Biochemical And Biophysical Characteristic Analysis Of Glucagon Receptor

Glucagon is a pancreatic hormone that plays pivotal roles in regulating glucose homeostasis and metabolism. Glucagon exerts its action by binding to its receptor, glucagon receptor (GCGR), a typical member of class B G-protein coupled receptors (GPCRs), which can interact with G protein upon activation to stimulate the second messanger system for biological functions. Diabetes is a bihormonal disease in which excessive glucagon secretion is a major contributor in the patho genesis of this disease. And antibodies or small molecules that can antagonize the glucagon receptor effect were demonstrated to have glucose-lowering effect in diabetes animal models. So elucidation of how glucagon binds to GCGR will facilitate the rational design of the GCGR antagonist for treating diabetic hyperglycemia.Here we report the successful expression and purification of the GCGR extracellular domain (GCGR-ECD) and its fusion protein (GCGR-ECD fused with glucagon, GCGR-ECD-Gc) with the glucagon peptide fused at its C-terminus. We utilized the maltose binding protein (MBP) fusion method for the success of the soluble expression of both GCGR-ECD and GCGR-ECD-Gc in E.coll We also obtained a high yield production of secretable GCGR-ECD with the baculovirus expression system by optimizing its N-terminal secreting signal. The final proteins purified from both systems can achieve purity up to90%with high homogeneity. The author first utilized isothermal titration calorimetry (ITC) method to determine the binding affinity of isolated GCGR-ECD for its authentic ligand in vitro. No significant differences were found between the binding affinity of the prokaryotic expressed GCGR-ECD (7.6μM) and eukaryotic glycosylated one (6.6μM) for glucagon.The binding analysis of GCGR-ECD-Gc with exogeneous glucagon by ITC suggestes intra ligand-receptor binding within the fusion protein.We employed several kinds of biophysical methods to detect the structural features or thermostability of recombinant GCGR-ECD proteins. Although trial of crystallizing GCGR-ECD proteins puoduced in both prokaryotic and eukaryotic systems failed under all kinds of conditions, the key obstacle in crystallization was found by the author. The auto-degradation was prominent for the MBP fused GCGR-ECD and sGCGR-ECD proteins from insect cells were difficult to crystallize without specific antibody. The author also shed new lights on the potential solvement of the problems which can be referred to by others.At the end of the thesis, a review of recent research on GPCR structure and function was presented.