SBI-115

A Critical Analysis of Molecular Mechanisms Underlying Membrane Cholesterol Sensitivity of GPCRs

Md. Jafurulla, G. Aditya Kumar, Bhagyashree D. Rao, and Amitabha Chattopadhyay

Abstract G protein-coupled receptors (GPCRs) are the largest and a diverse family of proteins involved in signal transduction across biological membranes. GPCRs mediate a wide range of physiological processes and have emerged as major targets for the development of novel drug candidates in all clinical areas. Since GPCRs are integral membrane proteins, regulation of their organization, dynamics, and func- tion by membrane lipids, in particular membrane cholesterol, has emerged as an exciting area of research. Cholesterol sensitivity of GPCRs could be due to direct interaction of cholesterol with the receptor (specific effect). Alternately, GPCR function could be influenced by the effect of cholesterol on membrane physical properties (general effect). In this review, we critically analyze the specific and gen- eral mechanisms of the modulation of GPCR function by membrane cholesterol, taking examples from representative GPCRs. While evidence for both the proposed mechanisms exists, there appears to be no clear-cut distinction between these two mechanisms, and a combination of these mechanisms cannot be ruled out in many cases. We conclude that classifying the mechanism underlying cholesterol sensitiv- ity of GPCR function merely into these two mutually exclusive classes could be somewhat arbitrary. A more holistic approach could be suitable for analyzing GPCR–cholesterol interaction.

Keywords GPCR–cholesterol interaction · Specific effect · General effect · Cholesterol binding motifs

Cholesterol is a crucial and representative lipid in higher eukaryotic cell membranes and plays a key role in membrane organization, dynamics, function, and sorting. The unique molecular structure of cholesterol has been intricately fine-tuned over a very long timescale of natural evolution [20, 21]. The chemical structure of choles- terol comprises of the 3β-hydroxyl group, the rigid tetracyclic fused ring, and the flexible isooctyl side chain (Fig. 1a). The 3β-hydroxyl group (sole polar group) helps cholesterol anchor at the membrane interface and is believed to form hydro- gen bonds with polar residues of membrane proteins. The tetracyclic fused ring and the isooctyl side chain constitute the apolar component of cholesterol. An inherent asymmetry about the plane of the sterol ring is generated by methyl substitutions on one of its faces (Fig. 1b). The protruding methyl groups (constituting the rough β face) are believed to participate in van der Waals interactions with the side chains of branched amino acids such as valine, leucine, and isoleucine. The other side of the sterol ring (constituting the smooth α face) exhibits favorable van der Waals interac- tion with the saturated fatty acyl chains of phospholipids (Fig. 1c; [22–24]). Cholesterol is nonrandomly distributed in specific domains (or pools) in biological and model membranes [22, 25–28]. Membrane cholesterol is essential for a range The mechanism of action of cholesterol on GPCRs has been explored using a battery of experimental strategies, each of which provides a unique perspective to address the molecular basis of these interactions. The strategies commonly used to study such interactions rely on the modulation of cholesterol content or its availability in membranes in order to probe its role in supporting the function and organization of GPCRs. These techniques, when used judiciously, could be helpful in delineating the specific and general effects of cholesterol on GPCR function. We discuss below a few important strategies that are used to explore the nature of the interaction of membrane cholesterol with GPCRs.

Solubilization and Reconstitution

Solubilization is an important method used to understand the structural and func- tional aspects of GPCRs. Solubilization involves the isolation of the receptor from its native membrane environment and dispersing it in a relatively purified state using suitable amphiphilic detergents. The process of solubilization leads to dissociation of proteins and lipids which are held together in the native membrane, ultimately resulting in the formation of small clusters of protein, lipid, and detergent in an aqueous solution [50–54]. Solubilization has been utilized as an effective strategy to study GPCR–lipid interactions and probe lipid specificity by reconstitution of the receptor with specific lipids [54, 55]. The process of reconstitution involves removal of detergent, followed by incorporation of the receptor into membrane-mimics such as micelles, bicelles, liposomes, nanodiscs, and planar lipid bilayers [55, 56]. This strategy has been earlier utilized to explore the role of cholesterol in the function of the serotonin1A receptor [54]. Using this strategy, we further explored the structural stringency of cholesterol in the function of the serotonin1A receptor by reconstitut- ing the solubilized receptor with close structural analogs (biosynthetic precursors and stereoisomers) of cholesterol [57–60].

Inhibition of Cholesterol Biosynthesis

Biosynthesis of cholesterol is carried out in a stringently regulated multi-step enzy- matic pathway [61]. A physiologically relevant approach to study the role of choles- terol in GPCR function is metabolic (chronic) depletion by inhibiting specific enzymes in its biosynthetic pathway. A common strategy that has been used to chronically deplete cellular cholesterol is the use of statins [62, 63]. Statins are competitive inhibitors of HMG-CoA reductase, the enzyme that catalyzes the rate- limiting step in the cholesterol biosynthetic pathway (Fig. 2a; [64]). In addition, distal inhibitors such as AY 9944 (trans-1,4-bis(2-chlorobenzylaminoethyl)cyclo- hexane dihydrochloride) that inhibits 3β-hydroxy-steroid-Δ7-reductase (7-DHCR), and triparanol which inhibits 3β-hydroxy-steroid-Δ24-reductase (24-DHCR) have been extensively utilized [65, 66]. Inhibition of 7-DHCR and 24-DHCR that cata- lyze final steps in the Kandutsch-Russell pathway [67] and Bloch pathway [68] results in the accumulation of 7-dehydrocholesterol (7-DHC) and desmosterol, respectively (Fig. 2a). Importantly, malfunctioning of 7-DHCR and 24-DHCR has been identified as major factors for lethal neuropsychiatric disorders such as Smith– Lemli–Opitz syndrome (SLOS) and desmosterolosis [69, 70]. Therefore, inhibitors of 7-DHCR and 24-DHCR have been successfully utilized to generate cellular and animal model systems to study these disease conditions [65, 66, 71, 72]. We previ- ously utilized this strategy to generate a cellular model for SLOS using AY 9944, and explored the function of the serotonin1A receptor (an important neurotransmitter receptor) in this neuropsychiatric disease condition [43].

Specific Carriers

A commonly utilized strategy for acute and specific modulation of membrane cholesterol content is by using specific carriers. Methyl-β-cyclodextrin (MβCD), a member of the cyclodextrin family, is an oligomer of seven methylated-glucose residues that exhibits specificity for cholesterol over other membrane lipids (see Fig. 2b; [34, 73, 74]). MβCD has been utilized as the carrier of choice to study the effect of cholesterol on GPCR function, organization, and dynamics in a large number of studies [36, 37]. The relatively small size and polar nature of MβCD allows its close interaction with membranes, thereby enabling efficient and selective modulation of cholesterol content. This strategy has been utilized to explore the cholesterol-dependent function of several GPCRs such as rhodopsin [75], oxytocin [76], galanin [77], serotonin1A [78, 79], cannabinoid [80–82], and bitter taste T2R4 receptors [83]. We have successfully utilized MβCD for controlled modulation of membrane cholesterol to study its role in the function of the serotonin1A receptor [78, 79, 84]. We further utilized MβCD to replace cholesterol with its various close structural analogs in order to explore the structural stringency of cholesterol for sup- porting receptor function [54]. Interestingly, we have recently shown that although both inhibition of cholesterol biosynthesis and specific carriers modulate choles- terol levels in cell membranes, the actual effect could differ a lot (even at same cholesterol concentrations), since the membrane dipolar environment in these cases turn out to be very different [85].

Enzymatic Oxidation

Specific modulation of membrane cholesterol could also be achieved by its oxida- tion using the enzyme cholesterol oxidase. Cholesterol oxidase catalyzes the oxida- tion of cholesterol to 4-cholestenone at the membrane interface [86], thereby modifying the chemical nature of cholesterol without physical depletion from mem- branes. Oxidation of cholesterol exhibits mild effect on global membrane properties relative to its physical depletion, and minimizes nonspecific effects of cholesterol modulation. This strategy has been earlier utilized to explore the structural speci- ficity of cholesterol (the hydroxyl group in particular) in the function of several GPCRs such as the serotonin1A receptor [87, 88], oxytocin and cholecystokinin (CCK) receptors [76], galanin-GalR2 receptors [77], rhodopsin [89], and chemo- kine receptors CXCR4 and CCR5 [90].

Complexing Agents

Modulating availability of cholesterol in the membrane, rather than physical deple- tion, is yet another method to explore the cholesterol sensitivity of GPCR function. Cholesterol-complexing agents such as digitonin, filipin, nystatin, amphotericin B, and perfringolysin O [91–95] at appropriate concentrations partition into mem- branes and sequester cholesterol, thereby making it unavailable for interaction with GPCRs. These agents could be used to address the interaction of cholesterol with GPCRs by restricting cholesterol availability. Figure 2c shows the chemical structure of nystatin, a representative complexing agent. This strategy has been earlier utilized to probe the requirement of membrane cholesterol for the function of the serotonin1A [96, 97], oxytocin [76], and galanin [77] receptors.

Mechanisms of Cholesterol Sensitivity of GPCRs

Cholesterol sensitivity of GPCRs is well documented. However, the underlying molecular mechanism remains elusive. The ongoing efforts to understand the struc- tural and functional correlates underlying cholesterol sensitivity of GPCR function have provided evidence in favor of both specific interaction and general (membrane) effects. We discuss below representative studies on cholesterol sensitivity of GPCRs. The serotonin1A receptor is a key neurotransmitter GPCR that is implicated in the generation and modulation of various cognitive, behavioral, and developmental func- tions [98–102]. The serotonin1A receptor is the most well-studied GPCR in terms of specificity of cholesterol in the organization, dynamics, and function of the receptor. Earlier work from our laboratory has comprehensively demonstrated the specific requirement of membrane cholesterol for the function of the serotonin1A receptor uti- lizing an array of experimental approaches. By modulating the availability of mem- brane cholesterol by employing (1) MβCD [57, 78], (2) biosynthetic inhibitors such
as statin [63] and AY 9944 [43], and (3) complexing agents such as nystatin [96] and digitonin [97], we have shown the requirement of cholesterol in receptor function. We generated a cellular model for SLOS (a fatal neuropsychiatric disorder) using AY 9944 and showed that the function of the serotonin1A receptor is compromised under this disease-like condition [43]. We have recently generated a rat model of SLOS by oral feeding of AY 9944 to dams for brain metabolic NMR studies. Importantly, enzy- matic oxidation of cholesterol [87, 88] led to a change in receptor function, without any appreciable effect on membrane order (as reported by fluorescence anisotropy measurements), thereby suggesting specific requirement of cholesterol for receptor function. We further demonstrated the structural stringency of cholesterol in support- ing the function of the serotonin1A receptor by replacing cholesterol with its immedi- ate biosynthetic precursors (7-DHC and desmosterol) [58, 59, 103] and stereoisomers of cholesterol ([60]; reviewed in [54]). In addition, we showed that the stability of the serotonin1A receptor is enhanced in the presence of cholesterol using biochemical approaches [104], molecular modeling [105], and all atom molecular dynamics simu- lations [106]. Taken together, these studies bring out the cholesterol sensitivity of the serotonin1A receptor function, which in some cases (such as treatment with cholesterol oxidase) could have a specific mechanism.

Oxytocin Receptor

The oxytocin receptor plays an important role in several neuronal functions and in reproductive biology [107]. Cholesterol dependence of oxytocin receptor function was explored using multiple approaches [76, 108]. Modulation of membrane cho- lesterol content using MβCD resulted in a change in the affinity state of the receptor for oxytocin, with the receptor in a high affinity state in the presence of cholesterol [108]. In addition, utilizing cholesterol-complexing agent filipin, mere complex- ation of cholesterol was shown to be sufficient to modulate receptor function [76]. Importantly, treatment with cholesterol oxidase modulated the function of the receptor without a significant change in membrane order. The structural stringency of cholesterol for the function of the oxytocin receptor was demonstrated by replac- ing cholesterol with an array of its structural analogs [76]. Further, the oxytocin receptor was shown to be more stable in the presence of cholesterol [109]. These results point out the role of specific mechanism in the cholesterol-dependent func- tion of the oxytocin receptor.

Galanin Receptor

Galanin receptors upon binding to the neuropeptide galanin mediate diverse physiolog- ical functions in the peripheral and central nervous systems. The requirement of cho- lesterol for galanin receptor (GalR2) function was shown by modulating cholesterol content in cellular membranes using MβCD or by culturing cells in lipoprotein-deficient serum [77]. Depletion of membrane cholesterol led to decrease in affinity of ligand binding to the receptor. In addition, complexation of cholesterol with filipin and enzy- matic oxidation of cholesterol led to significant reduction in ligand binding activity of the receptor. The mechanistic basis of cholesterol sensitivity was evident from experi- ments in which cholesterol was replaced with its structural analogs, thereby implying a possible specific mechanism responsible for cholesterol sensitivity of GalR2 [77].

Chemokine Receptors

Chemokine receptors are important GPCRs implicated in immunity and infection. A wide range of chemokines bind to these receptors and mediate specific immune responses. Membrane cholesterol has been shown to be essential for stabilizing the functional conformation and signaling of CCR5 and CXCR4 receptors, members of the chemokine receptor family [90, 110, 111]. The cholesterol sensitivity of the function of CCR5 was shown using conformation-specific antibodies, whose bind- ing to the receptor exhibited cholesterol dependence [110]. Treatment with choles- terol oxidase [90] resulted in reduction in binding of epitope-specific antibodies to CCR5 along with loss in receptor function. In addition, replacement of cholesterol with 4-cholesten-3-one showed reduction in specific ligand binding to the receptor [110]. Similar results were observed for CXCR4 where depletion or oxidation of membrane cholesterol resulted in reduction in binding of conformation-specific antibodies and signaling of the receptor [90, 111]. These effects were reversed upon replenishment with membrane cholesterol.

Bitter Taste Receptors

The human bitter taste receptors (T2Rs) are chemosensory receptors with signifi- cant therapeutic potential [112]. Earlier work from our laboratory has shown that the T2R4 receptor, a representative member of the bitter taste receptor family, exhibits cholesterol sensitivity in its signaling [83]. The molecular basis of such cholesterol dependence of receptor function could be attributed to the putative cho- lesterol recognition/interaction amino acid consensus (CRAC) motif (see below), since mutation of a lysine residue in the CRAC sequence led to loss of cholesterol sensitivity of the receptor [83].

Cannabinoid and Cholecystokinin Receptors

Cannabinoid receptors are activated by endocannabinoids which mediate a variety of physiological and neuroinflammatory processes, and are implicated in several neurodegenerative and neuroinflammatory disorders. The cholesterol sensitivity of type-1 cannabinoid (CB1) receptors was shown from dependence of specific ligand binding and signaling of the receptor on membrane cholesterol [80, 81, 113]. Importantly, such a sensitivity of CB1 receptor function to membrane cholesterol is lost upon mutation of a lysine residue in the putative CRAC sequence. Interestingly, the type-2 cannabinoid (CB2) receptor has glycine instead of lysine (as in CB1 receptor) in the CRAC sequence [113] and does not show cholesterol dependence for its function [82, 113]. These studies point toward the possible involvement of the CRAC motif in cholesterol sensitivity of CB1 receptors. Similar observations were reported for subtypes of cholecystokinin CCK1 and CCK2 receptors [114, 115]. CCK1 receptors were shown to be sensitive to mem- brane cholesterol by analyzing active conformation of the receptor, probed using fluorescence of a specific fluorescent ligand and intracellular calcium response [114]. Interestingly, a closely related subtype CCK2 receptor has been shown to be insensitive to membrane cholesterol [115]. Importantly, mutation in CRAC motif region in CCK1 receptor resulted in the loss of its cholesterol sensitivity.

Smoothened Receptor

One of the most compelling functional correlates of cholesterol interaction with GPCRs was shown in the recently reported structure of the sterol binding frizzled (class F) GPCR, smoothened (Smo) [138, 139, 159]. Smo is a component of the hedgehog signaling pathway involved in embryonic development and programmed cell death, and the role of cholesterol in this pathway is well documented [160]. Cholesterol acts as the endogenous activator of Smo by inducing conformational changes in the receptor that stimulates the hedgehog pathway. The structure of Smo showed a cholesterol molecule bound to the extracellular cysteine-rich domain of the receptor which is crucial for transduction of hedgehog signals (Fig. 3g). Importantly, the structure helped to predict key residues for this interaction, mutat- ing which impaired hedgehog signaling [159]. We would like to end this section with a cautionary note. Although crystallogra- phy is an excellent technique to resolve detailed high-resolution structures of GPCRs, it suffers from some inherent limitations. Despite the fact that the extra- membranous regions of GPCRs play crucial roles in receptor function and signaling [161–163], the flexible loops corresponding to these regions are generally stabilized using a monoclonal antibody or replaced with lysozyme [116, 164, 165], since the inherent conformational flexibility of the loops poses a problem for crystallography. In addition, crystallography is often carried out in detergent dispersions or lipidic cubic phases using a heavily engineered (mutated) and antibody-bound receptor. In spite of the popularity of lipidic cubic phase membranes for GPCR crystalliza- tion [166], the physiological significance of bound cholesterol molecules in GPCR crystal structures in lipidic cubic phases is not clear [167]. It is possible that the bound cholesterol molecules and the CCM site could be specific to membrane lipid environment (which is different in lipidic cubic phase relative to the lamellar phase). It would therefore be prudent to be careful in extrapolating bound cholesterol in crystal structures of GPCRs to their cholesterol-sensitive function.

Cholesterol Interaction Motifs

The specific association of cholesterol with GPCRs that could possibly mediate cholesterol-dependent function is proposed to be manifested through conserved sequence motifs on these receptors. We discuss here few putative cholesterol interaction motifs that have been identified in GPCRs.

Cholesterol Recognition/Interaction Amino Acid Consensus (CRAC) Motif

CRAC motif is one of the most well-studied sequence motifs proposed to be impli- cated in the interaction of proteins with cholesterol. The CRAC motif is character- ized by the sequence -L/V-(X)1-5-Y-(X)1-5-R/K- (from N-terminus to C-terminus of the protein), where (X)1-5 represents between one and five residues of any amino acid [24, 168]. Subsequent to the first report on the presence of CRAC motif in the peripheral-type benzodiazepine receptor [169], the motif has been identified in sev- eral membrane proteins such as HIV transmembrane protein gp41 [170], caveolin-1 [171], and receptors implicated in pathogen entry [35]. We reported, for the first time, the presence of CRAC motifs in representative GPCRs such as rhodopsin, β2- adrenergic receptor, and the serotonin1A receptor [172].
We have previously shown that the serotonin1A receptor consists of three CRAC motifs in transmembrane helices II, V, and VII ([172]; see Fig. 4a). Interestingly, coarse-grain molecular dynamics simulations identified high cholesterol occupancy at the CRAC motif in transmembrane helix V of the serotonin1A receptor ([173]; see Fig. 4b). A characteristic feature of these sites is the inherent dynamics exhibited by cholesterol, ranging from ns to μs timescale. The corresponding energy landscape of cholesterol association with GPCRs can be described as a series of shallow minima, interconnected by low energy barriers (see Fig. 4c; [40]). Ongoing work in our labo- ratory aims to elucidate the role of CRAC motifs in the function of the serotonin1A receptor. In addition, CRAC motifs have been identified and correlated to choles- terol-dependent function of GPCRs such as CB1 [113], CCK1 [115], and bitter taste T2R4 receptors [83]. Importantly, as described earlier (see Sect. 4.1), mutation of key residues in the respective CRAC motifs in these GPCRs led to the modulation of cholesterol sensitivity of their function.

CARC: An Inverted CRAC Motif

The search for cholesterol interaction sites led to the recent identification of CARC, a motif which is similar to CRAC sequence, but with opposite orientation along the polypeptide chain, i.e., -(K/R)-X1-5-(Y/F)-X1-5-(L/V)- [24, 174]. The CARC motif was first identified in the nicotinic acetylcholine receptor and was found to be conserved over natural evolution among members of this family of receptors [174]. Interestingly, the CARC motif was found in several GPCRs such as rhodopsin, β2- adrenergic receptor, δ-opioid receptor, galanin receptor type 1, metabotropic gluta- mate receptor, and chemokine receptor CXCR4 [174]. Some of these receptors display cholesterol sensitivity in their function. The simultaneous presence of the CARC and CRAC motifs in two leaflets of the membrane bilayer in membrane proteins has been proposed as a potential “mirror code” [175].

Cholesterol Consensus Motif (CCM)

CCM was one of the first putative cholesterol interaction sites identified in GPCRs from the crystal structure of the β2-adrenergic receptor [117]. On the basis of homology, the CCM site has been defined as [4.39-4.43(R,K)]-[4.50(W,Y)]- [4.46(I,V,L)]-[2.41(F,Y)] (according to the Ballesteros–Weinstein nomenclature [176]). We have previously shown high cholesterol occupancy at the CCM site located at the groove of transmembrane helices II and IV of the β2-adrenergic receptor using coarse-grain molecular dynamics simulations [177]. We have earlier identified a characteristic CCM in the serotonin1A receptor which was found to be evolutionarily conserved [49].
However, it should be noted that mere presence of cholesterol interaction motif(s) does not necessarily translate to cholesterol-dependence of receptor function. For example, the neurotensin receptor 1 does not exhibit cholesterol sensitivity for its function, although the receptor has CCM in its sequence [178].

The Accessibility Issue: Nonannular Binding Sites

In the context of cholesterol binding sites in GPCRs, we previously proposed that these sites could represent “nonannular” binding sites whose possible locations could be inter or intramolecular (interhelical) protein interfaces [49]. Transmembrane pro- teins are surrounded by a shell (or annulus) of lipid molecules, termed as “annular” lipids [179]. The rate of exchange of lipids between the annular lipid shell and the bulk lipid phase was shown to be approximately an order of magnitude slower than the rate of exchange of bulk lipids [37, 179]. In addition, it was previously proposed that cholesterol binding sites could be “nonannular” in nature [180, 181]. Nonannular sites are characterized by relative lack of accessibility (due to their location in deep clefts or cavities on the protein surface) to the annular lipids [182], and therefore it is proposed that lipids in these sites are difficult to be replaced by competition with annular lipids [181]. Binding to the nonannular sites is considered to be more specific compared to annular sites. Interestingly, a recent study, employing experimental and simulation approaches, has proposed that membrane cholesterol could enter the deep orthosteric ligand binding pocket in the adenosine A2A receptor [183]. The influence of cholesterol on bulk (global) membrane properties has been exten- sively studied. Cholesterol has been shown to modulate membrane physical properties such as fluidity, curvature, phase, elasticity, dipole potential, and thickness [184–193]. Such effects of cholesterol on general membrane properties have been shown to modulate the organization and function of GPCRs (see Fig. 5; [75, 76, 194–196]).

Acknowledgments A.C. gratefully acknowledges support from SERB Distinguished Fellowship (Department of Science and Technology, Govt. of India). G.A.K. and B.D.R. thank the Council of Scientific and Industrial Research and University Grants Commission for the award of Senior Research Fellowships, respectively. A.C. is a Distinguished Visiting Professor at Indian Institute of Technology, Bombay (Mumbai), and Adjunct Professor at Tata Institute of Fundamental Research (Mumbai), RMIT University (Melbourne, Australia), and Indian Institute of Science Education and Research (Kolkata). Some of the work described in this article was carried out by former members of A.C.’s research group whose contributions are gratefully acknowledged. We thank members of the Chattopadhyay laboratory, particularly Parijat Sarkar, for comments and discussions.

References

1. Pierce KL, Premont RT, Lefkowitz RJ. Seven-transmembrane receptors. Nat Rev Mol Cell Biol. 2002;3:639–50.
2. Rosenbaum DM, Rasmussen SGF, Kobilka BK. The structure and function of G-protein- coupled receptors. Nature. 2009;459:356–63.
3. Chattopadhyay A. GPCRs: lipid-dependent membrane receptors that act as drug targets. Adv Biol. 2014;2014:143023.
4. Zhang Y, DeVries ME, Skolnick J. Structure modeling of all identified G protein-coupled receptors in the human genome. PLoS Comput Biol. 2006;2:e13.
5. Gether U. Uncovering molecular mechanisms involved in activation of G protein-coupled receptors. Endocr Rev. 2000;21:90–113.
6. Weis WI, Kobilka BK. The molecular basis of G protein-coupled receptor activation. Annu Rev Biochem. 2018;87:897–919.
7. Heilker R, Wolff M, Tautermann CS, Bieler M. G-protein-coupled receptor-focused drug discovery using a target class platform approach. Drug Discov Today. 2009;14:231–40.
8. Cooke RM, Brown AJH, Marshall FH, Mason JS. Structures of G protein-coupled receptors reveal new opportunities for drug discovery. Drug Discov Today. 2015;20:1355–64.
9. Jacobson KA. New paradigms in GPCR drug discovery. Biochem Pharmacol. 2015;98:541–55.
10. Kumari P, Ghosh E, Shukla AK. Emerging approaches to GPCR ligand screening for drug discovery. Trends Mol Med. 2015;21:687–701.
11. Gutierrez AN, McDonald PH. GPCRs: emerging anti-cancer drug targets. Cell Signal. 2018;41:65–74.
12. Thomsen W, Frazer J, Unett D. Functional assays for screening GPCR targets. Curr Opin Biotechnol. 2005;16:655–65.
13. Schlyer S, Horuk R. I want a new drug: G-protein-coupled receptors in drug development. Drug Discov Today. 2006;11:481–93.
14. Hauser AS, Attwood MM, Rask-Andersen M, Schiöth HB, Gloriam DE. Trends in GPCR drug discovery: new agents, targets and indications. Nat Rev Drug Discov. 2017;16:829–42.
15. Lin SHS, Civelli O. Orphan G protein-coupled receptors: targets for new therapeutic inter- ventions. Ann Med. 2004;36:204–14.
16. Stockert JA, Devi LA. Advancements in therapeutically targeting orphan GPCRs. Front Pharmacol. 2015;6:100.
17. Huber T, Botelho AV, Beyer K, Brown MF. Membrane model for the G-protein-coupled receptor rhodopsin: hydrophobic interface and dynamical structure. Biophys J. 2004;86:2078–100.
18. van Meer G, Voelker DR, Feigenson GW. Membrane lipids: where they are and how they behave. Nat Rev Mol Cell Biol. 2008;9:112–24.
19. van Meer G, de Kroon AIPM. Lipid map of the mammalian cell. J Cell Sci. 2011;124:5–8.
20. Brown AJ, Galea AM. Cholesterol as an evolutionary response to living with oxygen. Evolution. 2010;64:2179–83.
21. Kumar GA, Chattopadhyay A. Cholesterol: an evergreen molecule in biology. Biomed Spectrosc Imaging. 2016;5:S55–66.
22. Chaudhuri A, Chattopadhyay A. Transbilayer organization of membrane cholesterol at low concentrations: implications in health and disease. Biochim Biophys Acta. 2011;1808:19–25.
23. Fantini J, Barrantes FJ. Sphingolipid/cholesterol regulation of neurotransmitter receptor conformation and function. Biochim Biophys Acta. 2009;1788:2345–61.
24. Fantini J, Barrantes FJ. How cholesterol interacts with membrane proteins: an exploration of cholesterol-binding sites including CRAC, CARC, and tilted domains. Front Physiol. 2013;4:31.
25. Simons K, Ikonen E. How cells handle cholesterol. Science. 2000;290:1721–6.
26. Xu X, London E. The effect of sterol structure on membrane lipid domains reveals how cholesterol can induce lipid domain formation. Biochemistry. 2000;39:843–9.
27. Mukherjee S, Maxfield FR. Membrane domains. Annu Rev Cell Dev Biol. 2004;20:839–66.
28. Lingwood D, Simons K. Lipid rafts as a membrane organizing principle. Science. 2010;327:46–50.
29. Simons K, van Meer G. Lipid sorting in epithelial cells. Biochemistry. 1988;27:6197–202.
30. Simons K, Toomre D. Lipid rafts and signal transduction. Nat Rev Mol Cell Biol. 2000;1:31–9.
31. van der Goot FG, Harder T. Raft membrane domains: from a liquid-ordered membrane phase to a site of pathogen attack. Semin Immunol. 2001;13:89–97.
32. Pucadyil TJ, Chattopadhyay A. Cholesterol: a potential therapeutic target in Leishmania
infection? Trends Parasitol. 2007;23:49–53.
33. Vieira FS, Corrêa G, Einicker-Lamas M, Coutinho-Silva R. Host-cell lipid rafts: a safe door for micro-organisms? Biol Cell. 2010;102:391–407.
34. Chattopadhyay A, Jafurulla M. Role of membrane cholesterol in leishmanial infection. Adv Exp Med Biol. 2012;749:201–13.
35. Kumar GA, Jafurulla M, Chattopadhyay A. The membrane as the gatekeeper of infection: cholesterol in host-pathogen interaction. Chem Phys Lipids. 2016;199:179–85.
36. Pucadyil TJ, Chattopadhyay A. Role of cholesterol in the function and organization of G-protein coupled receptors. Prog Lipid Res. 2006;45:295–333.
37. Paila YD, Chattopadhyay A. Membrane cholesterol in the function and organization of G-protein coupled receptors. Subcell Biochem. 2010;51:439–66.
38. Oates J, Watts A. Uncovering the intimate relationship between lipids, cholesterol and GPCR activation. Curr Opin Struct Biol. 2011;21:802–7.
39. Jafurulla M, Chattopadhyay A. Membrane lipids in the function of serotonin and adrenergic receptors. Curr Med Chem. 2013;20:47–55.
40. Sengupta D, Chattopadhyay A. Molecular dynamics simulations of GPCR-cholesterol inter- action: an emerging paradigm. Biochim Biophys Acta. 2015;1848:1775–82.
41. Gimpl G. Interaction of G protein coupled receptors and cholesterol. Chem Phys Lipids. 2016;199:61–73.
42. Sengupta D, Prasanna X, Mohole M, Chattopadhyay A. Exploring GPCR-lipid interactions by molecular dynamics simulations: excitements, challenges and the way forward. J Phys Chem B. 2018;122:5727–37.
43. Paila YD, Murty MRVS, Vairamani M, Chattopadhyay A. Signaling by the human sero- tonin1A receptor is impaired in cellular model of Smith–Lemli–Opitz syndrome. Biochim Biophys Acta. 2008;1778:1508–16.
44. Dietschy JM, Turley SD. Cholesterol metabolism in the brain. Curr Opin Lipidol. 2001;12:105–12.
45. Chattopadhyay A, Paila YD. Lipid-protein interactions, regulation and dysfunction of brain cholesterol. Biochem Biophys Res Commun. 2007;354:627–33.
46. Martin M, Dotti CG, Ledesma MD. Brain cholesterol in normal and pathological aging. Biochim Biophys Acta. 2010;1801:934–44.
47. Karnell FG, Brezski RJ, King LG, Silverman MA, Monroe JG. Membrane cholesterol con- tent accounts for developmental differences in surface B cell receptor compartmentalization and signaling. J Biol Chem. 2005;280:25621–8.
48. Paila YD, Chattopadhyay A. The function of G-protein coupled receptors and membrane cholesterol: specific or general interaction? Glycoconj J. 2009;26:711–20.
49. Paila YD, Tiwari S, Chattopadhyay A. Are specific nonannular cholesterol binding sites pres- ent in G-protein coupled receptors? Biochim Biophys Acta. 2009;1788:295–302.
50. Seddon AM, Curnow P, Booth PJ. Membrane proteins, lipids and detergents: not just a soap opera. Biochim Biophys Acta. 2004;1666:105–17.
51. Kalipatnapu S, Chattopadhyay A. Membrane protein solubilization: recent advances and challenges in solubilization of serotonin1A receptors. IUBMB Life. 2005;57:505–12.
52. Privé GG. Detergents for the stabilization and crystallization of membrane proteins. Methods. 2007;41:388–97.
53. Duquesne K, Sturgis JN. Membrane protein solubilization. Methods Mol Biol. 2010;601:205–17.
54. Chattopadhyay A, Rao BD, Jafurulla M. Solubilization of G protein-coupled receptors: a con- venient strategy to explore lipid-receptor interaction. Methods Enzymol. 2015;557:117–34.
55. Goddard AD, Dijkman PM, Adamson RJ, dos Reis RI, Watts A. Reconstitution of membrane proteins: a GPCR as an example. Methods Enzymol. 2015;556:405–24.
56. Serebryany E, Zhu GA, Yan ECY. Artificial membrane-like environments for in vitro studies of purified G-protein coupled receptors. Biochim Biophys Acta. 2012;1818:225–33.
57. Chattopadhyay A, Jafurulla M, Kalipatnapu S, Pucadyil TJ, Harikumar KG. Role of cho- lesterol in ligand binding and G-protein coupling of serotonin1A receptors solubilized from bovine hippocampus. Biochem Biophys Res Commun. 2005;327:1036–41.
58. Chattopadhyay A, Paila YD, Jafurulla M, Chaudhuri A, Singh P, Murty MRVS, et al. Differential effects of cholesterol and 7-dehydrocholesterol on ligand binding of solubilized hippocampal serotonin1A receptors: implications in SLOS. Biochem Biophys Res Commun. 2007;363:800–5.
59. Singh P, Jafurulla M, Paila YD, Chattopadhyay A. Desmosterol replaces cholesterol for ligand binding function of the serotonin1A receptor in solubilized hippocampal membranes: support for nonannular binding sites for cholesterol? Biochim Biophys Acta. 2011;1808:2428–34.
60. Jafurulla M, Rao BD, Sreedevi S, Ruysschaert J-M, Covey DF, Chattopadhyay
A. Stereospecific requirement of cholesterol in the function of the serotonin1A receptor. Biochim Biophys Acta. 2014;1838:158–63.
61. Nes WD. Biosynthesis of cholesterol and other sterols. Chem Rev. 2011;111:6423–51.
62. Levitt ES, Clark MJ, Jenkins PM, Martens JR, Traynor JR. Differential effect of membrane cholesterol removal on μ- and δ-opioid receptors: a parallel comparison of acute and chronic signaling to adenylyl cyclase. J Biol Chem. 2009;284:22108–22.
63. Shrivastava S, Pucadyil TJ, Paila YD, Ganguly S, Chattopadhyay A. Chronic cholesterol depletion using statin impairs the function and dynamics of human serotonin1A receptors. Biochemistry. 2010;49:5426–35.
64. Istvan ES, Deisenhofer J. Structural mechanism for statin inhibition of HMG-CoA reductase. Science. 2001;292:1160–4.
65. Mizuno GR, Chapman CJ, Chipault JR, Pfeiffer DR. Lipid composition and (Na++K+)- ATPase activity in rat lens during triparanol-induced cataract formation. Biochim Biophys Acta. 1981;644:1–12.
66. Kolf-Clauw M, Chevy F, Wolf C, Siliart B, Citadelle D, Roux C. Inhibition of 7-dehydrocholesterol reductase by the teratogen AY9944: a rat model for Smith-Lemli-Opitz syndrome. Teratology. 1996;54:115–25.
67. Kandutsch AA, Russell AE. Preputial gland tumor sterols. A metabolic pathway from lanos- terol to cholesterol. J Biol Chem. 1960;235:2256–61.
68. Bloch KE. Sterol structure and membrane function. CRC Crit Rev Biochem. 1983;14:47–92.
69. Porter FD, Herman GE. Malformation syndromes caused by disorders of cholesterol synthe- sis. J Lipid Res. 2011;52:6–34.
70. Kanungo S, Soares N, He M, Steiner RD. Sterol metabolism disorders and neurodevelop- ment-an update. Dev Disabil Res Rev. 2013;17:197–210.
71. Gaoua W, Wolf C, Chevy F, Ilien F, Roux C. Cholesterol deficit but not accumulation of aber- rant sterols is the major cause of the teratogenic activity in the Smith-Lemli-Opitz syndrome animal model. J Lipid Res. 2000;41:637–46.
72. Chevy F, Illien F, Wolf C, Roux C. Limb malformations of rat fetuses exposed to a distal inhibitor of cholesterol biosynthesis. J Lipid Res. 2002;43:1192–200.
73. Zidovetzki R, Levitan I. Use of cyclodextrins to manipulate plasma membrane choles- terol content: evidence, misconceptions and control strategies. Biochim Biophys Acta. 2007;1768:1311–24.
74. López CA, de Vries AH, Marrink SJ. Computational microscopy of cyclodextrin mediated cholesterol extraction from lipid model membranes. Sci Rep. 2013;3:2071.
75. Niu S-L, Mitchell DC, Litman BJ. Manipulation of cholesterol levels in rod disk membranes by methyl-β-cyclodextrin: effects on receptor activation. J Biol Chem. 2002;277:20139–45.
76. Gimpl G, Burger K, Fahrenholz F. Cholesterol as modulator of receptor function. Biochemistry. 1997;36:10959–74.
77. Pang L, Graziano M, Wang S. Membrane cholesterol modulates galanin-GalR2 interaction. Biochemistry. 1999;38:12003–11.
78. Pucadyil TJ, Chattopadhyay A. Cholesterol modulates ligand binding and G-protein coupling to serotonin1A receptors from bovine hippocampus. Biochim Biophys Acta. 2004;1663:188–200.
79. Pucadyil TJ, Chattopadhyay A. Cholesterol depletion induces dynamic confinement of the G-protein coupled serotonin1A receptor in the plasma membrane of living cells. Biochim Biophys Acta. 2007;1768:655–68.
80. Bari M, Battista N, Fezza F, Finazzi-Agrò A, Maccarrone M. Lipid rafts control signaling of type-1 cannabinoid receptors in neuronal cells. Implications for anandamide-induced apop- tosis. J Biol Chem. 2005;280:12212–20.
81. Bari M, Paradisi A, Pasquariello N, Maccarrone M. Cholesterol-dependent modulation of type 1 cannabinoid receptors in nerve cells. J Neurosci Res. 2005;81:275–83.
82. Bari M, Spagnuolo P, Fezza F, Oddi S, Pasquariello N, Finazzi-Agrò A, et al. Effect of lipid rafts on Cb2 receptor signaling and 2-arachidonoyl-glycerol metabolism in human immune cells. J Immunol. 2006;177:4971–80.
83. Pydi SP, Jafurulla M, Wai L, Bhullar RP, Chelikani P, Chattopadhyay A. Cholesterol modu- lates bitter taste receptor function. Biochim Biophys Acta. 2016;1858:2081–7.
84. Chattopadhyay A, Jafurulla M, Pucadyil TJ. Ligand binding and G-protein coupling of the serotonin1A receptor in cholesterol-enriched hippocampal membranes. Biosci Rep. 2006;26:79–87.
85. Sarkar P, Chakraborty H, Chattopadhyay A. Differential membrane dipolar orientation induced by acute and chronic cholesterol depletion. Sci Rep. 2017;7:4484.
86. Sampson NS, Vrielink A. Cholesterol oxidases: a study of nature’s approach to protein design. Acc Chem Res. 2003;36:713–22.
87. Pucadyil TJ, Shrivastava S, Chattopadhyay A. Membrane cholesterol oxidation inhib- its ligand binding function of hippocampal serotonin1A receptors. Biochem Biophys Res Commun. 2005;331:422–7.
88. Jafurulla M, Nalli A, Chattopadhyay A. Membrane cholesterol oxidation in live cells enhances the function of serotonin1A receptors. Chem Phys Lipids. 2017;203:71–7.
89. Boesze-Battaglia K, Albert AD. Cholesterol modulation of photoreceptor function in bovine retinal rod outer segments. J Biol Chem. 1990;265:20727–30.
90. Nguyen DH, Taub D. Inhibition of chemokine receptor function by membrane cholesterol oxidation. Exp Cell Res. 2003;291:36–45.
91. Holz RW. The effects of the polyene antibiotics nystatin and amphotericin B on thin lipid membranes. Ann N Y Acad Sci. 1974;235:469–79.
92. Nishikawa M, Nojima S, Akiyama T, Sankawa U, Inoue K. Interaction of digitonin and its analogs with membrane cholesterol. J Biochem. 1984;96:1231–9.
93. Bolard J. How do the polyene macrolide antibiotics affect the cellular membrane properties? Biochim Biophys Acta. 1986;864:257–304.
94. Coutinho A, Prieto M. Cooperative partition model of nystatin interaction with phospholipid vesicles. Biophys J. 2003;84:3061–78.
95. Savinov SN, Heuck AP. Interaction of cholesterol with perfringolysin O: what have we learned from functional analysis? Toxins. 2017;9:381.
96. Pucadyil TJ, Shrivastava S, Chattopadhyay A. The sterol-binding antibiotic nystatin differ- entially modulates ligand binding of the bovine hippocampal serotonin1A receptor. Biochem Biophys Res Commun. 2004;320:557–62.
97. Paila YD, Pucadyil TJ, Chattopadhyay A. The cholesterol-complexing agent digitonin mod- ulates ligand binding of the bovine hippocampal serotonin1A receptor. Mol Membr Biol. 2005;22:241–9.
98. Pucadyil TJ, Kalipatnapu S, Chattopadhyay A. The serotonin1A receptor: a representative member of the serotonin receptor family. Cell Mol Neurobiol. 2005;25:553–80.
99. Kalipatnapu S, Chattopadhyay A. Membrane organization and function of the serotonin1A receptor. Cell Mol Neurobiol. 2007;27:1097–116.
100. Müller CP, Carey RJ, Huston JP, De Souza Silva MA. Serotonin and psychostimulant addic- tion: focus on 5-HT1A-receptors. Prog Neurobiol. 2007;81:133–78.
101. Lacivita E, Leopoldo M, Berardi F, Perrone R. 5-HT1A receptor, an old target for new thera- peutic agents. Curr Top Med Chem. 2008;8:1024–34.
102. Fiorino F, Severino B, Magli E, Ciano A, Caliendo G, Santagada V, et al. 5-HT1A receptor: an old target as a new attractive tool in drug discovery from central nervous system to cancer. J Med Chem. 2014;57:4407–26.
103. Singh P, Paila YD, Chattopadhyay A. Differential effects of cholesterol and 7-dehydrocholesterol on the ligand binding activity of the hippocampal serotonin1A receptor: implications in SLOS. Biochem Biophys Res Commun. 2007;358:495–9.
104. Saxena R, Chattopadhyay A. Membrane cholesterol stabilizes the human serotonin1A recep- tor. Biochim Biophys Acta. 2012;1818:2936–42.
105. Paila YD, Tiwari S, Sengupta D, Chattopadhyay A. Molecular modeling of the human seroto- nin1A receptor: role of membrane cholesterol in ligand binding of the receptor. Mol BioSyst. 2011;7:224–34.
106. Patra SM, Chakraborty S, Shahane G, Prasanna X, Sengupta D, Maiti PK, et al. Differential dynamics of the serotonin1A receptor in membrane bilayers of varying cholesterol content revealed by all atom molecular dynamics simulation. Mol Membr Biol. 2015;32:127–37.
107. Burger K, Gimpl G, Fahrenholz F. Regulation of receptor function by cholesterol. Cell Mol Life Sci. 2000;57:1577–92.
108. Klein U, Gimpl G, Fahrenholz F. Alteration of the myometrial plasma membrane choles- terol content with β-cyclodextrin modulates the binding affinity of the oxytocin receptor. Biochemistry. 1995;34:13784–93.
109. Gimpl G, Fahrenholz F. Cholesterol as stabilizer of the oxytocin receptor. Biochim Biophys Acta. 2002;1564:384–92.
110. Nguyen DH, Taub D. Cholesterol is essential for macrophage inflammatory protein 1 beta binding and conformational integrity of CC chemokine receptor 5. Blood. 2002;99:4298–306.
111. Nguyen DH, Taub D. CXCR4 function requires membrane cholesterol: implications for HIV infection. J Immunol. 2002;168:4121–6.
112. Behrens M, Meyerhof W. Bitter taste receptors and human bitter taste perception. Cell Mol Life Sci. 2006;63:1501–9.
113. Oddi S, Dainese E, Fezza F, Lanuti M, Barcaroli D, De Laurenzi V, et al. Functional char- acterization of putative cholesterol binding sequence (CRAC) in human type-1 cannabinoid receptor. J Neurochem. 2011;116:858–65.
114. Harikumar KG, Puri V, Singh RD, Hanada K, Pagano RE, Miller LJ. Differential effects of modification of membrane cholesterol and sphingolipids on the conformation, func- tion, and trafficking of the G protein-coupled cholecystokinin receptor. J Biol Chem. 2005;280:2176–85.
115. Potter RM, Harikumar KG, Wu SV, Miller LJ. Differential sensitivity of types 1 and 2 chole- cystokinin receptors to membrane cholesterol. J Lipid Res. 2012;53:137–48.
116. Cherezov V, Rosenbaum DM, Hanson MA, Rasmussen SGF, Thian FS, Kobilka TS, et al. High-resolution crystal structure of an engineered human β2-adrenergic G protein-coupled receptor. Science. 2007;318:1258–65.
117. Hanson MA, Cherezov V, Griffith MT, Roth CB, Jaakola V-P, Chein YET, et al. A specific cholesterol binding site is established by the 2.8 Å structure of the human β2-adrenergic receptor. Structure. 2008;16:897–905.
118. Wacker D, Fenalti G, Brown MA, Katritch V, Abagyan R, Cherezov V, et al. Conserved bind- ing mode of human β2 adrenergic receptor inverse agonists and antagonist revealed by X-ray crystallography. J Am Chem Soc. 2010;132:11443–5.
119. Rosenbaum DM, Zhang C, Lyons JA, Holl R, Aragao D, Arlow DH, et al. Structure and func- tion of an irreversible agonist-β2 adrenoceptor complex. Nature. 2011;469:236–40.
120. Staus DP, Strachan RT, Manglik A, Pani B, Kahsai AW, Kim TH, et al. Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation. Nature. 2016;535:448–52.
121. Huang C-Y, Olieric V, Ma P, Howe N, Vogeley L, Liu X, et al. In meso in situ serial X-ray crys- tallography of soluble and membrane proteins at cryogenic temperatures. Acta Crystallogr D Struct Biol. 2016;72:93–112.
122. Liu X, Ahn S, Kahsai AW, Meng K-C, Latorraca NR, Pani B, et al. Mechanism of intracel- lular allosteric β2AR antagonist revealed by X-ray crystal structure. Nature. 2017;548:480–4.
123. Ma P, Weichert D, Aleksandrov LA, Jensen TJ, Riordan JR, Liu X, et al. The cubicon method for concentrating membrane proteins in the cubic mesophase. Nat Protoc. 2017;12:1745–62.
124. Liu W, Chun E, Thompson AA, Chubukov P, Xu F, Katritch V, et al. Structural basis for allo- steric regulation of GPCRs by sodium ions. Science. 2012;337:232–6.
125. Batyuk A, Galli L, Ishchenko A, Han GW, Gati C, Popov PA, et al. Native phasing of x-ray free-electron laser data for a G protein-coupled receptor. Sci Adv. 2016;2:e1600292.
126. Segala E, Guo D, Cheng RKY, Bortolato A, Deflorian F, Doré AS, et al. Controlling the disso- ciation of ligands from the adenosine A2A receptor through modulation of salt bridge strength. J Med Chem. 2016;59:6470–9.
127. Martin-Garcia JM, Conrad CE, Nelson G, Stander N, Zatsepin NA, Zook J, et al. Serial mil- lisecond crystallography of membrane and soluble protein microcrystals using synchrotron radiation. IUCrJ. 2017;4:439–54.
128. Weinert T, Olieric N, Cheng R, Brünle S, James D, Ozerov D, et al. Serial millisecond crystallography for routine room-temperature structure determination at synchrotrons. Nat Commun. 2017;8:542.
129. Cheng RKY, Segala E, Robertson N, Deflorian F, Doré AS, Errey JC, et al. Structures of human A1 and A2A adenosine receptors with xanthines reveal determinants of selectivity. Structure. 2017;25:1275–85.
130. Melnikov I, Polovinkin V, Kovalev K, Gushchin I, Shevtsov M, Shevchenko V, et al. Fast iodide-SAD phasing for high-throughput membrane protein structure determination. Sci Adv. 2017;3:e1602952.
131. Broecker J, Morizumi T, Ou W-L, Klingel V, Kuo A, Kissick DJ, et al. High-throughput in situ X-ray screening of and data collection from protein crystals at room temperature and under cryogenic conditions. Nat Protoc. 2018;13:260–92.
132. Eddy MT, Lee M-Y, Gao Z-G, White KL, Didenko T, Horst R, et al. Allosteric coupling of drug binding and intracellular signaling in the A2A adenosine receptor. Cell. 2018;172:68–80.
133. Rucktooa P, Cheng RKY, Segala E, Geng T, Errey JC, Brown GA, et al. Towards high throughput GPCR crystallography: in meso soaking of adenosine A2A receptor crystals. Sci Rep. 2018;8:41.
134. Che T, Majumdar S, Zaidi SA, Ondachi P, McCorvy JD, Wang S, et al. Structure of the nanobody-stabilized active state of the kappa opioid receptor. Cell. 2018;172:55–67.
135. Manglik A, Kruse AC, Kobilka TS, Thian FS, Mathiesen JM, Sunahara RK, et al. Crystal structure of the μ-opioid receptor bound to a morphinan antagonist. Nature. 2012;485: 321–6.
136. Huang W, Manglik A, Venkatakrishnan AJ, Laeremans T, Feinberg EN, Sanborn AL, et al. Structural insights into μ-opioid receptor activation. Nature. 2015;524:315–21.
137. Wu H, Wang C, Gregory KJ, Han GW, Cho HP, Xia Y, et al. Structure of a class C GPCR metabotropic glutamate receptor 1 bound to an allosteric modulator. Science. 2014;344:58–64.
138. Byrne EFX, Sircar R, Miller PS, Hedger G, Luchetti G, Nachtergaele S, et al. Structural basis of smoothened regulation by its extracellular domains. Nature. 2016;535:517–22.
139. Huang P, Zheng S, Wierbowski BM, Kim Y, Nedelcu D, Aravena L, et al. Structural basis of smoothened activation in hedgehog signaling. Cell. 2018;174:1–13.
140. Wacker D, Wang C, Katritch V, Han GW, Huang X-P, Vardy E, et al. Structural features for functional selectivity at serotonin receptors. Science. 2013;340:615–9.
141. Liu W, Wacker D, Gati C, Han GW, James D, Wang D, et al. Serial femtosecond crystallography of G protein-coupled receptors. Science. 2013;342:1521–4.
142. Wacker D, Wang S, McCorvy JD, Betz RM, Venkatakrishnan AJ, Levit A, et al. Crystal struc- ture of an LSD-bound human serotonin receptor. Cell. 2017;168:377–89.
143. Hua T, Vemuri K, Nikas SP, Laprairie RB, Wu Y, Qu L, et al. Crystal structures of agonist- bound human cannabinoid receptor CB1. Nature. 2017;547:468–71.
144. Oswald C, Rappas M, Kean J, Doré AS, Errey JC, Bennett K, et al. Intracellular allosteric antagonism of the CCR9 receptor. Nature. 2016;540:462–5.
145. Shihoya W, Nishizawa T, Yamashita K, Inoue A, Hirata K, Kadji FMN, et al. X-ray structures of endothelin ETB receptor bound to clinical antagonist bosentan and its analog. Nat Struct Mol Biol. 2017;24:758–64.
146. Burg JS, Ingram JR, Venkatakrishnan AJ, Jude KM, Dukkipati A, Feinberg EN, et al. Structural basis for chemokine recognition and activation of a viral G protein-coupled recep- tor. Science. 2015;347:1113–7.
147. Zhang D, Gao Z-G, Zhang K, Kiselev E, Crane S, Wang J, et al. Two disparate ligand-binding sites in the human P2Y1 receptor. Nature. 2015;520:317–21.
148. Zhang J, Zhang K, Gao Z-G, Paoletta S, Zhang D, Han GW, et al. Agonist-bound structure of the human P2Y12 receptor. Nature. 2014;509:119–22.
149. Zhang K, Zhang J, Gao Z-G, Zhang D, Zhu L, Han GW, et al. Structure of the human P2Y12 receptor in complex with an antithrombotic drug. Nature. 2014;509:115–8.
150. Yao Z, Kobilka B. Using synthetic lipids to stabilize purified 𝛽2 adrenoceptor in detergent micelles. Anal Biochem. 2005;343:344–6.
151. Pontier SM, Percherancier Y, Galandrin S, Breit A, Galés C, Bouvier M. Cholesterol- dependent separation of the β2-adrenergic receptor from its partners determines signal- ing efficacy: insight into nanoscale organization of signal transduction. J Biol Chem. 2008;283:24659–72.
152. Paila YD, Jindal E, Goswami SK, Chattopadhyay A. Cholesterol depletion enhances adrener- gic signaling in cardiac myocytes. Biochim Biophys Acta. 2011;1808:461–5.
153. Zocher M, Zhang C, Rasmussen SGF, Kobilka BK, Müller DJ. Cholesterol increases kinetic, energetic, and mechanical stability of the human β2-adrenergic receptor. Proc Natl Acad Sci U S A. 2012;109:E3463–72.
154. Lam RS, Nahirney D, Duszyk M. Cholesterol-dependent regulation of adenosine A2A receptor-mediated anion secretion in colon epithelial cells. Exp Cell Res. 2009;315:3028–35.
155. Xu W, Yoon S-I, Huang P, Wang Y, Chen C, Chong PL-G, et al. Localization of the κ opioid receptor in lipid rafts. J Pharmacol Exp Ther. 2006;317:1295–306.
156. Huang P, Xu W, Yoon S-I, Chen C, Chong PL-G, Liu-Chen LY. Cholesterol reduction by methyl-β-cyclodextrin attenuates the delta opioid receptor-mediated signaling in neuronal cells but enhances it in non-neuronal cells. Biochem Pharmacol. 2007;73:534–49.
157. Eroglu C, Brügger B, Wieland F, Sinning I. Glutamate-binding affinity of Drosophila metabo- tropic glutamate receptor is modulated by association with lipid rafts. Proc Natl Acad Sci U S A. 2003;100:10219–24.
158. Kumari R, Castillo C, Francesconi A. Agonist-dependent signaling by group I metabo- tropic glutamate receptors is regulated by association with lipid domains. J Biol Chem. 2013;288:32004–19.
159. Huang P, Nedelcu D, Watanabe M, Jao C, Kim Y, Liu J, et al. Cellular cholesterol directly activates smoothened in hedgehog signaling. Cell. 2016;166:1176–87.
160. Porter JA, Young KE, Beachy PA. Cholesterol modification of hedgehog signaling proteins in animal development. Science. 1996;274:255–9.
161. Turner JH, Gelasco AK, Raymond JR. Calmodulin interacts with the third intracellular loop of the serotonin 5-hydroxytryptamine1A receptor at two distinct sites. Putative role in receptor phosphorylation by protein kinase C. J Biol Chem. 2004;279:17027–37.
162. Wheatley M, Wootten D, Conner MT, Simms J, Kendrick R, Logan RT, et al. Lifting the lid on GPCRs: the role of extracellular loops. Br J Pharmacol. 2012;165:1688–703.
163. Pal S, Aute R, Sarkar P, Bose S, Deshmukh MV, Chattopadhyay A. Constrained dynamics of the sole tryptophan in the third intracellular loop of the serotonin1A receptor. Biophys Chem. 2018;240:34–41.
164. Day PW, Rasmussen SGF, Parnot C, Fung JJ, Masood A, Kobilka TS, et al. A monoclonal antibody for G protein-coupled receptor crystallography. Nat Methods. 2007;4:927–9.
165. Rosenbaum DM, Cherezov V, Hanson MA, Rasmussen SGF, Thian FS, Kobilka TS, et al. GPCR engineering yields high-resolution structural insights into β2-adrenergic receptor func- tion. Science. 2007;318:1266–73.
166. Caffrey M. A comprehensive review of the lipid cubic phase or in meso method for crystalliz- ing membrane and soluble proteins and complexes. Acta Crystallogr F Struct Biol Commun. 2015;71:3–18.
167. Khelashvili G, Albornoz PBC, Johner N, Mondal S, Caffrey M, Weinstein H. Why GPCRs behave differently in cubic and lamellar lipidic mesophases. J Am Chem Soc. 2012;134:15858–68.
168. Epand RM. Cholesterol and the interaction of proteins with membrane domains. Prog Lipid Res. 2006;45:279–94.
169. Li H, Papadopoulos V. Peripheral-type benzodiazepine receptor function in cholesterol trans- port. Identification of a putative cholesterol recognition/interaction amino acid sequence and consensus pattern. Endocrinology. 1998;139:4991–7.
170. Vincent N, Genin C, Malvoisin E. Identification of a conserved domain of the HIV-1 trans- membrane protein gp41 which interacts with cholesteryl groups. Biochim Biophys Acta. 2002;1567:157–64.
171. Epand RM, Sayer BG, Epand RF. Caveolin scaffolding region and cholesterol-rich domains in membranes. J Mol Biol. 2005;345:339–50.
172. Jafurulla M, Tiwari S, Chattopadhyay A. Identification of cholesterol recognition amino acid consensus (CRAC) motif in G-protein coupled receptors. Biochem Biophys Res Commun. 2011;404:569–73.
173. Sengupta D, Chattopadhyay A. Identification of cholesterol binding sites in the serotonin1A receptor. J Phys Chem B. 2012;116:12991–6.
174. Baier CJ, Fantini J, Barrantes FJ. Disclosure of cholesterol recognition motifs in transmem- brane domains of the human nicotinic acetylcholine receptor. Sci Rep. 2011;1:69.
175. Fantini J, Di Scala C, Evans LS, Williamson PTF, Barrantes FJ. A mirror code for protein- cholesterol interactions in the two leaflets of biological membranes. Sci Rep. 2016;6:21907.
176. Ballesteros JA, Weinstein H. Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors. Methods Neurosci. 1995;25:366–428.
177. Prasanna X, Chattopadhyay A, Sengupta D. Cholesterol modulates the dimer interface of the
β2-adrenergic receptor via cholesterol occupancy sites. Biophys J. 2014;106:1290–300.
178. Oates J, Faust B, Attrill H, Harding P, Orwick M, Watts A. The role of cholesterol on the activity and stability of neurotensin receptor 1. Biochim Biophys Acta. 2012;1818:2228–33.
179. Lee AG. Lipid-protein interactions in biological membranes: a structural perspective. Biochim Biophys Acta. 2003;1612:1–40.
180. Simmonds AC, East JM, Jones OT, Rooney EK, McWhirter J, Lee AG. Annular and non- annular binding sites on the (Ca2+ + Mg2+)-ATPase. Biochim Biophys Acta. 1982;693:398–406.
181. Jones OT, McNamee MG. Annular and nonannular binding sites for cholesterol associated with the nicotinic acetylcholine receptor. Biochemistry. 1988;27:2364–74.
182. Marius P, Zagnoni M, Sandison ME, East JM, Morgan H, Lee AG. Binding of anionic lipids to at least three nonannular sites on the potassium channel KcsA is required for channel open- ing. Biophys J. 2008;94:1689–98.
183. Guixà-González R, Albasanz JL, Rodriguez-Espigares I, Pastor M, Sanz F, Martí-Solano M, et al. Membrane cholesterol access into a G-protein-coupled receptor. Nat Commun. 2017;8:14505.
184. McIntosh TJ. The effect of cholesterol on the structure of phosphatidylcholine bilayers. Biochim Biophys Acta. 1978;513:43–58.
185. Simon SA, McIntosh TJ, Latorre R. Influence of cholesterol on water penetration into bilayers. Science. 1982;216:65–7.
186. Nezil FA, Bloom M. Combined influence of cholesterol and synthetic amphiphilic peptides upon bilayer thickness in model membranes. Biophys J. 1992;61:1176–83.
187. McMullen TPW, Lewis RNAH, McElhaney RN. Differential scanning calorimetric study of the effect of cholesterol on the thermotropic phase behavior of a homologous series of linear saturated phosphatidylcholines. Biochemistry. 1993;32:516–22.
188. Chen Z, Rand RP. The influence of cholesterol on phospholipid membrane curvature and bending elasticity. Biophys J. 1997;73:267–76.
189. Arora A, Raghuraman H, Chattopadhyay A. Influence of cholesterol and ergosterol on mem- brane dynamics: a fluorescence approach. Biochem Biophys Res Commun. 2004;318:920–6.
190. Bacia K, Schwille P, Kurzchalia T. Sterol structure determines the separation of phases and the curvature of the liquid-ordered phase in model membranes. Proc Natl Acad Sci U S A. 2005;102:3272–7.
191. Starke-Peterkovic T, Turner N, Vitha MF, Waller MP, Hibbs DE, Clarke RJ. Cholesterol effect on the dipole potential of lipid membranes. Biophys J. 2006;90:4060–70.
192. Haldar S, Kanaparthi RK, Samanta A, Chattopadhyay A. Differential effect of cholesterol and its biosynthetic precursors on membrane dipole potential. Biophys J. 2012;102:1561–9.
193. Yeagle P. The membranes of cells. 3rd ed. Orlando, FL: Academic Press; 2016. p. 200–7.
194. Brejchová J, Sýkora J, Dlouhá K, Roubalová L, Ostašov P, Vošahlíková M, et al. Fluorescence spectroscopy studies of HEK293 cells expressing DOR-Gi1α fusion protein; the effect of cholesterol depletion. Biochim Biophys Acta. 2011;1808:2819–29.
195. Soubias O, Gawrisch K. The role of the lipid matrix for structure and function of the GPCR rhodopsin. Biochim Biophys Acta. 2012;1818:234–40.
196. Pal S, Chakraborty H, Bandari S, Yahioglu G, Suhling K, Chattopadhyay A. Molecular rheology of neuronal membranes explored using a molecular rotor: implications for receptor function. Chem Phys Lipids. 2016;196:69–75.
197. Brown MF. Modulation of rhodopsin function by properties of the membrane bilayer. Chem Phys Lipids. 1994;73:159–80.
198. Brown MF. Soft matter in lipid-protein interactions. Annu Rev Biophys. 2017;46:379–410.
199. Mitchell DC, Straume M, Miller JL, Litman BJ. Modulation of metarhodopsin formation by cholesterol-induced ordering of bilayer lipids. Biochemistry. 1990;29:9143–9.
200. Falck E, Patra M, Karttunen M, Hyvönen MT, Vattulainen I. Impact of cholesterol on voids in phospholipid membranes. J Chem Phys. 2004;121:12676–89.
201. Jafurulla M, Chattopadhyay A. Structural stringency of cholesterol for membrane protein function utilizing stereoisomers as novel tools: a review. Methods Mol Biol. 2017;1583:21–39.
202. Singh P, Haldar S, Chattopadhyay A. Differential effect of sterols on dipole potential in hippocampal membranes: implications for receptor function. Biochim Biophys Acta. 2013;1828:917–23.
203. Clarke RJ. The dipole potential of phospholipid membranes and methods for its detection. Adv Colloid Interface Sci. 2001;89-90:263–81.
204. Duffin RL, Garrett MP, Busath DD. Modulation of lipid bilayer interfacial dipole potential by phloretin, RH421, and 6-ketocholestanol as probed by gramicidin channel conductance. Langmuir. 2003;19:1439–42.
205. Starke-Peterkovic T, Turner N, Else PL, Clarke RJ. Electric field strength of membrane lipids from vertebrate species: membrane lipid composition and Na+-K+-ATPase molecular activity. Am J Physiol Regul Integr Comp Physiol. 2005;288:R663–70.
206. Bandari S, Chakraborty H, Covey DF, Chattopadhyay A. Membrane dipole potential is sensi- tive to cholesterol stereospecificity: implications for receptor function. Chem Phys Lipids. 2014;184:25–9.
207. Oakes V, Domene C. Stereospecific interactions of cholesterol in a model cell membrane: implications for the membrane dipole potential. J Membr Biol. 2018;251:507–19.
208. Mickus DE, Levitt DG, Rychnovsky SD. Enantiomeric cholesterol as a probe of ion-channel structure. J Am Chem Soc. 1992;114:359–60.
209. Covey DF. ent-Steroids: novel tools for studies of signaling pathways. Steroids. 2009; 74:577–85.
210. D’Avanzo N, Hyrc K, Enkvetchakul D, Covey DF, Nichols CG. Enantioselective protein- sterol interactions mediate regulation of both prokaryotic and eukaryotic inward rectifier K+ channels by cholesterol. PLoS One. 2011;6:e19393.
211. Kristiana I, Luu W, Stevenson J, Cartland S, Jessup W, Belani JD, et al. SBI-115 Cholesterol through the looking glass: ability of its enantiomer also to elicit homeostatic responses. J Biol Chem. 2012;287:33897–904.
212. Westover EJ, Covey DF. The enantiomer of cholesterol. J Membr Biol. 2004;202:61–72.