Peptides Identify the Critical Hotspots Involved in the Biological Activation of the Insulin Receptor

Renuka C Pillutla,Ku-Chuan Hsiao,James R Beasley,Jakob Brandt,Søren Østergaard,Per Hertz Hansen,Jane C Spetzler,Gillian M Danielsen,Asser S Andersen,Renee E Brissette,Michael Lennick,Paul W Fletcher,Arthur J Blume,Lauge Schäffer,Neil I Goldstein

doi:10.1074/jbc.m202119200

Renuka C Pillutla, Ku-Chuan Hsiao + Show 13 more

Open Access

https://doi.org/10.1074/jbc.m202119200

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Abstract

We used phage display to generate surrogate peptides that define the hotspots involved in protein-protein interaction between insulin and the insulin receptor. All of the peptides competed for insulin binding and had affinity constants in the high nanomolar to low micromolar range. Based on competition studies, peptides were grouped into non-overlapping Sites 1, 2, or 3. Some Site 1 peptides were able to activate the tyrosine kinase activity of the insulin receptor and act as agonists in the insulin-dependent fat cell assay, suggesting that Site 1 marks the hotspot involved in insulin-induced activation of the insulin receptor. On the other hand, Site 2 and 3 peptides were found to act as antagonists in the phosphorylation and fat cell assays. These data show that a peptide display can be used to define the molecular architecture of a receptor and to identify the critical regions required for biological activity in a site-directed manner.

Highlights

We used phage display to generate surrogate peptides that define the hotspots involved in protein-protein interaction between insulin and the insulin receptor
Group 2 consisted of peptides with short and long cysteine loops that bound to a non-overlapping hotspot (Site 2). Both Group 1 and Group 2 peptides displaced insulin in radioreceptor binding assays (Table I). These data indicated that the insulin binding domain on the insulin receptor (IR) could be subdivided into at least two smaller contact regions marked by the Site 1 and Site 2 peptides
The Site 2 and Site 3 surrogates were found to bind almost exclusively to IR and not to its related receptor IGF-1R, whereas the Site 1 peptides bound to both receptors with varying degrees of specificity

Summary

EXPERIMENTAL PROCEDURES

Preparation of the Random Peptide Phage Display Libraries—DNA fragments coding for peptides containing 20 – 40 random amino acids were generated by the following protocol. Additional phage libraries of random 20-mer peptides were constructed in a similar manner, except for the size of the initial oligonucleotide The diversity of this cell library was found to be Ͼ1.1 ϫ 1011 clones, and sequencing revealed that 77% of the clones were in-frame [13]. 100 ␮l of a phage library was added to the antigen-coated wells, and the plates were incubated for 3 h at room temperature. Each well was washed 13 times with PBS-2% NFM, and the phage was eluted with 100 ␮l of 50 mM glycine/HCl containing 0.1% bovine serum albumin (pH 2.2) following a 5-min incubation. 2 ϫ 1011 plaque-forming units of the phage library (complexity, 2 ϫ 109) was added to plates with immobilized IR for 2– 4 h at room temperature. The reactions were transferred to streptavidin-coated 96-well microtiter plates

Primary DGI

RESULTS

DISCUSSION