Dual mechanisms of cholesterol-GPCR interactions that depend on membrane phospholipid composition

Highlights

• Specific cholesterol-A_2AAR interactions activate receptor in POPC lipid bilayers

• Influence of sterols on agonist-bound A_2AAR observed with cholesterol analogs

• Cholesterol and anionic lipids share complementary roles in A_2AAR activation

• Different cholesterol interaction sites distinctly impact structure and function

Summary

Cholesterol is a critical component of mammalian cell membranes and an allosteric modulator of G protein-coupled receptors (GPCRs), but divergent views exist on the mechanisms by which cholesterol influences receptor functions. Leveraging the benefits of lipid nanodiscs, i.e., quantitative control of lipid composition, we observe distinct impacts of cholesterol in the presence and absence of anionic phospholipids on the function-related conformational dynamics of the human A_2A adenosine receptor (A_2AAR). Direct receptor-cholesterol interactions drive activation of agonist-bound A_2AAR in membranes containing zwitterionic phospholipids. Intriguingly, the presence of anionic lipids attenuates cholesterol’s impact through direct interactions with the receptor, highlighting a more complex role for cholesterol that depends on membrane phospholipid composition. Targeted amino acid replacements at two frequently predicted cholesterol interaction sites showed distinct impacts of cholesterol at different receptor locations, demonstrating the ability to delineate different roles of cholesterol in modulating receptor signaling and maintaining receptor structural integrity.

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 serotonin_1A 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 A_2A adenosine receptor (A_2AAR), a class A GPCR. A_2AAR is a model receptor for investigating mechanisms underlying GPCR signaling phenomena, owing to a large number of structures^{42,43,44,45,46} and the availability of spectroscopic data in different membrane mimetics.^{47,48,49,50,51,52,53} A_2AAR 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 A_2AAR signaling and pharmacology, though there is no consensus on the precise role of cholesterol or mechanisms by which cholesterol may influence A_2AAR function. In HEK293 cells treated with methyl-β-cyclodextrin (MβCD) to deplete cholesterol, A_2AAR 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 A_2AAR antagonist, suggesting that cholesterol inhibited antagonist binding.¹⁵ Canonical cholesterol recognition motifs and cholesterol-binding “hotspots” have been predicted for A_2AAR as evidence for specific receptor-cholesterol interactions.^57,58,59 In contrast to this view, ¹⁹F-NMR experiments with A_2AAR 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 A_2AAR 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-A_2AAR 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 A_2AAR 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 A_2AAR 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.