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.


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