Receptor-receptor, ligand, and membrane interactions of the adenosine A2A receptor
Date
2016
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
G Protein-Coupled Receptors (GPCRs) comprise the largest superfamily of membrane proteins in the human genome, with ~800 GPCRs identified. These receptors are responsible for many essential functions in day-to-day life, including sight, taste, neural and cardiac activity, and the immune system. Though this receptor superfamily is known to play a critical role in everyday life, many details of GPCR activity remain unclear. Within the GPCR superfamily is the adenosine receptor family, comprised of four receptor subtypes (A 1R, A2AR, A2BR, and A3R). The adenosine receptors predominantly mediate signaling through Gαs and Gαi G-protein subtypes, and are involved in neural, cardiac, pulmonary, and immune systems, and currently being studied for their role in Parkinson’s and Huntington’s diseases. As such, the adenosine receptors provide a potentially rich therapeutic target.
In canonical GPCR-ligand interaction, an extracellular ligand binds to the receptor at the cell surface and initiates a downstream signaling cascade through an associated heterotrimeric G protein complex. In the complex world of cells, GPCR-mediated signaling is known to be rather more complex than the canonical model. GPCRs are exposed to numerous other proteins and lipids within the plasma membrane. Although most of the protein interactions are coincidental results of the constrained environment of the plasma membrane, experimental evidence has been building over the last ten years to suggest that GPCR oligomerization modulates signaling. In addition to modulation through receptor-receptor interactions, multiple ligands interact with GPCRs to upregulate, prevent, or reduce basal signaling. Some ligands also facilitate upregulation of signaling in certain pathways over others. The molecular details of how ligands orchestrate conformational changes in a receptor and which receptor conformations lead to biased signaling remain largely unknown.
A yeast strain with a fluorescent reporter of GPCR activation was developed to examine the interaction between A2A and A2B adenosine receptors, and the influence of these interactions on GPCR-mediated MAPK signaling. The interactions were also monitored using Forster Resonant Energy Transfer (FRET) between fluorescent tags on the receptor monomers. As expected, agonist addition upregulated the MAPK pathway, while antagonist addition prevented MAPK upregulation. Significant MAPK signaling cross-talk was observed between homo- and heterodimers, and the FRET signal was found to be dynamic depending on the ligand added. In addition to upregulating signaling independently, A2AR and A2BR appear to form a signaling-sensitized complex that responds to some ligands differently than either receptor singly.
Ligand binding plays a fundamental role in stimulating the response of receptors to affect the downstream signaling. Various natural and artificial ligands for A2AR have been identified, but the kinetic binding properties of the ligands are not well understood. The equilibrium affinity constant KD is frequently reported, and has been used to screen for small molecules in the early stages of drug development. However, the KD does not indicate the pharmacological kinetics of the ligand, including how rapidly it associates and dissociates from the receptor. Ligand binding kinetics of A2AR reconstituted in detergent micelles were measured using fluorescence anisotropy. Importantly, these experiments were conducted close to the ligand depletion regime, in which the analytical solutions for binding kinetics are expected to lose validity. Here we modeled the kinetic data using numerical as well as analytical solutions. The most accurate parameters required a combination of the one-ligand numerical solutions and the two-ligand analytical solution.
As membrane proteins, GPCRs are also exposed to the various lipid components of the plasma membrane. Over the years, many studies have shown that A 2AR is stabilized by cholesterol, which is also essential in the retention of ligand binding upon purification. Here we reconstitute A2AR into liposomes, and test the effect of cholesterol supplementation in the liposomes on ligand binding. A significant increase was found in ligand binding of A2AR in cholesterol-supplemented liposomes over micelle-solubilized A2AR, but interestingly, no significant change between the A2AR in cholesterol-supplemented liposomes compared to cholesterol-free liposomes. In the future, this system may be further used to explore the effects of various lipids on the ligand binding activity of A2AR.