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

摘要

The ultimate goal of the research project is to reconstruct the 3- dimensional quaternary structure of the IGF-II/isoform A (IR-A) of the insulin receptor by cryo scanning transmission electronmicroscopy (cryoSTEM) using the electron-dense gold-labeled IGF-II to visualize the complex. During the first year of the grant support we succeeded in preparing in micro-scale gold-labeled IGF-II by reacting IGF-II with sulfo-NHS-Nanogold. The gold labeling occurred at the N-terminal alanine of IGF-II. The gold-IGF-II, like native IGF-II, activated the autophosphorylation of the isoform A (IR-A) but not the isoform B (IR-B) of the insulin receptor. We began this second year of grant support to scale up the preparation of the gold-ICE-II. We have now prepared a sufficient quantity (approx. 100 microng) of gold-ICE-II which is biologically as active as ICE-II in activating the autophosphorylation of IR-A. In preparation for the formation of the ICE-II/IR-A complex we have begun the large-scale preparation of IR-A protein from cultures of transfectant cells expressing this receptor protein.