G protein-coupled-receptors (GPCRs) are the largest family of human proteins. They are involved in many physiological processes and implicated in numerous human diseases. GPCRs bind to extracellular ligands and transduce this signal to the intracellular G protein complex. Class-A GPCRs contain about 700 members and recognize a variety of ligands. The structures of two human class-A GPCRs: beta2-adrenergic and A2A adenosine, were solved in Dr. Ray Stevens'laboratory. These structures were solved with technological breakthroughs in baculovirus protein expression, receptor stabilization, and crystallization in cholesterol doped lipid cubic phase. Class-B GPCRs are distinctly different given the large extracellular N- terminal domain combined with the common 7-helix transmembrane domain. This subfamily recognizes hormone peptides that are of critical importance in biology and drug discovery (e.g. blood glucose regulation and bone homeostasis). The activation mechanisms of ligand binding and signal transduction for the class-B receptors are unclear given the critical role of the large extracellular N-terminal domain. The goal of this proposal is to reveal important details regarding the activation mechanisms of the glucagon receptor by determining the structure of the full-length class-B human glucagon receptor bound to its ligand using X-ray crystallography, and studying the effects of various amino acid residues in the receptor on ligand binding and induced signaling using site-directed mutagenesis. The processes of construct design, expression, and purification for the structural study of the glucagon receptor have been conducted. Encouraging preliminary results show high-level expression of the full-length glucagon receptor, and the purified receptor is bound and stabilized with small compound and peptide antagonists. These results demonstrate two of the most difficult structure determination hurdles have been overcome. Crystals of the glucagon receptor-ligand complex will be obtained using a cholesterol doped lipid cubic phase (LCP) crystallization method, and the structure solved using an X-ray mini-beam at the Advanced Photon Source at Argonne National Laboratory. Mutants of the transmembrane domain of the glucagon receptor will be used to measure ligand binding affinities and signal transduction levels. The mutagenesis study will reveal residues in the transmembrane domain of the glucagon receptor involved in the interaction of ligand binding and induced signaling. Together, the structure and mutagenesis study of the glucagon receptor will advance the understanding of class-B GPCRs, and facilitate drug development for diabetes and other class-B related diseases.

Public Health Relevance

Class-B G protein-coupled receptors (GPCRs) recognize hormone peptides that are critical to pathological conditions such as diabetes, osteoporosis, cancer, obesity, and neurological disorders, including depression and anxiety. The goal of this proposal is to elucidate details of ligand binding and induced signaling of the class-B glucagon receptor by 1) obtaining a high-resolution structure of the ligand-receptor complex using X-ray crystallography, and 2) studying the role of various amino acid residues in the receptor involved in ligand binding and signaling by site-directed mutagenesis. The structure and the mutagenesis study will reveal important details regarding the activation mechanisms upon ligand binding and induced signaling of the glucagon receptor that will advance the understanding of class-B GPCRs and ultimately, leading to better treatment of various prevalent diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32DK088392-03
Application #
8320780
Study Section
Special Emphasis Panel (ZDK1-GRB-W (O1))
Program Officer
Castle, Arthur
Project Start
2010-09-30
Project End
2013-09-29
Budget Start
2012-09-30
Budget End
2013-09-29
Support Year
3
Fiscal Year
2012
Total Cost
$55,670
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Siu, Fai Yiu; He, Min; de Graaf, Chris et al. (2013) Structure of the human glucagon class B G-protein-coupled receptor. Nature 499:444-9
Wang, Chong; Wu, Huixian; Katritch, Vsevolod et al. (2013) Structure of the human smoothened receptor bound to an antitumour agent. Nature 497:338-43