ABSTRACT
Insulin is thought to elicit its effects by crosslinking the two extracellular alpha-subunits of its receptor, thereby inducing a conformational change in the receptor, which activates the intracellular tyrosine kinase signaling cascade. Previously we identified a series of peptides binding to two discrete hotspots on the insulin receptor. Here we show that covalent linkage of such peptides into homodimers or heterodimers results in insulin agonists or antagonists, depending on how the peptides are linked. An optimized agonist has been shown, both in vitro and in vivo, to have a potency close to that of insulin itself. The ability to construct such peptide derivatives may offer a path for developing agonists or antagonists for treatment of a wide variety of diseases.
Subject(s)
Peptides/pharmacology , Receptor, Insulin/agonists , Receptor, Insulin/antagonists & inhibitors , Adipocytes/drug effects , Adipocytes/metabolism , Amino Acid Sequence , Animals , Dimerization , Humans , In Vitro Techniques , Insulin/pharmacology , Kinetics , Lipids/biosynthesis , Male , Mice , Molecular Sequence Data , Peptides/chemistry , Peptides/genetics , Protein Subunits , Rats , Rats, Wistar , Receptor, Insulin/genetics , Receptor, Insulin/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/pharmacologyABSTRACT
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.
Subject(s)
Receptor, Insulin/metabolism , Adipocytes/metabolism , Amino Acid Motifs , Animals , Binding Sites , Binding, Competitive , DNA/metabolism , Dose-Response Relationship, Drug , Enzyme-Linked Immunosorbent Assay , Humans , Kinetics , Mice , Mutagenesis, Site-Directed , Peptide Biosynthesis , Peptide Library , Peptides , Phosphorylation , Protein Binding , Protein Structure, Tertiary , Receptor, Insulin/chemistryABSTRACT
We have previously shown that a minimized insulin receptor (IR) consisting of the first 468 amino acids of the insulin receptor fused to 16 amino acids from the C terminus of the alpha-subunit (CT domain) bound insulin with nanomolar affinity (Kristensen, C., Wiberg, F. C., Schäffer, L., and Andersen, A. S. (1998) J. Biol. Chem. 273, 17780-17786). In the present study, we show that a smaller construct that has the first 308 residues fused to the CT domain also binds insulin. Insulin receptor fragments consisting of the first 468 or 308 residues did not bind insulin. However, when these fragments were mixed with a synthetic peptide corresponding to the CT domain, insulin binding was detectable. At concentrations of 10 microm CT peptide, insulin binding was fully reconstituted yielding apparent affinities of 9-11 nm. To further investigate the minimum requirement for the length of the N terminus of IR, we tested smaller receptor fragments for insulin binding in the presence of the CT peptide and found that a fragment consisting of the first 255 amino acids of IR was able to fully reconstitute the insulin binding site, yielding an apparent affinity of 11 +/- 4 nm for insulin.
