The long-term goal of my laboratory is to understand the mechanism of cellular signaling, beginning at hormone-mediated activation of the heterotrimeric family of G proteins. My laboratory endeavors to determine the molecular details relating hormone-receptor binding with guanine-nucleotide exchange. The current working paradigm is the beta2-adrenergic receptor (beta2R) and the stimulatory heterotrimeric G protein, Gsabg. Considerable progress has been made toward understanding the mechanism of rhodopsin activity, the prototypical G protein-coupled receptor (GPCR). In contrast, comparatively little is known about other GPCRs. Furthermore, while it is clear that the structures of rhodopsin and several G proteins reveal the molecular details of their independent function, little is known about their structure and function as a complex. Our understanding of how activation of rhodopsin, and all GPCRs, leads to activation of G proteins, through stimulation of nucleotide exchange is incomplete. I propose to take a biochemical and structural approach to delineate the hormone-binding site structure, the quaternary structure of the beta2R:Gsabg complex, and develop a model to elucidate how hormone-dependent nucleotide exchange occurs. I will develop an expression and purification system for the production of active beta2R in a complex with Gsabg. These reagents will be used to assess real time spectrophotometric measurements of beta2R:Gsabg activity. I will take various approaches to determine and characterize the site of interaction between beta2R and Gsabg using cross-linking, site-directed mutagenesis and fluorescence resonance energy transfer spectrometry. I further extend that the beta2R exists as an oligomer and that the function of dimerization relates inherently to the mechanism of receptor activation, and thus G protein activation. I will engage in an effort to delineate the low and high resolution structures of the beta2R:Gsabg complex in various activation states. Finally, I will integrate these data to formulate a model of how hormone activation of beta2R leads to activation of nucleotide exchange and propose that this model will extend to the entire family of GPCRs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM068603-02
Application #
6784230
Study Section
Pharmacology A Study Section (PHRA)
Program Officer
Lograsso, Philip
Project Start
2003-08-01
Project End
2008-07-31
Budget Start
2004-08-01
Budget End
2005-07-31
Support Year
2
Fiscal Year
2004
Total Cost
$325,159
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Pharmacology
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
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