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.
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