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.
| Cai, Yingying; Liu, Yuting; Culhane, Kelly J et al. (2017) Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor. PLoS One 12:e0179568 |
| DeVree, Brian T; Mahoney, Jacob P; Vélez-Ruiz, Gisselle A et al. (2016) Allosteric coupling from G protein to the agonist-binding pocket in GPCRs. Nature 535:182-6 |
| Mahoney, Jacob P; Sunahara, Roger K (2016) Mechanistic insights into GPCR-G protein interactions. Curr Opin Struct Biol 41:247-254 |
| Mallik, Leena; Dhakal, Soma; Nichols, Joseph et al. (2015) Electron Microscopic Visualization of Protein Assemblies on Flattened DNA Origami. ACS Nano 9:7133-41 |
| Irannejad, Roshanak; Tomshine, Jin C; Tomshine, Jon R et al. (2013) Conformational biosensors reveal GPCR signalling from endosomes. Nature 495:534-8 |
| Westfield, Gerwin H; Rasmussen, Søren G F; Su, Min et al. (2011) Structural flexibility of the G alpha s alpha-helical domain in the beta2-adrenoceptor Gs complex. Proc Natl Acad Sci U S A 108:16086-91 |
| Rasmussen, Søren G F; Choi, Hee-Jung; Fung, Juan Jose et al. (2011) Structure of a nanobody-stabilized active state of the ?(2) adrenoceptor. Nature 469:175-80 |
| Rosenbaum, Daniel M; Zhang, Cheng; Lyons, Joseph A et al. (2011) Structure and function of an irreversible agonist-ýý(2) adrenoceptor complex. Nature 469:236-40 |
| Vélez-Ruiz, Gisselle A; Sunahara, Roger K (2011) Reconstitution of G protein-coupled receptors into a model bilayer system: reconstituted high-density lipoprotein particles. Methods Mol Biol 756:167-82 |
| Rasmussen, Søren G F; DeVree, Brian T; Zou, Yaozhong et al. (2011) Crystal structure of the ?2 adrenergic receptor-Gs protein complex. Nature 477:549-55 |
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