Structure
ArticleDual mechanisms of cholesterol-GPCR interactions that depend on membrane phospholipid composition
Graphical abstract
Introduction
Cholesterol is a major component of mammalian cell membranes and an important regulator of the functions of membrane proteins, as documented extensively in the literature.1,2,3,4,5,6,7,8,9 Alterations in cholesterol distribution and metabolism, either through genetic or environmental causes, are highly correlated with numerous health disorders, including Alzheimer’s disease,10 heart disease,11 and diabetes.12 Cholesterol is also increasingly known for its role as an allosteric modulator of G protein-coupled receptors (GPCRs), integral membrane sensory proteins, and targets of over 35% of FDA-approved drugs.13
Literature data have reported the impacts of cholesterol on essentially every aspect of GPCR pharmacology and biology.14,15,16,17,18,19 Cholesterol has been shown experimentally to act as an allosteric modulator of GPCRs, altering the binding affinities of orthosteric ligands, as observed with, for example, the oxytocin receptor,20 the CXCR4 chemokine receptor,21 the serotonin1A receptor,22 the cannabinoid CB1 receptor,23 and the CCR5 chemokine receptor.24 Structural and biochemical evidence also support the role of cholesterol as a direct orthosteric agonist for some GPCRs, including the smoothened receptor (SMO).25 In addition to cholesterol’s impacts on receptor pharmacology, experimental and computational studies have identified roles of cholesterol in GPCR oligomerization,26,27,28,29 receptor sorting and trafficking,30,31,32,33 and formation of signaling complexes.19,34,35
Three general mechanisms have been proposed to understand the influence of cholesterol on GPCR function: direct cholesterol-GPCR interactions, indirectly via modulation of bulk membrane properties, or a combination of both.36 Support for direct receptor-cholesterol interactions has come, in part, from the observation of cholesterol or cholesterol analogs closely associated with the transmembrane regions of more than 60 GPCRs in X-ray crystallography and cryoelectron microscopy structures, as reviewed.37 Nuclear magnetic resonance (NMR) studies of the β2-adrenergic receptor (β2AR) indicated that the receptor showed a specific preference to associate with cholesterol over ergosterol,38 and high-pressure NMR studies reported that the cholesterol analog cholesteryl hemisuccinate (CHS) specifically associated with the β1AR to inhibit its activation.39 However, there is no consensus on the extent to which specific cholesterol-receptor interactions influence the function of GPCRs, and earlier studies of rhodopsin reported evidence for the influence of cholesterol on rhodopsin function both through direct interactions with rhodopsin40 and by modulating the properties of the lipid bilayer.41
We investigated the mechanism by which cholesterol modulates the function-related dynamics of the human A2A adenosine receptor (A2AAR), a class A GPCR. A2AAR is a model receptor for investigating mechanisms underlying GPCR signaling phenomena, owing to a large number of structures42,43,44,45,46 and the availability of spectroscopic data in different membrane mimetics.47,48,49,50,51,52,53 A2AAR is an important and validated drug target for Parkinson’s disease54 and cancers55 for which cholesterol may play a role. A growing body of literature has documented the impact of cholesterol on A2AAR signaling and pharmacology, though there is no consensus on the precise role of cholesterol or mechanisms by which cholesterol may influence A2AAR function. In HEK293 cells treated with methyl-β-cyclodextrin (MβCD) to deplete cholesterol, A2AAR signaling was observed to significantly decrease,56 indicating that cholesterol was required to achieve a maximum signaling response to agonist stimulation. In a separate study, treatment of C6 glioma cells with MβCD showed an increase in the specific binding of an A2AAR antagonist, suggesting that cholesterol inhibited antagonist binding.15 Canonical cholesterol recognition motifs and cholesterol-binding “hotspots” have been predicted for A2AAR as evidence for specific receptor-cholesterol interactions.57,58,59 In contrast to this view, 19F-NMR experiments with A2AAR labeled at position V229C on transmembrane helix VI (TM VI) concluded that cholesterol was a weak positive allosteric modulator and that impacts of cholesterol were likely due more to cholesterol-mediated changes in membrane bilayer fluidity rather than direct interactions.60
To reconcile these divergent views, we integrated 19F-NMR data with correlative functional and pharmacological assays to investigate the impact of cholesterol on signaling-related A2AAR conformational dynamics. We leveraged the benefits of lipid nanodiscs to quantitatively control lipid composition in all samples. By systematically exploring a wider range of lipid compositions than previously studied, we observed evidence for both the direct and indirect influences of cholesterol. The mechanism of cholesterol influence depended both on the presence of cholesterol and the composition of phospholipids in the membrane. Using conditions where we observe specific cholesterol-receptor interactions, we explored variations in the cholesterol chemical scaffold to test the extent to which cholesterol-A2AAR interactions depended on the structural details of cholesterol molecules. The same conditions also allowed us to investigate two of the most frequently predicted cholesterol interaction sites.61,62,63 Our findings reveal a clear and significant impact of cholesterol on the activation of agonist-bound A2AAR in nanodiscs that contain zwitterionic lipids. This effect is observable even for samples containing only 1–2 molecules of cholesterol per nanodisc. An investigation of cholesterol analogs also yielded comparable results. Interestingly, this response is obscured when A2AAR is embedded in nanodiscs containing mixtures of zwitterionic and anionic lipids, suggesting a potential interplay between anionic lipids and cholesterol. These results are discussed in the context of integrating in vitro and in vivo data to provide a more comprehensive understanding of the role of cholesterol in GPCR activation mechanisms.
Section snippets
Cholesterol manifests different impacts on the pharmacology of A2AAR antagonists and agonists
For biophysical and functional experiments, a variant of human A2AAR containing a single cysteine replacement at position 289 located at the intracellular surface of TM VII (Figure S1), A2AAR[A289C], was expressed in Pichia pastoris using previously described protocols.64 The location of the introduced cysteine was selected based on literature data from 19F-NMR studies of A2AAR, which demonstrated that NMR spectra of A2AAR labeled with 19F-2,2,2-trifluoroethanethiol at this position were
Discussion
The pharmacological data in Figure 1 show no correlation between measured binding affinities for antagonists and cholesterol and a potential positive correlation between binding affinities of agonists and cholesterol concentrations in lipid nanodiscs, albeit less than a factor of 10 over the range of cholesterol concentrations studied. Previous studies reported an increase in specific binding of the antagonist ZM241385 to A2AAR upon using MβCD to remove cholesterol from C6 glioma cells.15 As
Key resources table
REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Monoclonal ANTI-FLAG® M2-Alkaline Phosphatase antibody produced in mouse Sigma-Aldrich Cat#A9469; RRID: AB_439699 Bacterial and virus strains XL10-Gold ultracompetent cells Agilent Cat#200314 BL21 (DE3) cells Agilent Cat#200132 BL21(DE3)-RIL cells Agilent Cat#230245 Chemicals, peptides, and recombinant proteins n-Dodecyl-b-D-Maltopyranoside (DDM) Anatrace Cat#D310 Cholesteryl hemisuccinate (CHS) Sigma-Aldrich Cat#C6512 Adenosine 5′-triphosphate disodium salt hydrate
Acknowledgments
This work is supported by the National Institutes of Health, NIGMS MIRA grant R35GM138291 (M.T.E., N.T., A.P.R., and N.G.P). A portion of this work was supported by the McKnight Brain Institute at the National High Magnetic Field Laboratory’s AMRIS Facility, which is funded by National Science Foundation Cooperative Agreement No. DMR-1644779 and the state of Florida.
Author contributions
A.P.R., N.T., and N.G.P. performed protein production, purification, and nanodisc sample preparation. N.G.P. carried out molecular
References (92)
Non-covalent binding of membrane lipids to membrane proteins
Biochim. Biophys. Acta
(2014)Modulation of membrane function by cholesterol
Biochimie
(1991)Cholesterol and the interaction of proteins with membrane domains
Prog. Lipid Res.
(2006)- et al.
The influence of cholesterol on membrane protein structure, function, and dynamics studied by molecular dynamics simulations
Biochim. Biophys. Acta
(2015) - et al.
How bilayer lipids affect membrane protein activity
Trends Biochem. Sci.
(1986) - et al.
A century of cholesterol and coronaries: from plaques to genes to statins
Cell
(2015) - et al.
Cholesterol metabolism, pancreatic β-cell function and diabetes
Biochim. Biophys. Acta, Mol. Basis Dis.
(2019) - et al.
Cholesterol interaction sites on the transmembrane domain of the hedgehog signal transducer and class F G protein-coupled receptor smoothened
Structure
(2019) - et al.
Membrane cholesterol stabilizes the human serotonin1A receptor
Biochim. Biophys. Acta
(2012) - et al.
Ligand-independent dimerization of CXCR4, a principal HIV-1 coreceptor
J. Biol. Chem.
(2003)
Role of cholesterol in ligand binding and G-protein coupling of serotonin1A receptors solubilized from bovine hippocampus
Biochem. Biophys. Res. Commun.
Mechanisms of selective G protein–coupled receptor localization and trafficking
Curr. Opin. Cell Biol.
Uncovering the intimate relationship between lipids, cholesterol and GPCR activation
Curr. Opin. Struct. Biol.
Two classes of cholesterol binding sites for the β 2 AR revealed by thermostability and NMR
Biophys. J.
Rhodopsin-cholesterol interactions in bovine rod outer segment disk membranes
Biochim. Biophys. Acta
Delineating the conformational landscape of the adenosine A2A receptor during G protein coupling
Cell
A2A adenosine receptor partial agonism related to structural rearrangements in an activation microswitch
Structure
Allosteric coupling of drug binding and intracellular signaling in the A2A adenosine receptor
Cell
Co-Inhibition of CD73 and A2AR adenosine signaling improves anti-tumor immune responses
Cancer Cell
Membrane cholesterol depletion reduces downstream signaling activity of the adenosine A2A receptor
Biochim. Biophys. Acta. Biomembr.
Identification of two new cholesterol interaction sites on the A2A adenosine receptor
Biophys. J.
State-dependent lipid interactions with the A2a receptor revealed by MD simulations using in vivo-mimetic membranes
Structure
Predictable cholesterol binding sites in GPCRs lack consensus motifs
Structure
State-dependent lipid interactions with the A2a receptor revealed by MD simulations using in vivo-mimetic membranes
Structure
Molecular model for the solubilization of membranes into nanodisks by styrene maleic acid copolymers
Biophys. J.
Fluorometric method for the enzymatic determination of cholesterol
J. Biochem. Biophys. Methods
Erg6 affects membrane composition and virulence of the human fungal pathogen Cryptococcus neoformans
Fungal Genet. Biol.
Fluorescence generalized polarization of cell membranes: a two-photon scanning microscopy approach
Biophys. J.
Ligand-dependent cholesterol interactions with the human A2A adenosine receptor
Chem. Phys. Lipids
Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors
A specific cholesterol binding site is established by the 2.8 Å structure of the human β2-adrenergic receptor
Structure
Stabilization of the human β2-adrenergic receptor TM4–TM3–TM5 helix interface by mutagenesis of Glu1223.41, A critical residue in GPCR structure
J. Mol. Biol.
Production of human A2AAR in lipid nanodiscs for 19F-NMR and single-molecule fluorescence spectroscopy
STAR Protoc.
Production of human A2AAR in lipid nanodiscs for 19F-NMR and single-molecule fluorescence spectroscopy
STAR Protoc.
Slow conformational dynamics of the human A2A adenosine receptor are temporally ordered
Structure
Microscale fluorescent thermal stability assay for membrane proteins
Structure
Structural connection between activation microswitch and allosteric sodium site in GPCR signaling
Structure
Cholesterol-binding site of the influenza M2 protein in lipid bilayers from solid-state NMR
Proc. Natl. Acad. Sci. USA
How cholesterol interacts with membrane proteins: an exploration of cholesterol-binding sites including CRAC, CARC, and tilted domains
Front. Physiol.
Cholesterol as a co-solvent and a ligand for membrane proteins: cholesterol-Protein Interactions
Protein Sci.
The effect of membrane cholesterol content on ion transport processes in plasma membranes
Cardiovasc. Res.
Linking lipids to Alzheimer's disease: cholesterol and beyond
Nat. Rev. Neurosci.
Trends in GPCR drug discovery: new agents, targets and indications
Nat. Rev. Drug Discov.
Lipid-Dependent regulation of ion channels and G protein–coupled receptors: insights from structures and simulations
Annu. Rev. Pharmacol. Toxicol.
Membrane cholesterol access into a G-protein-coupled receptor
Nat. Comm.
Smoothened stimulation by membrane sterols drives Hedgehog pathway activity
Nature
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