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The molecular mechanism behind the action of local anesthetics is not well understood. Phenylethanol (PEtOH) is an ingredient of essential oils with a rose-like odor, and it has previously been used as a local anesthetic. In this work, we explored the effect of PEtOH on dipole potential in membranes representing biologically relevant phases, employing the dual-wavelength ratiometric method utilizing the potential-sensitive probe di-8-ANEPPS. Our results show that PEtOH reduces membrane dipole potential in membranes of all biologically relevant phases (gel, liquid-ordered, and fluid) in a concentration-dependent manner. To the best of our knowledge, these results constitute one of the early reports describing reduction of membrane dipole potential induced by local anesthetics, irrespective of membrane phase.

G protein-coupled receptors (GPCRs) are the largest family of transmembrane proteins that relay extracellular signals across the plasma membrane and elicit an intricate cascade of cellular signaling events. A significantly large fraction of available drugs target GPCRs in order to exert fine control over functional outcomes from these receptors in pathological conditions. In this context, endocytosis and intracellular trafficking of GPCRs stringently regulate signaling outcomes from GPCRs within physiologically relevant spatiotemporal regimes. The membrane microenvironment around GPCRs has recently emerged as a key player in receptor function. Cholesterol is the single most abundant lipid in the eukaryotic plasma membrane and plays a central role in membrane organization and dynamics, with far-reaching functional implications in cellular physiology. In this review, we discuss current excitements in GPCR endocytosis and trafficking, with an emphasis on the role of membrane cholesterol. We envision that a detailed understanding of the contribution of membrane lipids such as cholesterol in spatiotemporal regulation of GPCR signaling would enable the development of therapeutic interventions fine-tuned to receptors residing in specific membrane microenvironments.

Smith-Lemli-Opitz syndrome (SLOS) is a congenital and developmental malformation syndrome associated with defective cholesterol biosynthesis. It is characterized by accumulation of 7-dehydrocholesterol (the immediate biosynthetic precursor of cholesterol in the Kandutsch-Russell pathway) and an altered cholesterol to total sterol ratio. Because SLOS is associated with neurological malfunction, exploring the function and trafficking of neuronal receptors and their interaction with membrane lipids under these conditions assume significance. In this work, we generated a cellular model of SLOS in HEK-293 cells stably expressing the human serotonin1A receptor (an important neurotransmitter G-protein coupled receptor) using AY 9944, an inhibitor for the enzyme 3β-hydroxy-steroid-∆7 -reductase (7-DHCR). Using a quantitative flow cytometry based assay, we show that the plasma membrane population of serotonin1A receptors was considerably reduced under these conditions without any change in total cellular expression of the receptor. Interestingly, the receptors were trafficked to sterol-enriched LysoTracker positive compartments, which accumulated under these conditions. To the best of our knowledge, our results constitute one of the first reports demonstrating intracellular accumulation and misregulated traffic of a neurotransmitter GPCR in SLOS-like conditions. We believe these results assume relevance in our overall understanding of the molecular basis underlying the functional relevance of neurotransmitter receptors in SLOS.

The cell cycle is a sequential multi-step process essential for growth and proliferation of cells comprising multicellular organisms. Although a number of proteins are known to modulate the cell cycle, the role of lipids in regulation of cell cycle is still emerging. In our previous work, we monitored the role of cholesterol in cell cycle progression in CHO-K1 cells. Since sphingolipids enjoy a functionally synergistic relationship with membrane cholesterol, in this work, we explored whether sphingolipids could modulate the eukaryotic cell cycle using CHO-K1 cells. Sphingolipids are essential components of eukaryotic cell membranes and are involved in a number of important cellular functions. To comprehensively monitor the role of sphingolipids on cell cycle progression, we carried out metabolic depletion of sphingolipids in CHO-K1 cells using inhibitors (fumonisin B1, myriocin, and PDMP) that block specific steps of the sphingolipid biosynthetic pathway and examined their effect on individual cell cycle phases. Our results show that metabolic inhibitors led to significant reduction in specific sphingolipids, yet such inhibition in sphingolipid biosynthesis did not show any effect on cell cycle progression in CHO-K1 cells. We speculate that any role of sphingolipids on cell cycle progression could be context and cell-type dependent, and cancer cells could be a better choice for monitoring such regulation, since sphingolipids are differentially modulated in these cells.

Sphingolipids and their metabolites are increasingly implicated in the pathogenesis of many metabolic and neurological diseases. It has been postulated that sphingolipids coalesce with cholesterol ...

G protein-coupled receptors (GPCRs) constitute the largest class of membrane proteins that transduce signals across the plasma membrane and orchestrate a multitude of physiological processes within cells. The serotonin1A receptor is a crucial neurotransmitter receptor in the GPCR family involved in a multitude of neurological, behavioral and cognitive functions. We have previously shown, using a combination of experimental and simulation approaches, that membrane cholesterol acts as a key regulator of organization, dynamics, signaling and endocytosis of the serotonin1A receptor. In addition, we showed that membrane cholesterol stabilizes the serotonin1A receptor against thermal deactivation. In the present work, we explored the molecular basis of cholesterol-induced thermal stability of the serotonin1A receptor. For this, we explored the possible role of the K101 residue in a cholesterol recognition/interaction amino acid consensus (CRAC) motif in transmembrane helix 2 in conferring the thermal stability of the serotonin1A receptor. Our results show that a mutation in the K101 residue leads to loss in thermal stability of the serotonin1A receptor imparted by cholesterol, independent of membrane cholesterol content. We envision that our results could have potential implications in structural biological advancements of GPCRs and design of thermally-stabilized receptors for drug development.

The role of membrane cholesterol in modulating G protein-coupled receptor (GPCR) structure and function has emerged as a powerful theme in contemporary biology. In this paper, we report the subtlety and stringency involved in the interaction of sterols with the serotonin1A receptor. For this, we utilized two immediate biosynthetic precursors of cholesterol, 7-dehydrocholesterol (7-DHC) and desmosterol, which differ with cholesterol merely in a double bond in their chemical structures in a position-dependent manner. We show that whereas 7-DHC could not support the ligand binding function of the serotonin1A receptor in live cells, desmosterol could partially support it. Importantly, depletion and enrichment of membrane cholesterol over basal level resulted in an increase and reduction of the basal receptor activity, respectively. These results demonstrate the relevance of optimal membrane cholesterol in maintaining the activity of the serotonin1A receptor, thereby elucidating the relevance of cellular cholesterol homeostasis.

The cell cycle is a sequential multistep process essential for growth and proliferation of cells that make up multicellular organisms. A number of nuclear and cytoplasmic proteins are known to modulate the cell cycle. Yet, the role of lipids, membrane organization, and physical properties in cell cycle progression remains largely elusive. Membrane dipole potential is an important physicochemical property and originates due to the electrostatic potential difference within the membrane because of nonrandom arrangement of amphiphile dipoles and water molecules at the membrane interface. In this work, we explored the modulation of membrane dipole potential in various stages of the cell cycle in CHO-K1 cells. Our results show that membrane dipole potential is highest in the G1 phase relative to S and G2/M phases. This was accompanied by regulation of membrane cholesterol content in the cell cycle. The highest cholesterol content was found in the G1 phase with a considerable reduction in cholesterol in S and G2/M phases. Interestingly, we noted a similarity in the dependence of membrane dipole potential and cholesterol with progress of the cell cycle. In addition, we observed an increase in neutral lipid (which contains esterified cholesterol) content as cells progressed from the G1 to G2/M phase via the S phase of the cell cycle. Importantly, we further observed a cell cycle dependent reduction in ligand binding activity of serotonin1A receptors expressed in CHO-K1 cells. To the best of our knowledge, these results constitute the first report of cell cycle dependent modulation of membrane dipole potential and activity of a neurotransmitter receptor belonging to the G protein-coupled receptor family. We envision that understanding the basis of cell cycle events from a biophysical perspective would result in a deeper appreciation of the cell cycle and its regulation in relation to cellular function.

Previous work from us and others has suggested that cholesterol is an important lipid in the context of the organization of the actin cytoskeleton. However, reorganization of the actin cytoskeleton upon modulation of membrane cholesterol is rarely addressed in the literature. In this work, we explored the signaling crosstalk between cholesterol and the actin cytoskeleton by using a high-resolution confocal microscopic approach to quantitatively measure changes in F-actin content upon cholesterol depletion. Our results show that F-actin content significantly increases upon chronic cholesterol depletion, but not during acute cholesterol depletion. In addition, utilizing inhibitors targeting the cholesterol biosynthetic pathway at different steps, we show that reorganization of the actin cytoskeleton could occur due to the synergistic effect of multiple pathways, including prenylated Rho GTPases and availability of membrane phosphatidylinositol 4,5-bisphosphate. These results constitute one of the first comprehensive dissections of the mechanistic basis underlying the interplay between cellular actin levels and cholesterol biosynthesis. We envision these results will be relevant for future understating of the remodeling of the actin cytoskeleton in pathological conditions with altered cholesterol.

Macrophages are the primary hosts for Mycobacterium tuberculosis (M. tb), an intracellular pathogen, and the causative organism of tuberculosis (TB) in humans. While M. tb has the ability to enter and survive in host macrophages, the precise mechanism of its internalization, and factors that control this essential process are poorly defined. We have previously demonstrated that perturbations in levels of cholesterol and sphingolipids in macrophages lead to significant reduction in the entry of Mycobacterium smegmatis (M. smegmatis), a surrogate model for mycobacterial internalization, signifying a role for these plasma membrane lipids in interactions at the host-pathogen interface. In this work, we investigated the role of the host actin cytoskeleton, a critical protein framework underlying the plasma membrane, in the entry of M. smegmatis into human macrophages. Our results show that cytochalasin D mediated destabilization of the actin cytoskeleton of host macrophages results in a dose-dependent reduction in the entry of mycobacteria. Notably, the internalization of Escherichia coli remained invariant upon actin destabilization of host cells, implying a specific involvement of the actin cytoskeleton in mycobacterial infection. By monitoring the F-actin content of macrophages utilizing a quantitative confocal microscopy-based technique, we observed a close correlation between the entry of mycobacteria into host macrophages with cellular F-actin content. Our results constitute the first quantitative analysis of the role of the actin cytoskeleton of human macrophages in the entry of mycobacteria, and highlight actin-mediated mycobacterial entry as a potential target for future anti-TB therapeutics.

Leishmania donovani is an intracellular protozoan parasite that causes visceral leishmaniasis, a major cause of mortality and morbidity worldwide. The host plasma membrane serves as the portal of entry for Leishmania to gain access to the cellular interior. Although several host cell membrane receptors have been shown to be involved in the entry of Leishmania donovani into host cells, the endocytic pathway involved in the internalization of the parasite is not known. In this work, we explored the endocytic pathway involved in the entry of Leishmania donovani into host macrophages, utilizing specific inhibitors against two major pathways of internalization, i.e., clathrin- and caveolin-mediated endocytosis. We show that pitstop 2, an inhibitor for clathrin-mediated endocytosis, does not affect the entry of Leishmania donovani promastigotes into host macrophages. Interestingly, a significant reduction in internalization was observed upon treatment with genistein, an inhibitor for caveolin-mediated endocytosis. These results are supported by a similar trend in intracellular amastigote load within host macrophages. These results suggest that Leishmania donovani utilizes caveolin-mediated endocytosis to internalize into host cells. Our results provide novel insight into the mechanism of phagocytosis of Leishmania donovani into host cells and assume relevance in the development of novel therapeutics against leishmanial infection.

G protein-coupled receptors (GPCRs) are the largest class of membrane proteins involved in signal transduction and are characterized by seven transmembrane domain architecture interconnected by extra- and intracellular loops. These loops, along with the N- and C-terminal domains, constitute the extramembranous regions in GPCRs. These regions, accounting for ~ 40% or more amino acid residues across different GPCR classes, are distinct from the conserved transmembrane domains in terms of nonconservation of sequence, diversity in length, and conformational heterogeneity. Due to technical challenges in exploring the molecular basis underlying the relation between structure, dynamics, and function in these regions, their contribution to GPCR organization and signaling remain underappreciated. Despite existing literature on the involvement of GPCR loops in numerous aspects of GPCR biology, the functional relevance of GPCR loops in the context of their inherent conformational heterogeneity and probable membrane interaction are not well understood. This review focuses on highlighting these aspects of GPCR extramembranous regions in the overall context of GPCR organization, dynamics, and biology. We envision that a judicious combination of insights obtained from structured transmembrane domains and disordered extramembranous regions in GPCRs would be crucial in arriving at a comprehensive understanding of GPCR structure, function, and dynamics, thereby leading to efficient drug discovery.

The serotonin1A receptor is a representative member of the G protein-coupled receptor (GPCR) superfamily and acts as an important drug target. In our previous work, we comprehensively demonstrated that membrane cholesterol is necessary in the organization, dynamics and function of the serotonin1A receptor. In this context, analysis of high-resolution GPCR crystal structures in general and in silico studies of the serotonin1A receptor in particular, have suggested the presence of cholesterol interaction sites (hotspots) in various regions of the receptor. In this work, we have identified an evolutionarily conserved collage of four categories of cholesterol interaction motifs associated with transmembrane helix V and the adjacent intracellular loop 3 fragment of the vertebrate serotonin1A receptor. This collage of motifs represents a total of twenty diverse context-dependent cholesterol interaction configurations. We envision that the gamut of cholesterol interaction sites, characterized by sequence plasticity in cholesterol interaction, could be relevant in receptor-cholesterol interaction in membranes of varying cholesterol content and organization, as found in diverse cell types. We conclude that an evolutionarily conserved mechanism of GPCR-cholesterol interaction allows the serotonin1A receptor to adapt to diverse membrane cholesterol levels during natural evolution.

G protein-coupled receptors (GPCRs) represent the largest class of receptors involved in signal transduction across cell membranes and are major drug targets in all clinical areas. Endocytosis of GPCRs offers a regulatory mechanism for sustaining their signaling within a stringent spatiotemporal regime. In this work, we explored agonist-induced endocytosis of the human serotonin1A receptor stably expressed in HEK-293 cells and the cellular machinery involved in receptor internalization and intracellular trafficking. The serotonin1A receptor is a popular GPCR implicated in neuropsychiatric disorders such as anxiety and depression and serves as an important drug target. In spite of its pharmacological relevance, its mechanism of endocytosis and intracellular trafficking is less understood. In this context, we have utilized a combination of robust population-based flow cytometric analysis and confocal microscopic imaging to address the path and fate of the serotonin1A receptor during endocytosis. Our results, utilizing inhibitors of specific endocytosis pathways and intracellular markers, show that the serotonin1A receptor undergoes endocytosis predominantly via the clathrin-mediated pathway and subsequently recycles to the plasma membrane via recycling endosomes. These results would enhance our understanding of molecular mechanisms of GPCR endocytosis and could offer novel insight into the underlying mechanism of antidepressants that act via the serotonergic pathway. In addition, our results could be relevant in understanding cell (or tissue)-specific GPCR endocytosis.

Bound cholesterol molecules are emerging as important hallmarks of GPCR structures. In this commentary, we analyze their statistical prevalence and biological relevance.

An emerging feature of several high-resolution GPCR structures is the presence of closely bound cholesterol molecules. In this Perspective, we share the excitement of the recent advancements in GPCR structural biology. We further highlight our laboratory’s journey in comprehensively elucidating functional sensitivity of GPCRs (using the serotonin1A receptor as a representative neurotransmitter GPCR) to membrane cholesterol and validation using a variety of assays and molecular dynamics simulations. Although high-resolution structures of many GPCRs have been reported in the last few years, the structure of the serotoin1A receptor proved to be elusive for a long time. Very recently the cryo-EM structure of the serotoin1A receptor displaying 10 bound cholesterol molecules has been reported. We conclude by providing a critical analysis of caveats involved in GPCR structure determination.

Serotonin1A receptor senses membrane cholesterol via a lysine residue in a CRAC motif in transmembrane helix 2., The function of several G protein–coupled receptors (GPCRs) exhibits cholesterol sensitivity. Cholesterol sensitivity of GPCRs could be attributed to specific sequence and structural features, such as the cholesterol recognition/interaction amino acid consensus (CRAC) motif, that facilitate their cholesterol-receptor interaction. In this work, we explored the molecular basis of cholesterol sensitivity exhibited by the serotonin1A receptor, the most studied GPCR in the context of cholesterol sensitivity, by generating mutants of key residues in CRAC motifs in transmembrane helix 2 (TM2) and TM5 of the receptor. Our results show that a lysine residue (K101) in one of the CRAC motifs is crucial for sensing altered membrane cholesterol levels. Insights from all-atom molecular dynamics simulations showed that cholesterol-sensitive functional states of the serotonin1A receptor are associated with reduced conformational dynamics of extracellular loops of the receptor. These results constitute one of the first reports on the molecular mechanism underlying cholesterol sensitivity of GPCRs.

Although widely used clinically, the mechanism underlying the action of local anesthetics remains elusive. Direct interaction of anesthetics with membrane proteins and modulation of membrane physical properties by anesthetics are plausible mechanisms proposed, although a combination of these two mechanisms cannot be ruled out. In this context, the role of G protein-coupled receptors (GPCRs) in local anesthetic action is a relatively new area of research. We show here that representative tertiary amine local anesthetics induce a reduction in two-dimensional diffusion coefficient of the serotonin1A receptor, an important neurotransmitter GPCR. The corresponding change in mobile fraction is varied, with tetracaine exhibiting the maximum reduction in mobile fraction, whereas the change in mobile fraction for other local anesthetics was not appreciable. These results are supported by quantitation of cellular F-actin, using a confocal microscopic approach previously developed by us, which showed that a pronounced increase in F-actin level was induced by tetracaine. These results provide a novel perspective on the action of local anesthetics in terms of GPCR lateral diffusion and actin cytoskeleton reorganization.

Cholesterol is an indispensible component of cellular membranes in higher eukaryotes and plays a vital role in many cellular functions. 7-Dehydrocholesterol (7-DHC) and desmosterol represent two immediate biosynthetic precursors of cholesterol in the Kandutsch-Russell and Bloch pathways of cholesterol biosynthesis, respectively. Although 7-DHC and desmosterol differ from cholesterol merely by a double bond, accumulation of these two immediate biosynthetic precursors due to defective cholesterol biosynthesis leads to severe developmental and neurological disorders. In this context, we explored the role of cholesterol and its immediate biosynthetic precursors (7-DHC and desmosterol) on the dynamics and heterogeneity of fluid phase POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and gel phase DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) membranes, using fluorescence lifetime distribution analysis of Nile Red (9-diethylamino-5H-benzo[α]phenoxazine-5-one) using the maximum entropy method (MEM). We show here that the membrane interfacial dynamic heterogeneity, manifested as the width of the fluorescence lifetime distribution of Nile Red, exhibited by 7-DHC and desmosterol vastly differ from that displayed by cholesterol, particularly in fluid phase membranes. We conclude that a subtle alteration in sterol structure could considerably alter dynamic membrane heterogeneity, which could have implications in pathogenicity associated with defective cholesterol biosynthesis.

Plasma membrane (PM) lipid composition imbalances affect drug susceptibilities of the human pathogen Candida albicans. The PM fundamental structure is made up of phospholipid bilayer where phosphatidylethanolamine (PE) contributes as second major phospholipid moieties, which is asymmetrically distributed between the two leaflets of the bilayer. PSD1 and PSD2 genes encode phosphatidylserine decarboxylase which converts phosphatidylserine (PS) into PE in C. albicans cells. Genetic manipulation of PSD1 and PSD2 genes is known to impact virulence, cell wall thickness and mitochondrial function in C. albicans. In the present study, we have examined the impact of PSD1 and PSD2 deletion on physiochemical properties of PM. Our fluorescence recovery after photobleaching (FRAP) experiments point that the PM of psd1Δ/Δ psd2Δ/Δ mutant strain displays increased membrane fluidity and reduced PM dipole potential. Further, the result of PSD1 and PSD2 deletion on the thermotropic phase behavior monitored by differential scanning calorimetry (DSC) showed that in comparison to WT, the apparent phase transition temperature is reduced by ~3 °C in the mutant strain. The functional consequence of altered physical state of PM of psd1Δ/Δ psd2Δ/Δ mutant strain was evident from observed high diffusion of fluorescent dye rhodamine 6G and radiolabelled fluconazole (FLC). The higher diffusion of FLC resulted in an increased drug accumulation in psd1Δ/Δ psd2Δ/Δ mutant cells, which was manifested in an increased susceptibility to azoles. To the best of our knowledge, these results constitute the first report on the effect of the levels of phospholipid biosynthesis enzyme on physiochemical properties of membranes and drug susceptibilities of Candida cells.

Mycobacteria are intracellular pathogens that can invade and survive within host macrophages. Mycobacterial infections remain a major cause of mortality and morbidity worldwide, with serious concerns of emergence of multi and extensively drug-resistant tuberculosis. While significant advances have been made in identifying mycobacterial virulence determinants, the detailed molecular mechanism of internalization of mycobacteria into host cells remains poorly understood. Although several studies have highlighted the crucial role of sphingolipids in mycobacterial growth, persistence and establishment of infection, the role of sphingolipids in the entry of mycobacteria into host cells is not known. In this work, we explored the role of host membrane sphingolipids in the entry of Mycobacterium smegmatis into J774A.1 macrophages. Our results show that metabolic depletion of sphingolipids in host macrophages results in a significant reduction in the entry of M. smegmatis. Importantly, the entry of Escherichia coli into host macrophages under similar conditions remained invariant, implying the specificity of the requirement of sphingolipids in mycobacterial entry. To the best of our knowledge, our results constitute the first report demonstrating the role of host macrophage sphingolipids in the entry of mycobacteria. Our results could help in the development of novel therapeutic strategies targeting sphingolipid-mediated entry of mycobacteria into host cells.

International audience; ATP-binding cassette (ABC) transporters help export various substrates across the cell membrane and significantly contribute to drug resistance. However, a recent study reported an unusual case in which the loss of an ABC transporter in Candida albicans, orf19.4531 (previously named ROA1), increases resistance against antifungal azoles, which was attributed to an altered membrane potential in the mutant strain. To obtain further mechanistic insights into this phenomenon, here we confirmed that the plasma membrane-localized transporter (renamed CDR6/ROA1 for consistency with C. albicans nomenclature) could efflux xenobiotics such as berberine, rhodamine 123, and paraquat. Moreover, a CDR6/ROA1 null mutant, NKKY101, displayed increased susceptibility to these xenobiotics. Interestingly, fluorescence recovery after photobleaching (FRAP) results indicated that NKKY101 mutant cells exhibited increased plasma membrane rigidity, resulting in reduced azole accumulation and contributing to azole resistance. Transcriptional profiling revealed that ribosome biogenesis genes were significantly up-regulated in the NKKY101 mutant. As ribosome biogenesis is a well-known downstream phenomenon of target of rapamycin (TOR1) signaling, we suspected a link between ribosome biogenesis and TOR1 signaling in NKKY101. Therefore, we grew NKKY101 cells on rapamycin and observed TOR1 hyperactivation, which leads to Hsp90-dependent calcineurin stabilization and thereby increased azole resistance. This in vitro finding was supported by in vivo data from a mouse model of systemic infection in which NKKY101 cells led to higher fungal load after fluconazole challenge than wild-type cells. Taken together, our study uncovers a mechanism of azole resistance in C. albicans, involving increased membrane rigidity and TOR signaling.

Solubilization of membrane proteins by amphiphilic detergents represents a crucial step in studies of membrane proteins in which proteins and lipids in natural membranes are dissociated giving rise to mixed clusters of proteins, lipids and detergents in the aqueous dispersion. Although solubilization is a popular method, physicochemical principles underlying solubilization are not well understood. In this work, we monitored solubilization of the bovine hippocampal serotonin1A receptor, a representative member of the GPCR family, using membrane dipole potential measured by a dual fluorescence ratiometric approach with a potential-sensitive fluorophore. Our results show that membrane dipole potential is a good indicator of solubilization and reflects the change in dipolar environment upon solubilization due to dipolar reorganization associated with solubilization. To the best of our knowledge, these results constitute the first report linking membrane dipole potential with solubilization. We envision that these results are potentially useful in providing a molecular mechanism for membrane protein solubilization.

Sphingolipids are essential components of eukaryotic cell membranes and are known to modulate a variety of cellular functions. It is becoming increasingly clear that membrane lipids play a crucial role in modulating the function of integral membrane proteins such as G protein-coupled receptors (GPCRs). In this work, we utilized LY-B cells, that are sphingolipid-auxotrophic mutants defective in sphingolipid biosynthesis, to monitor the role of cellular sphingolipids in the function of an important neurotransmitter receptor, the serotonin1A receptor. Serotonin1A receptors belong to the family of GPCRs and are implicated in behavior, development and cognition. Our results show that specific ligand binding and G-protein coupling of the serotonin1A receptor exhibit significant enhancement under sphingolipid-depleted conditions, which reversed to control levels upon replenishment of cellular sphingolipids. In view of the reported role of sphingolipids in neuronal metabolism and pathogenesis of several neuropsychiatric disorders, exploring the role of serotonin1A receptors under conditions of defective sphingolipid metabolism assumes relevance, and could contribute to our overall understanding of such neuropsychiatric disorders. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.

The serotonin1A (5-HT1A) receptor is an important neurotransmitter receptor that belongs to the G protein-coupled receptor (GPCR) family. It is implicated in a variety of cognitive and behavioral functions and serves as an important drug target for neuropsychiatric disorders such as anxiety and depression. Previous work from our laboratory has demonstrated that membrane cholesterol plays an important role in the function of the serotonin1A receptor. Our earlier results highlighted several structural features of cholesterol essential for receptor function. In order to explore the importance of the hydroxyl group of cholesterol in the function of the serotonin1A receptor, we utilized cholesterol oxidase to oxidize the hydroxyl group of cholesterol to keto group. Our results show that the oxidation of the hydroxyl group of cholesterol in live cells resulted in enhancement of agonist binding and G-protein coupling to the receptor with no appreciable change in overall membrane order. These results extend our understanding of the structural requirements of cholesterol for receptor function.

pH (low) insertion peptide (pHLIP) is a polypeptide from the third transmembrane helix of bacteriorhodopsin. The pH-dependent membrane insertion of pHLIP has been conveniently exploited for translocation of cargo molecules and as a novel imaging agent in cancer biology due to low extracellular pH in cancer tissues. Although the application of pHLIP for imaging tumor and targeted drug delivery is well studied, literature on pHLIP-membrane interaction is relatively less studied. Keeping this in mind, we explored the differential interaction of pHLIP with ester and ether lipid membranes utilizing fluorescence and CD spectroscopy. We report, for the first time, higher binding affinity of pHLIP toward ether lipid relative to ester lipid membranes. There results gain relevance since Halobacterium halobium (source of bacteriorhodopsin) is enriched with ether lipids. In addition, we monitored the difference in microenvironment around pHLIP tryptophans utilizing red edge excitation shift and observed increased motional restriction of water molecules in the interfacial region in ether lipid membranes. These changes were accompanied with increase in helicity of pHLIP in ether lipid relative to ester lipid membranes. Our results assume further relevance since ether lipids are upregulated in cancer cells and have emerged as potential biomarkers of various diseases including cancer.

Simvastatin belongs to the statin family of cholesterol lowering drugs which act as competitive inhibitors of HMG-CoA reductase, the rate-determining enzyme in cholesterol biosynthesis pathway. Simvastatin is a semi-synthetic, highly lipophilic statin, and has several side effects. Since HMG-CoA reductase is localized in the endoplasmic reticulum, orally administered simvastatin needs to cross the cellular plasma membrane to be able to act on HMG-CoA reductase. With an overall goal of exploring the interaction of simvastatin with membranes, we examined the effect of simvastatin on the organization and dynamics in membranes of varying phase, in a depth-dependent manner. For this, we employed DPH and TMA-DPH, which represent fluorescent membrane probes localized at two different locations (depths) in the membrane. Analysis of fluorescence anisotropy and lifetime data of these depth-specific probes in membranes of varying phase (gel/fluid/liquid-ordered) showed that the maximum membrane disordering was observed in gel phase, while moderate effects were observed in liquid-ordered phase, with no significant change in membrane order in fluid phase membranes. We conclude that simvastatin induces change in membrane order in a depth-dependent and phase-specific manner. These results provide novel insight in the membrane interaction of simvastatin and could be crucial for understanding its pharmacological effect.

G protein-coupled receptors (GPCRs) are transmembrane receptors that mediate a large number of cellular responses. The organization of GPCRs into dimers and higher-order oligomers is known to allow a larger repertoire of downstream signaling events. In this context, a crosstalk between the adenosine and dopamine receptors has been reported, indicating the presence of heterodimers that are functionally relevant. In this paper, we explored the effect of membrane cholesterol on the adenosine2A (A2A) and dopamine D3 (D3) receptors using coarse-grain molecular dynamics simulations. We analyzed cholesterol interaction sites on the A2A receptor and were able to reproduce the sites indicated by crystallography and previous atomistic simulations. We predict novel cholesterol interaction sites on the D3 receptor that could be important in the reported cholesterol sensitivity in receptor function. Further, we analyzed the formation of heterodimers between the two receptors. Our results suggest that membrane cholesterol modulates the relative population of several co-existing heterodimer conformations. Both direct receptor-cholesterol interaction and indirect membrane effects contribute toward the modulation of heterodimer conformations. These results constitute one of the first examples of modulation of GPCR hetero-dimerization by membrane cholesterol, and could prove to be useful in designing better therapeutic strategies.

The dynamic interactions between G protein-coupled receptors (GPCRs) and their cognate protein partners are central to several cell signaling pathways. For example, the association of CXC chemokine receptor 1 (CXCR1) with its cognate chemokine, interleukin-8 (IL8 or CXCL8) initiates pathways leading to neutrophil-mediated immune responses. The N-terminal domain of chemokine receptors confers ligand selectivity, but unfortunately the conformational dynamics of this intrinsically disordered region remains unresolved. In this work, we have explored the interaction of CXCR1 with IL8 by microsecond time scale coarse-grain simulations, complemented by atomistic models and NMR chemical shift predictions. We show that the conformational plasticity of the apo-receptor N-terminal domain is restricted upon ligand binding, driving it to an open C-shaped conformation. Importantly, we corroborated the dynamic complex sampled in our simulations against chemical shift perturbations reported by previous NMR studies and show that the trends are similar. Our results indicate that chemical shift perturbation is often not a reporter of residue contacts in such dynamic associations. We believe our results represent a step forward in devising a strategy to understand intrinsically disordered regions in GPCRs and how they acquire functionally important conformational ensembles in dynamic protein-protein interfaces., Author summary How cells communicate with the outside environment is intricately controlled and regulated by a large family of receptors on the cell membrane (G protein-coupled receptors or GPCRs) that respond to external signals (termed ligands). Chemokine receptors belong to this GPCR family and regulate immune responses. We analyze here the first step of binding of a representative chemokine receptor (CXCR1) with its natural ligand, interleukin-8 (IL8) by an extensive set of molecular dynamics simulations. Our work complements previous mutational and NMR experiments which lack molecular-level resolution. We show that in the inactive state, one of the extracellular domains of the CXCR1 receptor, namely the N-terminal domain, is highly flexible and like a "shape-shifter" can exist in multiple conformational states. However, when IL8 binds, the N-terminal domain undergoes a conformational freezing, and acquires a C-shaped "claw-like" structure. The complex between the receptor and IL8 is still quite dynamic as this C-shaped N-terminal domain forms an extensive but slippery interface with the ligand. We further corroborated these results by quantitative comparison with NMR and mutagenesis studies. Our work helps clarify the inherent disorder in N-terminal domains of chemokine receptors and demonstrates how this domain can acquire functionally important conformational states in dynamic protein-protein interfaces.

Endocytosis and intracellular trafficking constitute important regulatory features associated with G protein-coupled receptor (GPCR) function. GPCR endocytosis involves several remodeling events at the plasma membrane orchestrated by a concerted interplay of a large number of proteins and membrane lipids. Although considerable literature exists on the protein framework underlying GPCR endocytosis, the role of membrane lipids in this process remains largely unexplored. In order to explore the role of membrane cholesterol (an essential and important lipid in higher eukaryotes) in GPCR endocytosis, we monitored the effect of acute cholesterol depletion using methyl-β-cyclodextrin (MβCD) on endocytosis and intracellular trafficking of the serotonin1A receptor, an important neurotransmitter GPCR. Our results show that the serotonin1A receptor exhibits agonist-induced clathrin-mediated endocytosis with a concentration-dependent inhibition in internalization with increasing concentrations of MβCD, which was restored upon cholesterol replenishment. Interestingly, subsequent to internalization under these conditions, serotonin1A receptors were re-routed toward lysosomal degradation, instead of endosomal recycling observed under normal conditions, thereby implicating membrane cholesterol in modulation of intracellular trafficking of the receptor. This raises the possibility of a novel cholesterol-dependent role of intracellular sorting proteins in GPCR trafficking. These results differ from our previous observations on the endocytosis of the serotonin1A receptor upon statin-induced chronic cholesterol depletion, in terms of endocytic pathway. We conclude that analysis of complex cellular trafficking events such as GPCR endocytosis under acute and chronic cholesterol depletion conditions should be carried out with caution due to fundamental differences underlying these processes.

The fundamental mechanism behind the action of local anesthetics is still not clearly understood. Phenylethanol (PEtOH) is a constituent of essential oils with a pleasant odor and can act as a local anesthetic. In this work, we have explored the effect of PEtOH on the function of the hippocampal serotonin1A receptor, a representative neurotransmitter receptor belonging to the G protein-coupled receptor (GPCR) family. Our results show that PEtOH induces reduction in ligand binding to the serotonin1A receptor due to lowering of binding affinity, along with a concomitant decrease in the degree of G-protein coupling. Analysis of membrane order using the environment-sensitive fluorescent probe DPH revealed decrease in membrane order with increasing PEtOH concentration, as evident from reduction in rotational correlation time of the probe. Analysis of results obtained shows that the action of local anesthetics could be attributed to the combined effects of specific interaction of the receptor with anesthetics and alteration of membrane properties (such as membrane order). These results assume relevance in the perspective of anesthetic action and could be helpful to achieve a better understanding of the possible role of anesthetics in the function of membrane receptors.

Glycosphingolipids are minor yet essential components of eukaryotic cell membranes and are involved in a variety of cellular processes. Although glycosphingolipids such as GM1 have been previously reported to influence the function of G protein-coupled receptors (GPCRs), the molecular mechanism remains elusive. In this paper, we have explored the interaction of GM1 with the serotonin1A receptor, an important neurotransmitter receptor that belongs to the GPCR family. To examine the molecular basis of the interaction of GM1 with the serotonin1A receptor, we performed a series of coarse-grain molecular dynamics simulations of the receptor embedded in membrane bilayers containing GM1. Our results show that GM1 interacts with the serotonin1A receptor predominantly at the extracellular loop 1 and specifically at the sphingolipid binding domain (SBD). The SBD motif consists of a characteristic combination of aromatic, basic and turn-inducing residues, and is evolutionarily conserved in case of the serotonin1A receptor. The interaction of the SBD site with GM1 appears to stabilize a ‘flip-out’ conformation in which W102 of the extracellular loop 1 flips out from the central lumen of the receptor toward the membrane. The population of the ‘flip-out’ conformation is increased in the presence of cholesterol. Our data strongly suggest that a direct interaction between GM1 and the SBD site of the serotonin1A receptor may occur in vivo. In view of the reported role of GM1 and the serotonin1A receptor in neurodegenerative diseases, GM1-receptor interaction assumes significance in the context of malfunctioning of neuronal GPCRs under such conditions.

The molecular mechanism underlying the action of local anesthetics remains elusive. Phenylethanol (PEtOH) is an ingredient of essential oils with a rose-like odor and has been used as a local anesthetic. In this work, we explored the effect of PEtOH on organization and dynamics in membranes representing various biologically relevant phases using differentially localized fluorescent membrane probes, DPH and TMA-DPH. We show here that PEtOH induces disorder in membranes of all phases (gel/fluid/liquid-ordered). However, the extent of membrane disorder varies in a phase-specific manner. Maximum membrane disordering was observed in gel phase, followed by liquid-ordered membranes. The disordering was minimal in fluid phase membranes. Interestingly, our results show that the disordering effect of PEtOH in gel phase is sufficiently large to induce phase change at higher PEtOH concentrations. Our results are relevant in the context of natural membranes and could be useful in understanding the role of anesthetics in membrane protein function.

The human serotonin1A receptor is a representative member of the superfamily of G protein-coupled receptors (GPCRs) and an important drug target for neurological disorders. Using a combination of biochemical, biophysical and molecular dynamics simulation approaches, we and others have shown that membrane cholesterol modulates the organization, dynamics and function of vertebrate serotonin1A receptors. Previous studies have shown that the cytoplasmic portion of transmembrane helix V (TM V) and the extramembraneous intracellular loop 3 are critical for G-protein coupling, phosphorylation and desensitization of the receptor. We have recently resolved a collage of putative cholesterol interaction motifs from the amino acid sequence overlapping this region. In this paper, we explore the sequence plasticity of this fragment that may have adapted to altered membrane lipidome, after vertebrates evolved from primordial invertebrates. Since invertebrates have lower levels of membrane cholesterol relative to vertebrates, we compared TM V sequence fragments from invertebrate serotonin1 receptors with vertebrate orthologs to infer the sequence plasticity in TM V. We report that the average number of cholesterol interaction motifs in TM V for diverse phyla represents an increasing trend that could mirror vertebrate evolution from primordial invertebrates. By statistical modeling, we propose that the collage of cholesterol interaction motifs in TM V of the human serotonin1A receptor may have evolved from rudimentary collages, reminiscent of primordial invertebrate orthologs. Taken together, we propose that a repertoire of cholesterol-philic nonsynonymous substitutions may have enhanced collage complexity in TM V during vertebrate evolution.

G protein-coupled receptors (GPCRs) constitute the largest superfamily of membrane proteins in higher eukaryotes, and facilitate information transfer from the extracellular environment to the cellular interior upon activation by ligands. Their role in diverse signaling processes makes them an attractive choice as drug targets. GPCRs are coupled to heterotrimeric G-proteins which represent an important interface through which signal transduction occurs across the plasma membrane upon activation by ligands. To obtain further insight into the molecular details of interaction of G-proteins with GPCRs, in this work, we explored the selectivity of binding of specific agonists and antagonists to the serotonin1A receptor under conditions of progressive G-protein inactivation. The serotonin1A receptor is an important neurotransmitter receptor belonging to the GPCR family and is a popular drug target. By use of a number of agents to inactivate G-proteins, we show here that the serotonin1A receptor displays differential discrimination between agonist and antagonist binding. Our results show a reduction in binding sites of the receptor upon treatment with G-protein inactivating agents. In addition, G-protein coupling efficiency was enhanced when G-proteins were inactivated using urea and alkaline pH. We envision that our results could be useful in achieving multiple signaling states of the receptor by fine tuning the conditions of G-protein inactivation and in structural biology of GPCRs bound to specific ligands.

Sphingolipids correspond to a major class of lipids which serve as indispensable structural components of membranes and play an important role in various cellular functions. They constitute ~10-20% of total membrane lipids and are known to form segregated domains in biological membranes. Sphingolipids have been shown to play a vital role in the function of various G protein-coupled receptors (GPCRs). We report here the presence of sphingolipid-binding motif (SBM) in representative GPCRs such as cholecystokinin, oxytocin and secretin receptors, and subtypes of human serotonin receptors. We previously reported the importance of sphingolipids in the function of the serotonin1A receptor, a representative member of the GPCR superfamily, involved in behavioral, cognitive, and developmental functions. In this work, we show that the serotonin1A receptor contains a putative SBM, corresponding to amino acids 205 to 213. In addition, our analysis shows that SBM is an intrinsic characteristic feature of the serotonin1A receptor and is conserved throughout the course of natural evolution. Our results represent the first report on the presence of SBM in serotonin1A receptors and provide novel insight on the molecular mechanism of GPCR-sphingolipid interaction.

Dipole potential is the potential difference within the membrane bilayer, which originates due to the nonrandom arrangement of lipid dipoles and water molecules at the membrane interface. Although dipole potential is generally used in the context of bilayer membranes, the nonrandom arrangement of amphiphiles and water dipoles would also contribute to dipole potential in organized molecular assemblies such as micelles. In this work, we show that the process of micelle formation from monomers for a representative variety of detergents is associated with dipolar rearrangement. We monitor the dipolar reorganization upon micellization as a change in dipole potential, measured by the dual wavelength ratiometric approach utilizing the potential-sensitive membrane probe di-8-ANEPPS. We further utilized this phenomenon to estimate the critical micelle concentration (CMC) of a variety of detergents. CMC determined by this method are in overall agreement with the literature values of CMC for these detergents. To the best of our knowledge, these results constitute the first report showing dipolar reorientation during micellization. We conclude that dipole potential measurements could provide a novel approach to explore micellar organization.

The function of membrane receptors in the nervous system depends on physicochemical characteristics of neuronal membranes such as membrane order and phase. In this work, we have monitored the changes in hippocampal membrane order and related parameters by cholesterol and protein content utilizing a Nile Red-based phase-sensitive fluorescent membrane probe NR12S. Since alteration of membrane cholesterol is often associated with membrane phase change, the phase-sensitive nature of NR12S fluorescence becomes useful in these experiments. Our results show that fluorescence spectroscopic parameters such as emission maximum, anisotropy, and lifetime of NR12S display characteristic dependence on membrane cholesterol content. Interestingly, cholesterol-dependent red edge excitation shift is displayed by NR12S under these conditions. Hippocampal membranes exhibited reduction in liquid-ordered phase upon cholesterol depletion. These results provide insight into changes in hippocampal membrane order in the overall context of cholesterol and protein modulation.

Mycobacteria are intracellular pathogens that can invade and survive within host macrophages, and are a major cause of mortality and morbidity worldwide. The molecular mechanism involved in the internalization of mycobacteria is poorly understood. In this work, we have explored the role of host membrane cholesterol in the entry of the avirulent surrogate mycobacterial strain Mycobacterium smegmatis into THP-1 macrophages. Our results show that depletion of host membrane cholesterol using methyl-β-cyclodextrin results in a significant reduction in the entry of M. smegmatis into host cells. More importantly, we show that the inhibition in the ability of M. smegmatis to enter host macrophages could be reversed upon replenishment of membrane cholesterol. To the best of our knowledge, these results constitute the first report showing that membrane cholesterol replenishment can reverse the inhibition in the entry of mycobacteria into host cells. In addition, we demonstrate that cholesterol complexation using amphotericin B (without physical depletion) is sufficient to inhibit mycobacterial entry. Importantly, we observed a significant reduction in mycobacterial entry upon enrichment of host membrane cholesterol. Taken together, our results demonstrate, for the first time, that an optimum host plasma membrane cholesterol is necessary for the entry of mycobacteria. These results assume relevance in the context of developing novel therapeutic strategies targeting cholesterol-mediated mycobacterial host cell entry.

The molecular mechanism underlying the action of local anesthetics is still elusive. Phenylethanol (PEtOH) is an ingredient of essential oils with a rose-like odor and has been used as a local anesthetic. In this work, we have explored the effect of PEtOH on thermotropic behavior and organization of dipalmitoylphosphatidylcholine (DPPC) membranes utilizing differential scanning calorimetry (DSC) and small angle X-ray scattering (SAXS). Our results indicate that the phase transition temperature of DPPC exhibited decrease with increasing PEtOH concentration. This is accompanied by hysteresis (difference in phase transition between the heating and cooling scans). We defined the threshold concentration of PEtOH as the concentration at which the difference in phase transition temperature between the heating and cooling thermograms is maximum. Interestingly, changes in enthalpy, entropy, and full width at half maximum displayed biphasic behavior beyond the threshold concentration of PEtOH. The biphasic change in thermodynamic parameters corresponding to phase transtition, coupled with hysteresis, is indicative of interdigitation in DPPC bilayers. We confirmed this proposition by SAXS measurements which show formation of the interdigitated phase in DPPC bilayers at and above the threshold concentration of PEtOH. To the best of our knowledge, these results constitute the first report describing the interdigitation of membrane bilayers induced by PEtOH. We further show that the formation of interdigitated phase in DPPC bilayers depends on PEtOH concentration and temperature. Our results could be useful in ongoing efforts to address the mechanism of action of local anesthetics in model and biological membranes.

The CXC chemokine receptor 1 (CXCR1) is an important member of the G protein-coupled receptor (GPCR) family in which the extracellular N-terminal domain has been implicated in ligand binding and selectivity. The structure of this domain has not yet been elucidated due to its inherent dynamics, but experimental evidence points toward membrane-dependent organization and dynamics. To gain molecular insight into the interaction of the N-terminal domain with the membrane bilayer, we performed a series of microsecond time scale atomistic simulations of the N-terminal domain of CXCR1 in the presence and absence of POPC bilayers. Our results show that the peptide displays a high propensity to adopt a β-sheet conformation in the presence of the membrane bilayer. The interaction of the peptide with the membrane bilayer was found to be transient in our simulations. Interestingly, a scrambled peptide, containing the same residues in a randomly varying sequence, did not exhibit membrane-modulated structural dynamics. These results suggest that sequence-dependent electrostatics, modulated by the membrane, could play an important role in folding of the N-terminal domain. We believe that our results reinforce the emerging paradigm that cellular membranes could be important modulators of function of G protein-coupled receptors such as CXCR1.

Membrane fusion, one of the most fundamental processes in life, occurs when two separate lipid membranes merge into a single continuous bilayer. Membrane fusion is essential for the entry of lipid-sheathed viruses such as influenza and HIV. Influenza virus is internalized via receptor-mediated endocytosis and then fuses with the endosomal membrane at low pH. Hemagglutinin, a glycoprotein found on the surface of influenza virus, is responsible for the fusion of the viral sheath with the endosomal membrane. The ∼20 amino acid long N-terminus of hemagglutinin, known as the fusion peptide, plays a crucial role in the viral fusion process. Although there exists vast literature on the importance and role of the fusion peptide in promoting membrane fusion, there is no consensus on the mechanism by which it promotes fusion. A recent report suggested that the fusion peptide occupies and orders space in the outer leaflets of contacting bilayers so as to promote acyl chain protrusion into interbilayer space and promote fusion "stalk" formation. We report here the effect of the wild type, G1S, G1V, and W14A mutants of hemagglutinin fusion peptide on depth-dependent ordering of model membranes along the bilayer normal. We utilized fluorescence anisotropy, lifetime measurements, and lifetime distribution analyses of different anthroyloxy stearic acid probes (n-AS) in order to examine the effect of fusion peptides at various depths along the bilayer normal. Wild type peptide uniquely ordered a region ∼12 Å from the bilayer midpoint, W14A and G1S mutants mainly ordered the bilayer interface, while G1V had little ordering influence. On the basis of recent analysis of the effects of these peptides on fusion, ordering of the mid-upper region of the bilayer appears to promote fusion pore formation, while ordering of the bilayer interface inhibits it.

Dipole potential is the potential difference within the membrane bilayer, which originates due to the nonrandom arrangement of lipid dipoles and water molecules at the membrane interface. Cholesterol, an essential lipid in higher eukaryotic membranes, has previously been shown to increase membrane dipole potential. In this work, we explored the effect of stereoisomers of cholesterol, ent-cholesterol and epi-cholesterol, on membrane dipole potential, monitored by the dual wavelength ratiometric approach utilizing the probe di-8-ANEPPS. Our results show that cholesterol and ent-cholesterol share comparable ability in increasing membrane dipole potential. In contrast, epi-cholesterol displays a slight reduction in membrane dipole potential. Our results constitute the first report on the effect of stereoisomers of cholesterol on membrane dipole potential, and imply that an extremely subtle change in sterol structure can significantly alter the dipolar field at the membrane interface. These results assume relevance in the context of differential abilities of these stereoisomers of cholesterol in supporting the activity of the serotonin1A receptor, a representative G protein-coupled receptor. The close correlation between membrane dipole potential and receptor activity provides new insight in receptor-cholesterol interaction in terms of stereospecificity. We envision that membrane dipole potential could prove to be a sensitive indicator of lipid-protein interactions in biological membranes.

G protein-coupled receptors (GPCRs) are the largest family of proteins involved in signal transduction across cell membranes, and they represent major drug targets in all clinical areas. Oligomerization of GPCRs and its implications in drug discovery constitute an exciting area in contemporary biology. In this Review, we have highlighted the application of fluorescence resonance energy transfer (FRET) in exploring GPCR oligomerization, with special emphasis on possible pitfalls and experimental complications involved. Based on FRET photophysics, we discuss some of the possible complications, and recommend that FRET results in complex cellular environments should be interpreted with caution. Although both hetero- and homo-FRET are used in measurements of GPCR oligomerization, we suggest that homo-FRET enjoys certain advantages over hetero-FRET. Given the seminal role of GPCRs as current drug targets, we envision that methodological progress in studying GPCR oligomerization would result in better therapeutic strategies.

G protein-coupled receptors (GPCRs) are the largest class of molecules involved in signal transduction across cell membranes and represent major targets in the development of novel drug candidates in all clinical areas. Although there have been some recent leads, structural information on GPCRs is relatively rare due to the difficulty associated with crystallization. A specific reason for this is the intrinsic flexibility displayed by GPCRs, which is necessary for their functional diversity. Since GPCRs are integral membrane proteins, interaction of membrane lipids with them constitutes an important area of research in GPCR biology. In particular, membrane cholesterol has been reported to have a modulatory role in the function of a number of GPCRs. The role of membrane cholesterol in GPCR function is discussed with specific example of theserotonin1Areceptor. Recent results show that GPCRs are characterized with structural motifs that preferentially associate with cholesterol. An emerging and important concept is oligomerization of GPCRs and its role in GPCR function and signaling. The role of membrane cholesterol in GPCR oligomerization is highlighted. Future research in GPCR biology would offer novel insight in basic biology and provide new avenues for drug discovery.