G protein-coupled receptors (GPCRs) are the largest known family of membrane receptors in humans. All GPCRs span the cell membrane, allowing them to bind extracellular stimuli and initiate intracellular downstream signaling cascades. These characteristics make GPCRs ideal targets for novel pharmaceutical development with approximately one third of approved drugs targeting GPCRs; however, only 10% of known GPCRs are targets of approved drugs, so further characterization of GPCRs is essential to development of novel therapeutics. Environmental factors, such as cell membrane composition, affect GPCR activation and signaling. One well-characterized GPCR, the adenosine A2A receptor (A2AR), contains cholesterol binding motifs, which are estimated amino acids where cholesterol allosterically interacts with the receptor. Here, we explore the effects of one such binding motif, the cholesterol consensus motif (CCM), by using simulations to approximate single amino acids where cholesterol interacts with A2AR. Amino acids in the CCM (S47, K122, W129) were mutated to alanine to characterize effects on receptor activity. Radiolabeled ligand binding experiments suggest all three variants affect agonist and antagonist binding to A2AR. Downstream signaling was characterized by observation of cAMP in HEK cells transfected with A2AR variants. When compared to WT-A2AR, S47A demonstrated an increase in constitutive cAMP, but K122A and W129A produced a decrease in both constitutive and agonist-induced cAMP signaling. Further effects of variants on downstream signaling was characterized by addition of MβCD to observe the effects of removing membrane cholesterol from HEK cells on cAMP. In these studies, S47A was most affected by addition of MβCD, and W129A was unaffected by MβCD treatment, suggesting little cholesterol interaction with the W129A variant. To further characterize the variant effects on the first step in the downstream signaling pathway, receptor association with G protein, receptor variants and Gs were purified and protein-protein interaction was observed via surface plasmon resonance. Initial results comparing WT-A2AR association to Gs to variant association to Gs suggest S47A has a faster dissociation rate constant and, therefore, a larger K<sub>D</sub>. This suggests the loss of constitutive cAMP for this variant stems from a decrease in G protein association in the pre-activated receptor state, likely due to a model with cholesterol affecting the pre-activation.

Effects of allosteric cholesterol association to A<sub>2A</sub>R and cholesterol consensus motif variants on experimental ligand binding, G protein coupling, and further downstream signaling markers

ACS Spring 2022

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Effects of allosteric cholesterol association to A2AR and cholesterol consensus motif variants on experimental ligand binding, G protein coupling, and further downstream signaling markers

Effects of allosteric cholesterol association to A_2AR and cholesterol consensus motif variants on experimental ligand binding, G protein coupling, and further downstream signaling markers