Abstract:
: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.
journal_name
Proc Natl Acad Sci U S Aauthors
Zhu J,Kahn CRdoi
10.1073/pnas.94.24.13063subject
Has Abstractpub_date
1997-11-25 00:00:00pages
13063-8issue
24eissn
0027-8424issn
1091-6490journal_volume
94pub_type
杂志文章abstract::Accumulation of (45)Ca(++) was found to occur in membrane vesicles of E. coli prepared by lysis with a French pressure cell. The uptake occurs by active transport, requiring an energy source. Substrates of the electron transport chain, including D-lactate, reduced phenazine methosulfate, and NADH, stimulated accumulat...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
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