Identification of IGF-II-Binding Site on the Quaternary 3-D Structure of the Insulin Receptor

摘要

The purpose of the project is to obtain the 3D reconstruction structure of the IGP-Il/insulin receptor isoform A(IR-A) complex by crySTEM to elucidate the laP-Il binding site on IR-A. Gold-labeled IGF-II has been prepared by reacting IGP-II with sulfo-NHS-Nanogold, and purified by size-exclusion HPThC. The purified product, Nanogold-IGF-lI, was labeled with gold at its N-terminal alanine. Insulin receptor proteins, IR-A and IR-B have been extracted and purified from transfectant cells. Nanogold-IGF-II, like IGE-lI, stimulated the autophosphorylation of IR-A. A complex of Nanogold-IGP-II and IR-A was prepared for cryoSTEM 3D reconstruction. Preliminary STEM of the complex showed images of particles of the expected size with electron-dense gold marker. These results demonstrate that it is possible to prepare a biologically active gold-labeled IGE-Il, and that a complex formed between it and the insulin receptor isoform IR-A can be used in cryoSTEM for the 3D reconstruction of the laP-Il/IR-A complex for the purpose of elucidating the binding site of laP-Il in the insulin receptor isoform IR-A.