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.     

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.     

Fixation alters fluorescence lifetime and anisotropy of cells expressing EYFP-tagged serotonin1A receptor

Fluorescence microscopic approaches represent powerful techniques to monitor molecular interactions in the cellular milieu. Measurements of fluorescence lifetime and anisotropy enjoy considerable popularity in this context. These measurements are often performed on live as well as fixed cells. We report here that formaldehyde-induced cell fixation introduces heterogeneities in the fluorescence emission of serotonin_1A receptors tagged to enhanced yellow fluorescent protein, and alters fluorescence lifetime and anisotropy significantly. To the best of our knowledge, our results constitute the first report on the effect of formaldehyde fixation on fluorescence parameters of cellular proteins. We conclude that fluorescence parameters derived from fixed cells should be interpreted with caution.     

Metabolic depletion of sphingolipids enhances the mobility of the human serotonin1A receptor

Sphingolipids are essential components of eukaryotic cell membranes. We recently showed that the function of the serotonin_1A receptor is impaired upon metabolic depletion of sphingolipids using fumonisin B₁ (FB₁), a specific inhibitor of ceramide synthase. Serotonin_1A receptors belong to the family of G-protein coupled receptors and are implicated in the generation and modulation of various cognitive, behavioral and developmental functions. Since function and dynamics of membrane receptors are often coupled, we monitored the lateral dynamics of the serotonin_1A receptor utilizing fluorescence recovery after photobleaching (FRAP) under these conditions. Our results show an increase in mobile fraction of the receptor upon sphingolipid depletion, while the diffusion coefficient of the receptor did not exhibit any significant change. These novel results constitute the first report on the effect of sphingolipid depletion on the mobility of the serotonin_1A receptor. Our results assume greater relevance in the broader context of the emerging role of receptor mobility in understanding cellular signaling.     

Cholesterol depletion modulates detergent resistant fraction of human serotonin1A receptors

Abstract

Insolubility of membrane components in non-ionic detergents such as Triton X-100 at low temperature is a widely used biochemical criterion to identify, isolate and characterize membrane domains. In this work, we monitored the detergent insolubility of the serotonin_1A receptor in CHO cell membranes and its modulation by membrane cholesterol. The serotonin_1A receptor is an important member of the G-protein coupled receptor family. It is implicated in the generation and modulation of various cognitive, behavioral and developmental functions and serves as a drug target. Our results show that a significant fraction (～ 28%) of the serotonin_1A receptor resides in detergent-resistant membranes (DRMs). Interestingly, the fraction of the serotonin_1A receptor in DRMs exhibits a reduction upon membrane cholesterol depletion. In addition, we show that contents of DRM markers such as flotillin-1, caveolin-1 and GM₁ are altered in DRMs upon cholesterol depletion. These results assume significance since the function of the serotonin_1A receptor has previously been shown to be affected by membrane lipids, specifically cholesterol. Our results are relevant in the context of membrane organization of the serotonin_1A receptor in particular, and G-protein coupled receptors in general.     

The cholesterol-complexing agent digitonin modulates ligand binding of the bovine hippocampal serotonin1A receptor

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 of the bovine hippocampal 5-HT_1A receptor by cholesterol complexation in native membranes using digitonin. Complexation of cholesterol from bovine hippocampal membranes using digitonin results in a concentration-dependent reduction in specific binding of the agonist 8-OH-DPAT and antagonist p-MPPF to 5-HT_1A receptors. The corresponding changes in membrane order were monitored by analysis of fluorescence polarization data of the membrane depth-specific probes, DPH and TMA-DPH. Taken together, our results point out the important role of membrane cholesterol in maintaining the function of the 5-HT_1A receptor. An important aspect of these results is that non-availability of free cholesterol in the membrane due to complexation with digitonin rather than physical depletion is sufficient to significantly reduce the 5-HT_1A receptor function. These results provide a comprehensive understanding of the effects of the sterol-complexing agent digitonin in particular, and the role of membrane cholesterol in general, on the 5-HT_1A receptor function.     

Membrane cholesterol oxidation inhibits ligand binding function of hippocampal serotonin(1A) receptors

We have monitored the ligand binding function of the bovine hippocampal 5-HT(1A) receptor following treatment of native membranes with cholesterol oxidase. Cholesterol oxidase is a water soluble enzyme that acts on the membrane interface to catalyze the conversion of cholesterol to cholestenone. Oxidation of membrane cholesterol significantly inhibits the specific binding of the agonist and antagonist to 5-HT(1A) receptors. Fluorescence polarization measurements of membrane probes incorporated at different locations in the membrane revealed no appreciable effect on membrane order due to the oxidation of cholesterol to cholestenone. These results therefore suggest that the ligand binding function of the 5-HT(1A) receptor is a cholesterol-dependent phenomenon that is not related to the ability of cholesterol to modulate membrane order. Importantly, these results represent the first report on the effect of a cholesterol-modifying agent on the ligand binding function of this important neurotransmitter receptor.     

Characterization of serotonin 5-hydroxytryptamine-1A receptor activation using a phospho-extracellular-signal regulated kinase 2 sensor

The activation of G-protein-coupled receptors (GPCRs) can result in the stimulation of numerous signaling networks that extend beyond canonical secondary messenger-dependent pathways. It is well-established that many of these diverse networks converge on the MAPK pathway, resulting in the activation of extracellular-signal regulated kinase 1/2 (ERK). Since the link between GPCRs and ERK can be modulated via both G-protein-dependent and -independent mechanisms, measurement of ERK phosphorylation may serve as an ideal surrogate for GPCR activation. We have combined BacMam-mediated gene delivery of the GFP-ERK2 with a time-resolved Foerster resonance energy transfer (TR-FRET) immunoassay for the measurement of intracellular phospho-ERK2 levels. Together these technologies enable a flexible platform for measuring GPCR and MAPK activation in the cell line of interest. This technology has been applied to the measurement of activation of the serotonin 5-hydroxytryptamine-1A (5-HT_1A) receptor expressed in CHO-K1 cells. In addition to demonstrating the flexibility of this assay platform, we provide the first reported profile for 5-HT_1A receptor-mediated ERK activation using a panel of known Parkinson’s disease drugs. Our results demonstrate the value of using ERK activation as a downstream sensor for GPCR function, providing an attractive complement to upstream endpoints such as ligand occupancy and binding of GTPγS.     

The sterol-binding antibiotic nystatin differentially modulates ligand binding of the bovine hippocampal serotonin1A receptor

We have monitored the ligand binding of the bovine hippocampal 5-HT1A receptor following treatment with the sterol-binding antifungal antibiotic nystatin. Nystatin considerably inhibits the specific binding of the antagonist to 5-HT1A receptors in a concentration-dependent manner. However, the specific agonist binding does not show significant changes. Fluorescence polarization measurements of membrane probes incorporated at different locations in the membrane revealed a substantial decrease in the membrane order in the interior of the bilayer. Experiments with cholesterol-depleted membranes indicate that the action of nystatin is mediated through membrane cholesterol. These results represent the first report on the effect of a cholesterol-perturbing agent on the ligand-binding activity of this important neurotransmitter receptor.     

Monitoring of detergent binding to amphiphilic proteins by means fo micelles containing the hydrophobic dye Sudan Black B

A simple method for detecting micellar binding of Triton X-100 to amphiphilic proteins is described. The hydrophobic dye Sudan Black B is incorporated into Triton micelles. Binding of the coloured micelles to serum apoliproteins, as well as to amphiphilic proteins, of erythrocyte and fat globule membranes renders these visible as dark bands after sucrose density gradient centrifugation. In contrast, the hydrophilic proteins present in lipoprotein-free serum do not show detergent binding. The method does not permit accurate quantification of detergent binding, but may serve as a pilot procedure for initial detection of amphiphilic proteins and for monitoring their isolation from crude solubilized membrane material. The sensitivity of the assay corresponds to that obtained with [³H]Triton X-100.     

Predicting cortisol stress responses in older individuals: influence of serotonin receptor 1A gene (HTR1A) and stressful life events

Considerable variability in the activity of the hypothalamus-pituitary-adrenal (HPA) axis in response to stress has been found in quantitative genetic studies investigating healthy individuals suggesting that at least part of this variance is due to genetic factors. Since the HPA axis is regulated by a neuronal network including amygdala, hippocampus, prefrontal cortex as well as brainstem circuits, the investigation of candidate genes that impact neurotransmitter systems related to these brain regions might further elucidate the genetic underpinnings of the stress response. However, aside from genetic risk factors, past stressful life events might also result in long-term adjustments of HPA axis reactivity. Here, we investigated the effects of the − 1019 G/C polymorphism in the HTR1A gene encoding the serotonin (5-HT) receptor 1A (5-HT_1A) and stressful life events experienced during childhood and adolescence on changes in cortisol levels in response to the Trier Social Stress Test (TSST) in a sample of healthy older adults (N = 97). Regression analyses revealed a significant effect of HTR1A genotype with the G allele being associated with a less pronounced stress response. In addition, an inverse relationship between past stressful life events and cortisol release but no gene × environment interaction was detected. The results further underscore the crucial role of functional serotonergic genetic variation as well as stressful events during critical stages of development on the acute stress response later in life.     

Differential dynamics of the serotonin1A receptor in membrane bilayers of varying cholesterol content revealed by all atom molecular dynamics simulation

Abstract

The serotonin_1A receptor belongs to the superfamily of G protein-coupled receptors (GPCRs) and is a potential drug target in neuropsychiatric disorders. The receptor has been shown to require membrane cholesterol for its organization, dynamics and function. Although recent work suggests a close interaction of cholesterol with the receptor, the structural integrity of the serotonin_1A receptor in the presence of cholesterol has not been explored. In this work, we have carried out all atom molecular dynamics simulations, totaling to 3?μs, to analyze the effect of cholesterol on the structure and dynamics of the serotonin_1A receptor. Our results show that the presence of physiologically relevant concentration of membrane cholesterol alters conformational dynamics of the serotonin_1A receptor and, on an average lowers conformational fluctuations. Our results show that, in general, transmembrane helix VII is most affected by the absence of membrane cholesterol. These results are in overall agreement with experimental data showing enhancement of GPCR stability in the presence of membrane cholesterol. Our results constitute a molecular level understanding of GPCR-cholesterol interaction, and represent an important step in our overall understanding of GPCR function in health and disease.     

Effects of a Chronic Lithium Treatment on Cortical Serotonin Uptake Sites and 5-HT1A Receptors

The objectives of this study were to characterize the effects of a chronic lithium (Li⁺) treatment on serotonin (5-HT) uptake sites and on 5-HT_1A receptors, and to determine the eventual reversibility of the treatment. The experiments were carried out with membranes from rat cerebral cortex using 8-hydroxy-2-(propylamino)tetralin, or [³H]8-OH-DPAT, and [³H]citalopram to label 5-HT_1A receptors and 5-HT uptake sites, respectively. Endogenous levels of 5-HT and 5-hydroxyindole-3-acetic acid (5-HIAA) were measured by high-performance liquid chromatography in the cingulate cortex. The saturation curves with [³H]8-OH-DPAT were always best fitted a two-site model. After a treatment with Li⁺ for 28 days, no alterations in the binding parameters of [³H]8-OH-DPAT to the high- and low-affinity binding sites could be documented. However, competition curves with 5-HT to inhibit [³H]8-OH-DPAT binding revealed a decreased proportion of sites with high affinity for the agonist, together with an increased density of sites with low affinity for 5-HT, suggesting an alteration in the coupling efficacy between 5-HT_1A receptors and their transduction systems. Saturation studies with [³H]citalopram showed an increase (>40%) in the density of 5-HT uptake sites after chronic Li⁺, suggesting a more efficient 5-HT uptake process for the treated animals, in accord with clinical observations. Although 5-HT contents in cingulate cortex remained unchanged after the treatment, 5-HIAA levels decreased (>30%), leading to a diminished (almost 50%) 5-HT turnover; and also reflecting a more efficient uptake in the treated rats, so that less 5-HT could be degraded by extracellular monoamine oxidase. All the effects revealed by [³H]8-OH-DPAT and [³H]citalopram were reversed following a recovery period of two days without Li⁺. Since symptoms of bipolar affective disorders may reappear if the chronic Li⁺ treatment is interrupted, the reversibility of the observed effects further supports the importance of central 5-HT synaptic transmission in the pathophysiology and treatment of human affective disorders.     

Attenuation of ischemic efflux of endogenous amino acids by the novel 5-HT1A/5-HT2 receptor ligand adatanserin

Using sodium azide (NaN3)-induced anoxia plus aglycaemia as a model of chemically-induced ischemia in the hippocampal slice, we have evaluated the effects of the novel 5-HT(1A) partial agonist/5-HT(2) receptor antagonist adatanserin and the 5-HT(1A) receptor agonist BAYx3702 on the efflux of endogenous glutamate, aspartate and GABA. BAYx3702 (10-1000 nM) produced a significant (P<0.05) dose-related attenuation of ischemic efflux of both glutamate and GABA with maximum decrease being observed at 100 nM (73 and 69%, respectively). This attenuation was completely reversed by the addition of the 5-HT(1A) antagonist, WAY-100635 (100 nM). Similarly, adatanserin (10-1000 nM) produced a significant (P<0.05) dose-related attenuation in glutamate and GABA efflux with a maximum of 72 and 81% at 100 nM, respectively. This effect was completely reversed by the 5-HT(2A/C) receptor agonist, DOI but unaffected by WAY-100635. The 5-HT(2A) receptor antagonist MDL-100907 produced a comparable attenuation of glutamate when compared to adatanserin, while the 5-HT(2C) receptor antagonist, SB-206553, had no effect on ischemic efflux. None of these compounds significantly altered aspartate efflux from this preparation. In conclusion, the 5-HT(1A) receptor partial agonist 5-HT(2) receptor antagonist, adatanserin is able to attenuate ischemic amino acid efflux in a comparable manner to the full 5-HT(1A) agonist BAYx3702. However, in contrast to BAYx3702, adatanserin appears to produce it effects via blockade of the 5-HT(2A) receptor. This suggests that adatanserin may be an effective neuroprotectant, as has been previously demonstrated for full 5-HT(1A) receptor agonists such as BAYx3702.     

Hypothalamic 5-HT functional regulation through 5-HT1A and 5-HT2C receptors during pancreatic regeneration

5-HT receptors are predominantly located in the brain and are involved in pancreatic function and cell proliferation through sympathetic nervous system. The objective of this study was to investigate the role of hypothalamic 5-HT, 5-HT1A and 5-HT2C receptor binding and gene expression in rat model of pancreatic regeneration using 60% pancreatectomy. The pancreatic regeneration was evaluated by 5-HT content, 5-HT1A and 5-HT2C receptor gene expression in the hypothalamus of sham operated, 72 h and 7 days pancreatectomised rats. 5-HT content was quantified by HPLC. 5-HT1A receptor assay was done by using specific agonist [3H]8-OH DPAT. 5-HT2C receptor assay was done by using specific antagonist [3H]mesulergine. The expression of 5-HT1A and 5-HT2C receptor gene was analyzed by RT-PCR. 5-HT content was higher in the hypothalamus of 72 h pancreatectomised rats. 5-HT1A and 5-HT2C receptors were down-regulated in the hypothalamus. RT-PCR analysis revealed decreased 5-HT1A and 5-HT2C receptor mRNA expression. The 5-HT1A and 5-HT2C receptors gene expression in the 7 days pancreatectomised rats reversed to near sham level. This study is the first to identify 5-HT1A and 5-HT2C receptor gene expression in the hypothalamus during pancreatic regeneration in rats. Our results suggest the hypothalamic serotonergic receptor functional regulation during pancreatic regeneration.     

5-Hydroxytryptamine 2A receptor signaling cascade modulates adiponectin and plasminogen activator inhibitor 1 expression in adipose tissue

Knowledge of the regulatory factors associated with down-regulation of adiponectin gene expression and up-regulation of PAI-1 gene expression is crucial to understand the pathophysiological basis of obesity and metabolic diseases, and could establish new treatment strategies for these conditions. We showed that expression of 5-HT(2A) receptors was up-regulated in hypertrophic 3T3-L1 adipocytes, which exhibited decreased expression of adiponectin and increased expression of PAI-1. 5-HT(2A) receptor antagonists and suppression of 5-HT(2A) receptor gene expression enhanced adiponectin expression. Activation of Gq negatively regulated adiponectin expression, and inhibition of mitogen-activated protein kinase reversed the Gq-induced effect. Moreover, the 5-HT(2A) receptor blockade reduced PAI-1 expression. These findings indicate that antagonism of 5-HT(2A) receptors in adipocytes could improve the obesity-linked decreases in adiponectin expression and increases in PAI-1 expression.     

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.     

Dipeptide Tyr-Leu (YL) exhibits anxiolytic-like activity after oral administration via activating serotonin 5-HT1A, dopamine D1 and GABAA receptors in mice

We found that Tyr-Leu (YL) dose-dependently exhibits potent anxiolytic-like activity (0.1-1 mg/kg, i.p.) comparable to diazepam in the elevated plus-maze test in mice. YL was orally active (0.3-3 mg/kg). A retro-sequence peptide or a mixture of Tyr and Leu was inactive. The anxiolytic-like activity of YL was inhibited by antagonists for serotonin 5-HT_1A, dopamine D₁ and GABA_A receptors; however, YL had no affinity for them. We also determined the order of their activation is 5-HT_1A, D₁ and GABA_A receptors using selective agonists and antagonists. Taken together, YL may exhibit anxiolytic-like activity via activation of 5-HT_1A, D₁ and GABA_A receptors.     

The C-terminus of Gi family G-proteins as a determinant of 5-HT(1A) receptor coupling

Using a universal signaling assay employing G-protein chimeras comprising the C-terminal five amino acids of Gi1/2, Gi3, Go, and Gz fused to Gq, the calcium mobilizing G-protein, we explored the role of the C-terminus of Gi family G-proteins as a determinant for 5-HT(1A) receptor functional coupling. Co-expression of the 5-HT(1A) receptor with each of the Gq/Gi family chimeras resulted in a concentration-dependent increase in calcium upon addition of 5-HT, although the coupling efficiency differed dramatically. Gq/Gi3 resulted in the most efficient coupling based on both potency and relative maximum response to 5-HT. Gq/Go also produced efficient coupling in terms of relative 5-HT efficacy (76% of the Gq/Gi3 maximum response), although 5-HT exhibited 4-fold lower agonist potency, and Gq/Gz and Gq/Gi1/2 conferred poor functional coupling. Agonist potencies and relative efficacies determined for a number of 5-HT(1A) receptor agonists using Gq/Gi3 coupling were significantly weaker than those described previously for coupling through the native G-protein. These results indicate the C-terminus of Gi3 as an important determinant for coupling to the 5-HT(1A) receptor, while the reduced functional agonist activities suggest additional motifs participate in receptor/G-protein coupling.     

The serotonin 5-HT1D receptor: A progress review

Most of the known neurotransmitters interact with more than one type of receptor. Some of them even dispose of receptor subtypes to exert their actions. Serotonin, far from being an exception to that, possesses at least 3 classes of receptors, which have all been reported to be heterogeneous, although convincing data only exist for the 5-HT₁ class. This name has been proposed in 1979, two years before the introduction of A and B in the nomenclature to account for the observed heterogeneity of these cites. The 5-HT_1C receptor subtype was first described in 1984 and the last member of the family, named 5-HT_1D, was characterized in 1987. The pharmacological profiles, the signal transducing systems and the anatomical localizations, both at the regional and cellular levels, of all these subtypes have been investigated and possible functions have been proposed for each of them. Moreover, last and most definitive demonstration of the subtype individuality, the gene or complementary DNA coding for the 5-HT_1A and 5-HT_1C (and 5-HT₂) receptors have been cloned and sequenced. Such data are still missing for 5-HT_1D (and 5-HT_1B) receptors, but will certainly be provided in the next few years. However and waiting for this decisive clue, the characterization of the 5-HT_1D subtype leaves no doubt concerning its significance as a functional 5-HT receptor. This review will concentrate on the characteristics of this subtype of 5-HT receptor.Abbreviations 5-CT 5-carboxamidotryptamine - 5-MeOT 5-methoxy-tryptamine - 5-MeODMT N,N-dimethyl-5-methoxytryptamine - 8-OH-DPAT 8-hydroxy-2[di-n-propylamino]tetralin - CYP cyanopindolol - DHE dihydroergotamine - DOI 2,5-dimethoxy-4-iodophenylisopropylamine - DP-5-CT N,N-dipropyl 5-carboxamidotryptamine - ICPY 2-iodo-cyanopindolol - mCPP m-chloro-phenyl-piperazine - TFMPP m-trifluoro-methyl-phenyl-piperazine - E_MAX Maximal effect - EC₅₀ Half maximal effective concentration - K_D Dissociation constant - K_B Antagonist dissociation constant