Signaling by the human serotonin(1A) receptor is impaired in cellular model of Smith-Lemli-Opitz Syndrome

The Smith-Lemli-Opitz Syndrome (SLOS) is a congenital and developmental malformation syndrome associated with defective cholesterol biosynthesis. SLOS is clinically diagnosed by reduced plasma levels of cholesterol along with elevated levels of 7-dehydrocholesterol (and its positional isomer 8-dehydrocholesterol) and the ratio of their concentrations to that of cholesterol. Since SLOS is associated with neurological deformities and malfunction, exploring the function of neuronal receptors and their interaction with membrane cholesterol under these conditions assumes significance. We have earlier shown the requirement of membrane cholesterol for the ligand binding function of an important neurotransmitter G-protein coupled receptor, the serotonin(1A) receptor. In the present work, we have generated a cellular model of SLOS using CHO cells stably expressing the human serotonin(1A) receptor. This was achieved by metabolically inhibiting the biosynthesis of cholesterol, utilizing a specific inhibitor (AY 9944) of the enzyme required in the final step of cholesterol biosynthesis. We utilized this cellular model to monitor the function of the human serotonin(1A) receptor under SLOS-like condition. Our results show that ligand binding activity, G-protein coupling and downstream signaling of serotonin(1A) receptors are impaired in SLOS-like condition, although the membrane receptor level does not exhibit any reduction. Importantly, metabolic replenishment of cholesterol using serum partially restored the ligand binding activity of the serotonin(1A) receptor. These results are potentially useful in developing strategies for the future treatment of the disease since intake of dietary cholesterol is the only feasible treatment for SLOS patients.     

Membrane organization of the human serotonin(1A) receptor monitored by detergent insolubility using GFP fluorescence

Insolubility in non-ionic detergents such as Triton X-100 at low temperature is a widely used biochemical criterion for characterization of membrane domains. In view of the emerging role of membrane organization in the function of G-protein coupled receptors, we have examined detergent insolubility of the 5-HT(1A) receptor in CHO cells using a novel GFP fluorescence approach developed by us. Using this approach, we have explored the membrane organization of the serotonin(1A) receptor tagged to enhanced yellow fluorescent protein (5-HT(1A)R-EYFP) stably expressed in CHO-K1 cells under conditions of varying detergent concentration, reduced membrane cholesterol and agonist stimulation. Our results show that a small yet significant fraction of the 5-HT(1A) receptor exhibits detergent insolubility, which increases upon depletion of membrane cholesterol. Stimulation of 5-HT(1A)R-EYFP by its endogenous ligand, serotonin, did not cause a significant change in the detergent insolubility of the receptor. Taken together, our results on detergent insolubility of 5-HT(1A)R-EYFP provide new insights into the membrane organization of the 5-HT(1A) receptor and could be relevant in the analysis of membrane organization of other G-protein coupled receptors.     

Cholesterol depletion induces dynamic confinement of the G-protein coupled serotonin(1A) receptor in the plasma membrane of living cells

Cholesterol is an essential constituent of eukaryotic membranes and plays a crucial role in membrane organization, dynamics, function, and sorting. It is often found distributed non-randomly in domains or pools in biological and model membranes and is thought to contribute to a segregated distribution of membrane constituents. Signal transduction events mediated by seven transmembrane domain G-protein coupled receptors (GPCRs) are the primary means by which cells communicate with and respond to their external environment. We analyzed the role of cholesterol in the plasma membrane organization of the G-protein coupled serotonin(1A) receptor by fluorescence recovery after photobleaching (FRAP) measurements with varying bleach spot sizes. Our results show that lateral diffusion parameters of serotonin(1A) receptors in normal cells are consistent with models describing diffusion of molecules in a homogenous membrane. Interestingly, these characteristics are altered in cholesterol-depleted cells in a manner that is consistent with dynamic confinement of serotonin(1A) receptors in the plasma membrane. Importantly, analysis of ligand binding and downstream signaling of the serotonin(1A) receptor suggests that receptor function is affected in a significantly different manner when intact cells or isolated membranes are depleted of cholesterol. These results assume significance in the context of interpreting effects of cholesterol depletion on diffusion characteristics of membrane proteins in particular, and cholesterol-dependent cellular processes in general.     

Molecular modeling of the human serotonin(1A) receptor: role of membrane cholesterol in ligand binding of the receptor

Serotonin(1A) receptors are important neurotransmitter receptors and belong to the superfamily of G-protein coupled receptors (GPCRs). Although it is an important drug target, the crystal structure of the serotonin(1A) receptor has not been solved yet. Earlier homology models of the serotonin(1A) receptor were generated using rhodopsin as a template. We have used two recent crystal structures of the human β(2)-adrenergic receptor, one of which shows specific cholesterol binding site(s), as templates to model the human serotonin(1A) receptor. Since the sequence similarity between the serotonin(1A) receptor and β(2)-adrenergic receptor is considerably higher than the similarity between the serotonin(1A) receptor and rhodopsin, our model is more reliable. Based on these templates, we generated models of the serotonin(1A) receptor in the absence and presence of cholesterol. The receptor model appears more compact in the presence of cholesterol. We validated the stability of 'compactness' using coarse-grain MD simulation. Importantly, all ligands exhibit higher binding energies when docked to the receptor in the presence of cholesterol, thereby implying that membrane cholesterol facilitates ligand binding to the serotonin(1A) receptor. To the best of our knowledge, this is one of the first reports in which lipid-specific receptor conformations have been modeled by homology modeling.     

Membrane organization and dynamics of the serotonin1A receptor in live cells

The G-protein coupled receptor (GPCR) superfamily is one of the largest classes of molecules involved in signal transduction across the plasma membrane. The serotonin(1A) receptor is a representative member of the GPCR superfamily and serves as an important target in the development of therapeutic agents for neuropsychiatric disorders such as anxiety and depression. In the context of the pharmacological relevance of the serotonin(1A) receptor, the membrane organization and dynamics of this receptor in the cellular environment assume relevance. We have highlighted results, obtained from fluorescence microscopy-based approaches, related to domain organization and dynamics of the serotonin(1A) receptor. A fraction of serotonin(1A) receptors displays detergent insolubility, monitored using green fluorescent protein, that increases upon depletion of membrane cholesterol. Fluorescence recovery after photobleaching measurements with varying bleach spot sizes show that lateral diffusion parameters of serotonin(1A) receptors in normal cells are consistent with models describing diffusion of molecules in a homogenous membrane. Interestingly, these characteristics are altered in cholesterol-depleted cells. Taken together, we conclude that the serotonin(1A) receptor exhibits dynamic confinement in the cellular plasma membranes. Progress in understanding GPCR organization and dynamics would result in better insight into our overall understanding of GPCR function in health and disease.     

Differential effects of cholesterol and 7-dehydrocholesterol on ligand binding of solubilized hippocampal serotonin1A receptors: implications in SLOS

The serotonin1A receptor is an important member of the G-protein coupled receptor family, and is involved in the generation and modulation of a variety of cognitive, behavioral, and developmental functions. Solubilization of the hippocampal serotonin1A receptor by 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) is accompanied by loss of membrane cholesterol which results in a reduction in specific agonist binding activity. Replenishment of cholesterol to solubilized membranes restores the cholesterol content of the membrane and significantly enhances specific agonist binding activity. In order to test the stringency of the requirement of cholesterol in this process, we solubilized native hippocampal membranes followed by replenishment with 7-dehydrocholesterol (7-DHC). 7-DHC is an immediate biosynthetic precursor of cholesterol differing only in a double bond at the 7th position in its sterol ring. Our results show, for the first time, that replenishment of solubilized hippocampal membranes with 7-DHC does not restore ligand binding activity of the serotonin1A receptor, in spite of recovery of the overall membrane order. This observation shows that the requirement for restoration of ligand binding activity is more stringent than the requirement for the recovery of overall membrane order. These novel results have potential implications in understanding the interaction of membrane sterols with this important neuronal receptor under pathogenic conditions such as the Smith-Lemli-Opitz syndrome.     

Identification of cholesterol recognition amino acid consensus (CRAC) motif in G-protein coupled receptors

G-protein coupled receptors (GPCRs) are the largest class of molecules involved in signal transduction across membranes, and represent major targets in the development of novel drug candidates in all clinical areas. Membrane cholesterol has been reported to have an important role in the function of a number of GPCRs. Several structural features of proteins, believed to result in preferential association with cholesterol, have been recognized. Cholesterol recognition/interaction amino acid consensus (CRAC) sequence represents such a motif. Many proteins that interact with cholesterol have been shown to contain the CRAC motif in their sequence. We report here the presence of CRAC motifs in three representative GPCRs, namely, rhodopsin, the β(2)-adrenergic receptor, and the serotonin(1A) receptor. Interestingly, the function of these GPCRs has been previously shown to be dependent on membrane cholesterol. The presence of CRAC motifs in GPCRs indicates that interaction of cholesterol with GPCRs could be specific in nature. Further analysis shows that CRAC motifs are inherent characteristic features of the serotonin(1A) receptor and are conserved over natural evolution. These results constitute the first report of the presence of CRAC motifs in GPCRs and provide novel insight in the molecular nature of GPCR-cholesterol interaction.     

Role of cholesterol in ligand binding and G-protein coupling of serotonin1A receptors solubilized from bovine hippocampus

The serotonin(1A) (5-HT(1A)) receptor is an important member of the superfamily of seven transmembrane domain G-protein-coupled receptors. We report here that solubilization of the hippocampal 5-HT(1A) receptor by the zwitterionic detergent CHAPS is accompanied by loss of membrane cholesterol which results in a reduction in specific agonist binding activity and extent of G-protein coupling. Importantly, replenishment of cholesterol to solubilized membranes using MbetaCD-cholesterol complex restores the cholesterol content of the membrane and significantly enhances the specific agonist binding activity and G-protein coupling. These novel results provide useful information on the role of cholesterol in solubilization of G-protein-coupled receptors, an important step for molecular characterization of these receptors.     

Cholesterol modulates ligand binding and G-protein coupling to serotonin(1A) receptors from bovine hippocampus

The serotonin(1A) (5-HT(1A)) receptor is an important member of the superfamily of seven-transmembrane domain G-protein-coupled receptors. We have examined the modulatory role of cholesterol on the ligand binding activity and G-protein coupling of the bovine hippocampal 5-HT(1A) receptor by depleting cholesterol from native membranes using methyl-beta-cyclodextrin (MbetaCD). Removal of cholesterol from bovine hippocampal membranes using varying concentrations of MbetaCD results in a concentration-dependent reduction in specific binding of the agonist 8-OH-DPAT to 5-HT(1A) receptors. This is accompanied by alterations in binding affinity and sites obtained from analysis of binding data. Importantly, cholesterol depletion affected G-protein-coupling of the receptor as monitored by the GTP-gamma-S assay. The concomitant changes in membrane order were reported by changes in fluorescence polarization of membrane probes such as DPH and TMA-DPH, which are incorporated at different locations (depths) in the membrane. Replenishment of membranes with cholesterol led to recovery of ligand binding activity as well as membrane order to a considerable extent. Our results provide evidence, for the first time, that cholesterol is necessary for ligand binding and G-protein coupling of this important neurotransmitter receptor. These results could have significant implications in understanding the influence of the membrane lipid environment on the activity and signal transduction of other G-protein-coupled transmembrane receptors.     

Cholesterol modulates the antagonist-binding function of hippocampal serotonin1A receptors

The serotonin_1A receptor is the most extensively studied member of the family of seven transmembrane domain G-protein coupled serotonin receptors. Serotonergic signaling appears to play a key role in the generation and modulation of various cognitive and behavioral functions such as sleep, mood, pain, addiction, locomotion, sexual activity, depression, anxiety, alcohol abuse, aggression and learning. Since a significant portion of the protein lies embedded in the membrane and the ligand-binding pocket is defined by the transmembrane stretches in such receptors, membrane composition and organization represent a crucial parameter in the structure-function analysis of G-protein coupled receptors. In this paper, we have monitored the role of membrane cholesterol in the ligand-binding function of the hippocampal serotonin_1A receptor. Our results demonstrate that the reduction of membrane cholesterol significantly attenuates the antagonist-binding function of the serotonin_1A receptor. Based on prior pharmacological knowledge regarding the requirements for the antagonist to bind the receptor, our results indicate that membrane cholesterol modulates receptor function independently of its ability to interact with G-proteins. These effects on ligand-binding function of the receptor are predominantly reversed upon cholesterol-replenishment of cholesterol-depleted membranes. When viewed in the light of our earlier results on the effect of cholesterol depletion on the serotonin_1A receptor/G-protein interaction, these results comprehensively demonstrate the importance of cholesterol in the serotonin_1A receptor function and form the basis for understanding lipid-protein interactions involving this important neuronal receptor.     

Membrane cholesterol stabilizes the human serotonin1A receptor

A number of recently solved crystal structures of G-protein coupled receptors reveal the presence of closely associated cholesterol molecules in the receptor structure. We have previously shown the requirement of membrane cholesterol in the organization, dynamics and function of the serotonin_1A receptor, a representative G‐protein coupled receptor. In this work, we explored the role of membrane cholesterol in the stability of the human serotonin_1A receptor. Analysis of sensitivity of the receptor to thermal deactivation, pH, and proteolytic digestion in control, cholesterol-depleted and cholesterol-enriched membranes comprehensively demonstrate that membrane cholesterol stabilizes the serotonin_1A receptor. We conclude that these results could have potential implications in future efforts toward crystallizing the receptor.     

Removal of sphingomyelin headgroup inhibits the ligand binding function of hippocampal serotonin1A receptors

Sphingolipids are essential components of eukaryotic cell membranes and are thought to be involved in a variety of cellular functions. Sphingomyelin is the most abundant sphingolipid in the nervous system. In this work, we explored the ligand binding function of the hippocampal serotonin(1A) receptor upon hydrolyzing sphingomyelin to ceramide and phosphocholine using sphingomyelinase. The serotonin(1A) receptor is an important neurotransmitter receptor and belongs to the superfamily of G-protein coupled receptors. It is involved in the generation and modulation of various cognitive, behavioral and developmental functions. We show here that specific agonist binding to serotonin(1A) receptors in native hippocampal membranes is considerably reduced upon sphingomyelinase treatment. Interestingly, the overall membrane order does not exhibit any appreciable change under these conditions. Our results show the importance of sphingomyelin (specifically, the sphingomyelin headgroup) for the function of serotonin(1A) receptors. These novel results constitute the first report on the effect of enzymatic hydrolysis of sphingomyelin on the ligand binding function of this important neurotransmitter receptor in native hippocampal membranes. Our results assume greater relevance in the broader perspective of the influence of the membrane lipid environment on the function of the serotonin(1A) receptor in particular, and other G-protein coupled receptors in general.     

Membrane organization of the human serotonin1A receptor monitored by detergent insolubility using GFP fluorescence

Insolubility in non-ionic detergents such as Triton X-100 at low temperature is a widely used biochemical criterion for characterization of membrane domains. In view of the emerging role of membrane organization in the function of G-protein coupled receptors, we have examined detergent insolubility of the 5-HT_1A receptor in CHO cells using a novel GFP fluorescence approach developed by us. Using this approach, we have explored the membrane organization of the serotonin_1A receptor tagged to enhanced yellow fluorescent protein (5-HT_1AR-EYFP) stably expressed in CHO-K1 cells under conditions of varying detergent concentration, reduced membrane cholesterol and agonist stimulation. Our results show that a small yet significant fraction of the 5-HT_1A receptor exhibits detergent insolubility, which increases upon depletion of membrane cholesterol. Stimulation of 5-HT_1AR-EYFP by its endogenous ligand, serotonin, did not cause a significant change in the detergent insolubility of the receptor. Taken together, our results on detergent insolubility of 5-HT_1AR-EYFP provide new insights into the membrane organization of the 5-HT_1A receptor and could be relevant in the analysis of membrane organization of other G-protein coupled receptors.     

Ligand Binding and G-protein Coupling of the Serotonin1A Receptor in Cholesterol-enriched Hippocampal Membranes

The serotonin_1A receptor is the most extensively studied member of the family of seven transmembrane domain G-protein coupled serotonin receptors. Since a large portion of such transmembrane receptors remains in contact with the membrane lipid environment, lipid–protein interactions assume importance in the structure-function analysis of such receptors. We have earlier reported the requirement of cholesterol for serotonin_1A receptor function in native hippocampal membranes by specific depletion of cholesterol using methyl- β-cyclodextrin. In this paper, we monitored the serotonin_1A receptor function in membranes that are enriched in cholesterol using a complex prepared from cholesterol and methyl-β-cyclodextrin. Our results indicate that ligand binding and receptor/G-protein interaction of the serotonin_1A receptor do not exhibit significant difference in native and cholesterol-enriched hippocampal membranes indicating that further enrichment of cholesterol has little functional consequence on the serotonin_1A receptor function. These results therefore provide new information on the effect of cholesterol enrichment on the hippocampal serotonin_1A receptor function.     

The human serotonin 1A receptor expressed in neuronal cells: toward a native environment for neuronal receptors

1. The serotonin(1A) (5-HT(1A)) receptor is an important representative of G-protein coupled family of receptors. It is the most extensively studied among the serotonin receptors, and appears to be involved in various behavioral and cognitive functions. 2. We report here the pharmacological and functional characterization of the human serotonin(1A) receptor stably expressed in HN2 cell line, which is a hybrid cell line between hippocampal cells and mouse neuroblastoma. 3. Our results show that serotonin(1A) receptors in HN2-5-HT(1A)R cells display ligand-binding properties that closely mimic binding properties observed with native receptors. We further demonstrate that the differential discrimination of G-protein coupling by the specific agonist and antagonist, a hallmark of the native receptor, is maintained for the receptor in HN2-5-HT(1A)R cells. Importantly, the serotonin(1A) receptor in HN2-5-HT(1A)R cells shows efficient downstream signalling by reducing cellular cyclic AMP levels. 4. We conclude that serotonin(1A) receptors expressed in HN2-5-HT(1A)R cells represent a useful model system to study serotonin(1A) receptor biology, and is a potential system for solubilization and purification of the receptor in native-like membrane environment.     

A GFP fluorescence-based approach to determine detergent insolubility of the human serotonin1A receptor

Insolubility in non-ionic detergents such as Triton X-100 is a widely used biochemical criterion for characterization of membrane domains. We report here a novel green fluorescent protein fluorescence-based approach to directly determine detergent insolubility of specific membrane proteins. We have applied this method to explore the detergent resistance of an important G-protein coupled receptor, the serotonin1A (5-HT1A) receptor. Our results show, for the first time, that a small yet significant fraction of the 5-HT1A receptor exhibits detergent insolubility. These results are validated by control experiments involving fluorescent lipid probes and protein markers. Our results assume relevance in the context of localization of the 5-HT1A receptor in membrane domains and its significance in receptor function and signaling.     

Effect of cholesterol on lateral diffusion of fluorescent lipid probes in native hippocampal membranes

Cholesterol is an abundant lipid of mammalian membranes and plays a crucial role in membrane organization, dynamics, function and sorting. The role of cholesterol in membrane organization has been a subject of intense investigation that has largely been carried out in model membrane systems. An extension of these studies in natural membranes, more importantly in neuronal membranes, is important to establish a relationship between disease states and changes in membrane physical properties resulting from an alteration in lipid composition. We have monitored the lateral diffusion of lipid probes, DiIC(18)(3) and FAST DiI which are similar in their intrinsic fluorescence properties but differ in their structure, in native and cholesterol-depleted hippocampal membranes using the fluorescence recovery after photobleaching (FRAP) approach. Our results show that the mobility of these probes is in general higher in hippocampal membranes depleted of cholesterol. Interestingly, the increase in mobility of these probes does not linearly correlate with the extent of cholesterol depletion. These results assume significance in the light of recent reports on the requirement of cholesterol to support the function of the G-protein coupled serotonin(1A) receptor present endogenously in hippocampal membranes.     

Effect of sphingomyelinase treatment on ligand binding activity of human serotonin1A receptors

The serotonin1A receptor is an important member of the G-protein coupled receptor family, and is involved in the generation and modulation of a variety of cognitive, behavioral, and developmental functions. We have monitored the ligand binding of the human serotonin1A receptor stably expressed in CHO cells (termed CHO-5-HT1AR) following treatment with sphingomyelinase (SMase), an enzyme that specifically catalyzes the hydrolysis of sphingomyelin into ceramide and phosphorylcholine. Our results show, for the first time, that the specific ligand binding activity of the serotonin1A receptor in membranes isolated from CHO-5-HT1AR cells is increased upon sphingomyelinase treatment. Saturation binding analysis reveals increase in binding affinity of the receptor under these conditions. This is accompanied by a reduction in membrane order, as monitored by fluorescence anisotropy of the membrane probe 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) in intact cells. These results represent the first report on the effect of sphingomyelinase treatment on the ligand binding activity of this important neurotransmitter receptor.     

Solubilization of human serotonin 1A receptors expressed in neuronal cells

The serotonin_1A receptor is an important member of the G-protein coupled receptor family, and is involved in a variety of cognitive, behavioral, and developmental functions. None of the subtypes of G-protein coupled serotonin receptors have yet been purified to homogeneity from natural sources. We report here, for the first time, the solubilization of human serotonin_1A receptors stably expressed in neuronal (HN2) cells. Importantly, ligand binding assay shows that the serotonin_1A receptor solubilized this way is functionally active. The effective solubilization of the serotonin_1A receptor from neuronal cells represents an important step toward the purification of the receptor in native-like membrane environment.     

Interaction of Serotonin1A Receptors from Bovine Hippocampus with Tertiary Amine Local Anesthetics

1. We have examined the interaction of tertiary amine local anesthetics with the bovine hippocampal serotonin1A (5-HT1A) receptor, an important member of the G-protein-coupled receptor superfamily. 2. The local anesthetics inhibit specific agonist and antagonist binding to the 5-HT1A receptor at a clinically relevant concentration range of the anesthetics. This is accompanied by a concomitant reduction in the binding affinity of the 5-HT1A receptor to the agonist. Interestingly, the extent of G-protein coupling of the receptor is reduced in the presence of the local anesthetics. 3. Fluorescence polarization measurements using depth-dependent fluorescent probes show that procaine and lidocaine do not show any significant change in membrane fluidity. On the other hand, tetracaine and dibucaine were found to alter fluidity of the membrane as indicated by a fluorescent probe which monitors the headgroup region of the membrane. 4. The local anesthetics showed inhibition of agonist binding to the 5-HT1A receptor in membranes depleted of cholesterol more or less to the same extent as that of control membranes in all cases. This suggests that the inhibition in ligand binding to the 5-HT1A receptor brought about by local anesthetics is independent of the membrane cholesterol content. 5. Our results on the effects of the local anesthetics on the ligand binding and G-protein coupling of the 5-HT1A receptor support the possibility that G-protein-coupled receptors could be involved in the action of local anesthetics.