Insulin Receptors Methods For The Study Of Structure And Function

Author : Robert Zhao-Tian Luo
Publisher :
ISBN 13 :
Total Pages : 0 pages
Book Rating : 4.:/5 (133 download)

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Download or read book Structure and Function of the Human Insulin Receptor, Quaternary 3D Reconstruction of the Insulin-insulin Receptor Complex and the Structural Localization of the Insulin-binding Site written by Robert Zhao-Tian Luo and published by . This book was released on 2000 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: The elucidation of the mechanism of cellular signal transduction is one of the key challenges in biology. The first step in this pathway has been particularly refractory to study. In the case of insulin action, it is the binding of insulin to its cell surface receptor, a large integral transmembrane glycoprotein. The binding of insulin results in the activation of the cytoplasmic domain of the receptor as a tyrosine kinase. While much is known about the interaction of the activated receptor with its substrates leading to downstream signal transduction, how the binding of insulin to the extracellular domain of the receptor can lead to receptor activation remains unknown. The specific goal of this study is to localize the insulin-binding site of the insulin receptor (IR), and, if possible, to elucidate in molecular details the insulin-IR interaction and the structure-function relationship of the insulin receptor. To achieve this goal with biochemical approach, I used the baculovirus expression system to produce in large quantity the extracellular domain of the human insulin receptor, and used various specific photoaffinity probes to identify the insulin-contacting sites. Peptide identification methods of high sensitivity were developed to detect the labeled IR fragment containing the putative insulin-binding site by HPLC peptide mapping analysis. A small fragment of 1-3 kDa that might contain the putative insulin-binding or contacting site on the receptor à subunit was detected. However, partly due to the lack of an insulin photoprobe with a high efficiency of cross-linking, and a high efficiency peptide mapping method, I failed to obtain a sufficient quantity of the fragment for amino acid sequencing. In an alternative approach, using gold-labeled insulin as a ligand, and sets of electron micrographs obtained by low-dose low temperature dark field scanning transmission electron microscopy (STEM) of the insulin-IR complex, I successfully reconstructed the 3D quaternary structure of the whole 480-kDa human insulin receptor (HIR), complexed with insulin, at a resolution of 20 Å. Contiguous high densities within the 3D structure indicated a two-fold symmetry for this dimeric receptor and the presence of subdomain structures which begin with the extracellular subdomain involved in insulin binding and terminate with the intracellular tyrosine kinase subdomains. These subdomains were confirmed by fitting known high-resolution crystal domain substructures. In addition, the 3D insulin-HIR complex provides evidence for the involvement of only a single insulin molecule in receptor activation. It also explains many of the known characteristics of insulin binding to its receptor and many biochemical findings in the studies of the insulin receptor.