High resolution structural information is not available for G protein- coupled receptors (GPCRs), an exceedingly important family of cell surface receptors. Our long-range objective is to delineate the molecular mechanism of signal transduction by peptide hormone receptors of the GPCR family. We have chosen the glucagon receptor as a model system for study, and we will attempt to obtain site-specific structural information about the receptor using an interdisciplinary approach. The structural features of the glucagon receptor that dictate ligand-binding affinity, G protein coupling, and downstream signaling will be determined.
Specific Aim 1 is to identify the structural basis for glucagon binding affinity and specificity for the glucagon receptor. We will prepare mutant receptors and glucagon analogues and characterize their molecular properties. Detailed information about the chemical features of the hormone binding site are important for the subsequent study of the activation mechanism of the receptor.
Specific Aim 2 is to identify the structural determinants of the glucagon receptor that mediate glucagon-dependent signaling pathways. Glucagon causes both cAMP and intracellular calcium concentrations to increase. Definitive experiments to explain this dual signaling pathway and to identify the G proteins that mediate the calcium flux remain to be performed. Therefore, we have established stable cell lines and we have developed a reliable assay to measure calcium flux to address the mechanism of the glucagon-dependent calcium response.
Specific Aim 3 is to determine how glucagon binding leads to receptor activation using biochemical and biophysical approaches. To accomplish this aim, a procedure to isolate sufficient functional receptor from an insect cell expression system, or stable cell line, will be developed. We will study the receptors using fluorescence and electron paramagnetic resonance spectroscopy to monitor conformational changes that accompany ligand binding and receptor activation. Results from this interdisciplinary approach will advance our knowledge of the molecular events that occur during cellular signaling by glucagon receptor, a model system for the study of peptide-hormone-binding GPCRs.
