1) with its receptor by using GAL4 transcr iptional regulation with a beta-galactosidase assay as readout, In thi s system, IGF-1 and proIGF-1 bound to the cysteine-rich domain, extrac ellular domain, or entire IGF-1 proreceptor, This interaction was spec ific, Thus, proinsulin showed no significant interaction with the IGF- 1 receptor, while a chimeric proinsulin containing the C-peptide of IG F-1 had an intermediate interaction, consistent with its affinity for the IGF-1 receptor, Over 2000 IGF-1 mutants were generated by PCR and screened for interaction with the color assay, About 40% showed a stro ng interaction, 20% showed an intermediate interaction, and 40% give l ittle or no signal, Of 50 mutants that were sequenced, several (Leu-5 --> His, Glu-9 --> Val, Arg-37 --> Gly, and Met-59 --> Leu) appeared t o enhance receptor association, others resulted in weaker receptor int eraction (Tyr-31 --> Phe and Ile-43 --> Phe), and two gave no detectab le signal (Leu-14 --> Arg and Glu-46 --> Ala), Using PCR-based mutagen esis with proinsulin, we also identified a gain of function mutant (pr oinsulin Leu-17 --> Pro) that allowed for a strong IGF-1-receptor inte raction, These data demonstrate that the specificity of the interactio n between a hormone and its 0receptor can be characterized with high e fficiency in the two-hybrid system and that novel hormone analogues ma y be found by this method.