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Adenosine Receptor Oligomerization and G-Protein Complex Stoichiometry Revealed by Electron Microscopy

Wingard, Jennifer
Thesis/Dissertation; Online
Wingard, Jennifer
Yeager, Mark
The A2A adenosine receptor (A2AAR) belongs to a large superfamily of receptors (G-Protein Coupled Receptors), which are responsible for conveying signals from numerous external stimuli through their seven transmembrane helices to the membrane-associated heterotrimeric G-proteins. Drugs that target GPCRs comprise approximately 40% of the pharmaceutical market. Expressed highly in the brain, where it is thought to heterodimerize with the dopamine receptor, the A2AAR is the target of drugs treating Parkinson’s disease [1]. In the cardiovasculature, A2AAR blockade yields protection from ischemia and hypoxia [2]. Additional studies have delineated the roles of A2AAR in immunomodulation [2]. Detailing the A2AAR molecular architecture with a thorough understanding of GPCR dimerization is crucial to understanding mechanisms of transmembrane signaling and the development of new therapeutics that target the dimer interface. The publication of the X-ray crystal structure and three-dimensional (3D) single particle electron microscopy (EM) reconstructions of the β2 adrenergic receptor (β2AR) in a complex with Gs by 2012 Nobel prize-winner Dr. Brian Kobilka and colleagues provided surprising new insight into the flexibility of the helical domain of Gs and the direct mechanism of nucleotide exchange. Additional GPCR complex structures are necessary to corroborate this novel finding and determine whether the exchange mechanism is common to other G-proteins. Two-dimensional electron crystallography and atomic force microscopy studies suggest that the GPCR rhodopsin exists as a physiological dimer in native rod outer segment disc membranes; single particle 3D reconstructions of detergent-solubilized rhodopsin also suggest that the receptor is a dimer when associated with the G-protein. In contrast, 3D reconstructions of detergent-solubilized β2AR-Gs complexes suggest that the G-protein-bound receptor exists as a monomer. To date, rhodopsin is the only GPCR that has been crystallized within the milieu of the lipid bilayer. Unlike other Class A GPCRs, it is unusual in that it has a covalently bound ligand, 11-cis retinal. It remains unclear how GPCRs that bind diffusible ligands are organized within the lipid bilayer. To delineate the topology of the adenosine receptor within the lipid bilayer and with respect to the G-protein, my research has utilized electron microscopy and the complementary approaches of single particle image analysis and two-dimensional (2D) electron crystallography. The results provide insights about adenosine receptor-G-protein complex formation and suggest parallel dimerization of A2AAR that is mediated by interactions between helices H1 and H8 in the near-native environment of a lipid bilayer.
University of Virginia, Department of Biophysics, PHD (Doctor of Philosophy), 2015
Published Date
PHD (Doctor of Philosophy)
1. Ferre, S., C. Quiroz, A.S., et al., An update on adenosine A2A-dopamine D2 receptor interactions: implications for the function of G protein-coupled receptors. Curr Pharm Des, 2008. 14(15): p. 1468-74. 2. Link, A.A., T. Kino, J.A., et al., Ligand-activation of the adenosine A2a receptors inhibits IL-12 production by human monocytes. J Immunol, 2000. 164(1): p. 436-42.
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