The long range goal of this project is to investigate the mechanism of action of the peptide hormone glucagon and to contribute to the understanding of its role in the pathophysiology of diabetes mellitus. The principal approach towards this goal is to design and synthesize peptide analogs that will be potent antagonists of the hormone, using solid phase peptide synthesis. Such inhibitors are expected to provide insight into the structural basis of glucagon action at the molecular level, and might provide potentially therapeutic agents for the clinical management of hyperglycemia and ketoacidosis associated with diabetes. A second complementary approach is to investigate structure-function relationships in the glucagon receptor by site-directed mutagenesis and the biochemical and pharmacological characterization of mutant receptors. Specifically, the residues of the receptor that participate directly in glucagon recognition and mediate the subsequent transduction of the hormonal signal to intracellular effectors, will be identified. Information on the chemical features of the glucagon binding site is crucial for the conception of three-dimensional receptor models to be used in the rational design of glucagon antagonists of potential clinical value. To augment both approaches, glucagon interaction with its receptor will also be investigated using biophysical methods. Fluorescence and electron paramagnetic resonance spectroscopy will be used to monitor changes that accompany receptor activation. Nuclear magnetic resonance studies of glucagon bound to its receptor will be initiated. The combination of synthetic chemical approaches with molecular biology and biophysical methods is very likely to advance the understanding of glucagon-mediated signal transduction.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK024039-24
Application #
6380417
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Sechi, Salvatore
Project Start
1978-09-15
Project End
2003-08-31
Budget Start
2001-09-01
Budget End
2002-08-31
Support Year
24
Fiscal Year
2001
Total Cost
$287,279
Indirect Cost
Name
Rockefeller University
Department
Biochemistry
Type
Other Domestic Higher Education
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
Sachdev, Pallavi; Tirunagari, Laxmi Manohar; Kappei, Dennis et al. (2009) Monitoring glucagon and glucagon antagonist-mediated internalization: a useful approach to study glucagon receptor pharmacology. Adv Exp Med Biol 611:325-6
Unson, Cecilia G (2008) Expression of glucagon receptors in tetracycline-inducible HEK293S GnT1- stable cell lines: an approach toward purification of receptor protein for structural studies. Biopolymers 90:287-96
Sapse, Anne-Marie; Rothchild, Robert; Jain, Duli C et al. (2002) The role of salt bridge formation in glucagon: an experimental and theoretical study of glucagon analogs and peptide fragments of glucagon. Mol Med 8:251-62
Unson, Cecilia G; Wu, Cui-Rong; Jiang, Youwei et al. (2002) Roles of specific extracellular domains of the glucagon receptor in ligand binding and signaling. Biochemistry 41:11795-803
Jiang, Y; Cypess, A M; Muse, E D et al. (2001) Glucagon receptor activates extracellular signal-regulated protein kinase 1/2 via cAMP-dependent protein kinase. Proc Natl Acad Sci U S A 98:10102-7
Unson, C G; Wu, C R; Sakmar, T P et al. (2000) Selective stabilization of the high affinity binding conformation of glucagon receptor by the long splice variant of Galpha(s). J Biol Chem 275:21631-8
Cypess, A M; Unson, C G; Wu, C R et al. (1999) Two cytoplasmic loops of the glucagon receptor are required to elevate cAMP or intracellular calcium. J Biol Chem 274:19455-64
Unson, C G; Wu, C R; Cheung, C P et al. (1998) Positively charged residues at positions 12, 17, and 18 of glucagon ensure maximum biological potency. J Biol Chem 273:10308-12
Unson, C G; Cypess, A M; Wu, C R et al. (1996) Antibodies against specific extracellular epitopes of the glucagon receptor block glucagon binding. Proc Natl Acad Sci U S A 93:310-5
Unson, C G; Cypess, A M; Kim, H N et al. (1995) Characterization of deletion and truncation mutants of the rat glucagon receptor. Seven transmembrane segments are necessary for receptor transport to the plasma membrane and glucagon binding. J Biol Chem 270:27720-7

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