Solubilization of human serotonin<SUB>1A</SUB> receptors expressed in neuronal cells

The serotonin1A 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 serotonin1A receptors stably expressed in neuronal (HN2) cells. Importantly, ligand binding assay shows that the serotonin1A receptor solubilized this way is functionally active. The effective solubilization of the serotonin1A receptor from neuronal cells represents an important step toward the purification of the receptor in native-like membrane environment

Prolonged treatment with ligands affects ligand binding to the human serotonin<SUB>1A</SUB> receptor in chinese hamster ovary cells

1. The serotonin1A receptors are members of a superfamily of seven transmembrane domain receptors that couple to G-proteins, and appear to be involved in several behavioral and cognitive functions. 2. We monitored the effect of prolonged treatment of the human serotonin1A receptor expressed in Chinese hamster ovary (CHO) cells with pharmacologically well-characterized ligands on its binding to the agonist 8-hydroxy-2(di-N-propylamino)tetralin (8-OH-DPAT) and antagonist 4-(2'-methoxy)-phenyl-1-[2'-(N-2"-pyridinyl)-p-fluorodobenzamido]ethyl-piperazine (p-MPPF). 3. Our results indicate that prolonged treatment with the specific agonist (8-OH-DPAT) differentially affects subsequent binding of the agonist and antagonist to the receptor in a manner independent of receptor-G-protein coupling. Importantly, our results show that prolonged treatment with the commonly used antagonist p-MPPF, and its iodinated analogue 4-(2'-methoxy)-phenyl-1-[2'-(N-2"-pyridinyl)-p-iodobenzamido]ethyl-piperazine (p-MPPI), which have earlier been reported to display similar binding properties to serotonin1A receptors, induces significantly different effects on the ligand binding function of serotonin1A receptors

Solubilization of serotonin<SUB>1A</SUB> receptors heterologously expressed in chinese hamster ovary cells

1. The serotonin1A (5-HT1A) receptors are members of a superfamily of seven transmembrane domain receptors that couple to G-proteins. They appear to be involved in various behavioral and cognitive functions. 2. We report here, for the first time, the solubilization of 5-HT1A receptors stably expressed in Chinese Hamster Ovary (CHO) cells using the zwitterionic detergent CHAPS in presence of NaCl followed by polyethylene glycol (PEG) precipitation. We show by ligand-binding assay that the 5-HT1A receptor solubilized this way is functionally active. We have optimized the efficiency of solubilization with respect to total protein and NaCl concentration. 3.Our results show that careful control of salt and protein concentration is crucial in optimal solubilization of membrane receptors heterologously expressed in cells in culture. The effective solubilization of important neurotransmitter receptors such as 5-HT1A receptors which are present in very low amounts in the native tissue may represent an important step in characterizing membrane receptors expressed in mammalian cells in culture

Ligand binding and G-protein coupling of the serotonin<SUB>1A</SUB> receptor in cholesterol-enriched hippocampal membranes

The serotonin1A 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 serotonin1A receptor function in native hippocampal membranes by specific depletion of cholesterol using methyl-&#946; -cyclodextrin. In this paper, we monitored the serotonin1Areceptor function in membranes that are enriched in cholesterol using a complex prepared from cholesterol and methyl-&#946;-cyclodextrin. Our results indicate that ligand binding and receptor/G-protein interaction of the serotonin1A 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 serotonin1A receptor function. These results therefore provide new information on the effect of cholesterol enrichment on the hippocampal serotonin1A receptor function

Differential effects of cholesterol and 7-dehydrocholesterol on ligand binding of solubilized hippocampal serotonin<SUB>1A</SUB> 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

Mechanism of allosteric regulation of β2-adrenergic receptor by cholesterol

There is evidence that lipids can be allosteric regulators of membrane protein structure and activation. However, there are no data showing how exactly the regulation emerges from specific lipid-protein interactions. Here we show in atomistic detail how the human b2- adrenergic receptor (b2AR) – a prototypical G protein-coupled receptor – is modulated by cholesterol in an allosteric fashion. Extensive atomistic simulations show that cholesterol regulates b2AR by limiting its conformational variability. The mechanism of action is based on the binding of cholesterol at specific high-affinity sites located near the transmembrane helices 5–7 of the receptor. The alternative mechanism, where the b2AR conformation would be modulated by membrane-mediated interactions, plays only a minor role. Cholesterol analogues also bind to cholesterol binding sites and impede the structural flexibility of b2AR, however cholesterol generates the strongest effect. The results highlight the capacity of lipids to regulate the conformation of membrane receptors through specific interactions.There is evidence that lipids can be allosteric regulators of membrane protein structure and activation. However, there are no data showing how exactly the regulation emerges from specific lipid-protein interactions. Here we show in atomistic detail how the human b2- adrenergic receptor (b2AR) – a prototypical G protein-coupled receptor – is modulated by cholesterol in an allosteric fashion. Extensive atomistic simulations show that cholesterol regulates b2AR by limiting its conformational variability. The mechanism of action is based on the binding of cholesterol at specific high-affinity sites located near the transmembrane helices 5–7 of the receptor. The alternative mechanism, where the b2AR conformation would be modulated by membrane-mediated interactions, plays only a minor role. Cholesterol analogues also bind to cholesterol binding sites and impede the structural flexibility of b2AR, however cholesterol generates the strongest effect. The results highlight the capacity of lipids to regulate the conformation of membrane receptors through specific interactions.Peer reviewe

A novel mechanism for an old drug: amphotericin B in the treatment of Visceral leishmaniasis

Visceral leishmaniasis (VL) is caused by various species of the genus Leishmania. Internalization of Leishmania into host cells is facilitated by a large number of receptors, and therefore no panacea is available for the treatment of leishmaniasis. We previously demonstrated the requirement of host membrane cholesterol in the entry of Leishmania into macrophages by cholesterol depletion using methyl-β-cyclodextrin (MβCD). We recently showed that leishmanial infection is inhibited upon sequestration of host membrane cholesterol using amphotericin B (AmB), considered as the best existing drug against VL. The reason for the antileishmanial activity of AmB is generally believed to be its ability to bind ergosterol in parasite membranes. Our recent results offer the opportunity to reexamine the mechanism behind the effectiveness of current AmB-based therapeutic strategies to treat leishmaniasis. We propose here a novel mechanism in which the effectiveness of AmB treatment could be partly based on its ability to sequester cholesterol in the host membrane, thereby abrogating macrophage-parasite interaction

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 β2-adrenergic receptor, and the serotonin1A 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 serotonin1A 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

Membrane cholesterol depletion from live cells enhances the function of human serotonin<SUB>1A</SUB> receptors

Work from our laboratory has previously demonstrated the requirement of membrane cholesterol in the function of the serotonin1A receptor, a member of the G-protein coupled receptor (GPCR) superfamily. In order to monitor the effect of cellular organization on the function of human serotonin1A receptors, we explored receptor function following cholesterol depletion in live cells and membranes isolated from cholesterol-depleted cells. We report here the novel observation that while ligand binding of serotonin1A receptors displays an increase in membranes isolated from cholesterol-depleted cells, such trend is absent when binding is performed on cholesterol-depleted intact cells. Importantly, we show here, for the first time, that G-protein coupling of the serotonin1A receptor is enhanced in membranes isolated from cholesterol-depleted cells. These results assume pharmacological relevance in view of the recently described structural evidence of specific cholesterol binding sites in GPCRs, and may help in designing better therapeutic strategies for diseases related to GPCRs