Cholesterol Dependent Activity of the Adenosine A2A Receptor Is Modulated via the Cholesterol Consensus Motif

Abstract

Background: Membrane cholesterol dysregulation has been shown to alter the activity of the adenosine A2A receptor (A2AR), a G protein-coupled receptor, thereby implicating cholesterol levels in diseases such as Alzheimer’s and Parkinson’s. A limited number of A2AR crystal structures show the receptor interacting with cholesterol, as such molecular simulations are often used to predict cholesterol interaction sites. Methods: Here, we use experimental methods to determine whether a specific interaction between amino acid side chains in the cholesterol consensus motif (CCM) of full length, wild-type human A2AR, and cholesterol modulates activity of the receptor by testing the effects of mutational changes on functional consequences, including ligand binding, G protein coupling, and downstream activation of cyclic AMP. Results and conclusions: Our data, taken with previously published studies, support a model of receptor state-dependent binding between cholesterol and the CCM, whereby cholesterol facilitates both G protein coupling and downstream signaling of A2AR.

Cholesterol in Class C GPCRs: Role, Relevance, and Localization

“…A double CARC-CRAC motif has been identified within the transmembrane domains of some membrane proteins [ 10 , 98 ]; however, the limiting factor of the CRAC/CARC sequence is that they are based on a linear (1D) sequence motif, as opposed to cholesterol-binding sites which consist of a three-dimensional (3D) structure [ 100 ]. Another common motif is the CCM, which is defined by four spatially distributed interactions with cholesterol: an aromatic Trp158, conserved in 94% of class A GPCRs; a hydrophobic Ile154 conserved in 35% of class A GPCRs (both residues in helix IV); an aromatic Tyr70 from helix II, which forms a hydrogen bond with Arg151 from helix IV [ 101 , 102 , 103 ]. This motif was established from the analysis of the human 2-adrenergic receptor in a complex with timolol and two molecules of cholesterol [ 101 ].…”

Allosteric Regulation of G-Protein-Coupled Receptors: From Diversity of Molecular Mechanisms to Multiple Allosteric Sites and Their Ligands

“…Among lipids, cholesterol is the most important allosteric regulator of GPCRs, and its effects on receptor activity are highly dependent on the content of cholesterol in the membrane, the type of GPCR, and the nature of the orthosteric ligand [ 93 , 128 , 129 ]. A significant number of GPCRs contain consensus motifs for cholesterol binding (Cholesterol Recognition/Interaction Amino Acid Consensus motif, CRAC), which were first identified in β 2 -AR [ 130 ] and then found in other GPCRs, although often in a modified form [ 93 , 131 , 132 ]. Along with cholesterol, other lipids, such as phosphatidylserines [ 91 ] and phosphoinositides, including PI(4,5)P 2 [ 92 ], are also involved in the allosteric regulation of GPCRs.…”

Allosteric Regulation of G-Protein-Coupled Receptors: From Diversity of Molecular Mechanisms to Multiple Allosteric Sites and Their Ligands

“…Among lipids, cholesterol is the most important allosteric regulator of GPCRs, and its effects on receptor activity are highly dependent on the content of cholesterol in the membrane, the type of receptor, and the nature of the orthosteric ligand [91,132,133]. A significant number of GPCRs contain consensus motifs for cholesterol binding (Cholesterol Recognition/Interaction Amino Acid Consensus motif, CRAC), which were first identified in β2-AR [134] and then found in other GPCRs, although often in modified form [91,135,136]. Along with cholesterol, other lipids are involved in the allosteric regulation of GPCRs, including phosphatidylinositol-4,5-bisphosphate, phosphoinositols [90] and phosphoserines [89].…”