A Thermodynamic Study of Ligand Binding to the First Three Domains of the Human Insulin Receptor: Relationship between the Receptor R-Chain C-Terminal Peptide and the Site 1 Insulin Mimetic Peptides

摘要

The C-terminal segment of the insulin receptor (IR) R-chain plays a critical role in insulin binding.nThis 16-residue peptide together with the central β-sheet of the receptor L1 domain forms one of the insulinnbinding surfaces of the IRmonomer.Here we use isothermal titration calorimetry to assay directly the bindingnof the IR RCT peptide to an IR construct (IR485) consisting of the three N-terminal domains of the receptornmonomer. Our measurements show further that the binding of the IR RCT peptide to IR485 competes withnthe binding of a prototypical “Site 1” insulin mimetic peptide to the same receptor fragment. The competitivennature of their binding appears to be reflected in a previously undetected sequence similarity between the IRnRCT peptide and the Site 1 mimetic peptide. In contrast, a prototypical “Site 2” peptide has very limitednaffinity for IR485. Taken together, these results complement our recent observation that there is a possiblenstructural relationship between these mimetic peptides and insulin itself. They also add support to the viewnthat the segment of unexplained electron density lying on the surface of the central β-sheet of the L1 domain innthe IR ectodomain crystal structure arises from the IR RCT peptide. Finally, we show that mutation ofnthe critical IR RCT peptide residue Phe714 to alanine does not affect the peptide’s affinity for IR485nand conclude that the resultant loss of insulin binding with thismutation results fromloss of interaction of thenphenylalanine side chain with insulin.