Analysis of a peptide hormone-receptor interaction in the yeast two-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 insulin-like growth factor 1 (IGF-1) with its receptor by using GAL4 transcriptional regulation with a beta-galactosidase assay as readout. In this system, IGF-1 and proIGF-1 bound to the cysteine-rich domain, extracellular domain, or entire IGF-1 proreceptor. This interaction was specific. Thus, proinsulin showed no significant interaction with the IGF-1 receptor, while a chimeric proinsulin containing the C-peptide of IGF-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 strong interaction, 20% showed an intermediate interaction, and 40% give little or no signal. Of 50 mutants that were sequenced, several (Leu-5 --> His, Glu-9 --> Val, Arg-37 --> Gly, and Met-59 --> Leu) appeared to enhance receptor association, others resulted in weaker receptor interaction (Tyr-31 --> Phe and Ile-43 --> Phe), and two gave no detectable signal (Leu-14 --> Arg and Glu-46 --> Ala). Using PCR-based mutagenesis with proinsulin, we also identified a gain of function mutant (proinsulin Leu-17 --> Pro) that allowed for a strong IGF-1-receptor interaction. These data demonstrate that the specificity of the interaction between a hormone and its receptor can be characterized with high efficiency in the two-hybrid system and that novel hormone analogues may be found by this method.
Zhu
J
J
Joslin Diabetes Center, and Department of Medicine, Harvard Medical School, Boston, MA 02215, USA.
Kahn
C R
CR
D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011384:Proinsulin / Q000378:metabolism; D011485:Protein Binding; D017526:Receptor, IGF Type 1 / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
D007334:Insulin-Like Growth Factor I; D011384:Proinsulin; D017526:Receptor, IGF Type 1