Mapping the Antagonist Binding Site of the Serotonin Type 3 Receptor by Fluorescence Resonance Energy Transfer

Vallotton, Pascal; Tairi, Ana-Paula; Wohland, Thorsten; Friedrich-Benet, Kirstin; Pick, Horst; Hovius, Ruud; Vogel, Horst

doi:10.1021/bi0106989

Cited by 16 publications

(6 citation statements)

References 23 publications

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“…In earlier work from our group, the binding of a fluoresceinlabeled, high affinity competitive antagonist 1,2,3,9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl]-9-(3-amino-(N-fluorescein-thiocarbamoyl)-propyl)-4H-carbazol-4-one (GR-flu) to the 5-HT 3 receptor has been characterized by steady state (9,10) and time-resolved (11) fluorescence spectroscopy. These studies indicated that receptor-bound GR-flu senses a local environment, which is ϳ0.8 pH units more acidic than the bulk, suggesting that acidic residues, glutamate or aspartate, are located close to the binding site of GR-flu.…”

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confidence: 99%

Characterization of the Ligand-binding Site of the Serotonin 5-HT3 Receptor

Schreiter

Hovius

Costioli

et al. 2003

Journal of Biological Chemistry

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The question of how ligand binding to certain receptor proteins eventually gates ion channels is of central importance in cellular signaling but still unresolved at a molecular level. In order to enhance our understanding of the molecular mechanisms, we used a combined biomolecular and biophysical approach to study a serotonin-gated ion channel.The 5-hydroxytryptamine (5-HT 1 ; serotonin) type 3 receptor (5-HT 3 receptor (5-HT 3 R)) is the only ligand-gated ion channel found among the serotonin receptors. Its gene structure (1) and amino acid sequence are similar to those of other members of the Cys loop receptor family including the nicotinic acetylcholine (nAChR), ionotropic ␥-aminobutyric acid, and glycine receptors. They are composed of five homologous or identical subunits, each comprising four predicted transmembrane regions and a large extracellular N-terminal domain containing the ligand-binding site. Among them, the nAChR is most closely related to the 5-HT 3 receptor. Both receptors form ion channels that are permeable to cations and share about 20 -30% amino acid sequence identity.So far, three different 5-HT 3 receptor subunits, A, B, and C, have been cloned as well as a short splicing variant of the A subunit. Expression of only A subunits results in functional ion channels of similar properties as for 5-HT 3 receptors in native tissues, suggesting that this receptor is active as a homopentamer (2). Expression of solely the B (3) or C (4) subunits did not result in functional receptors; however, their coexpression with the A subunit yields in functional receptor proteins with slightly different channel properties.Site-directed mutagenesis and biochemical studies, combined with amino acid sequence alignments, have identified amino acid residues and sequence regions (the so-called loops A-F; see Fig. 1) in the N-terminal extracellular domain implicated in the ligand-binding site of the nicotinic acetylcholine and 5-HT 3 receptors (for reviews, see Refs. 5-7 and references therein).For the nAChR, these experiments together with the recently resolved three-dimensional structure of the acetylcholine-binding protein (AChBP) (8) indicate that the binding site is located at the interface between two adjacent subunits and that the binding loops A-F are forming the binding pocket. According to this model, loops A-C would form the binding site on one subunit, whereas loops D-F of the adjacent subunit would contribute to a lesser extent to ligand binding. In the case of the 5-HT 3 receptor, only few details are known about the ligand-binding site.

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confidence: 99%

Characterization of the Ligand-binding Site of the Serotonin 5-HT3 Receptor

Schreiter

Hovius

Costioli

et al. 2003

Journal of Biological Chemistry

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“…It has been used previously to analyze such distances between interacting soluble proteins (Garzon-Rodriguez et al, 1997;Rye, 2001) and between a soluble molecule and a purified, reconstituted membrane protein (Li et al, 1999;Vallotton et al, 2001). Site-directed spin-labeling has been used in a similar manner (Hubbell et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

Measurement of Intermolecular Distances for the Natural Agonist Peptide Docked at the Cholecystokinin Receptor Expressed in Situ Using Fluorescence Resonance Energy Transfer

Harikumar¹,

Pinon²,

Wessels³

et al. 2004

Mol Pharmacol

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Fluorescence resonance energy transfer is a powerful biophys-ical technique used to analyze the structure of membrane proteins. Here, we used this tool to determine the distances between a distinct position within a docked agonist and a series of distinct sites within the intramembranous confluence of helices and extracellular loops of the cholecystokinin (CCK) receptor. Pseudo-wild-type CCK receptor constructs having single reactive cysteine residues inserted into each of these sites were developed. The experimental strategy included the use of the full agonist, Alexa 488 -CCK, bound to these receptors as donor, with Alexa 568 covalently bound to the specific sites within the CCK receptor as acceptor. Site-labeling was achieved by derivatization of intact cells with a novel fluorescent methanethiosulfonate reagent. A high degree of spectral overlap was observed between receptor-bound donor and receptor-derivatized acceptors, with no transfer observed for a series of controls representing saturation of the receptor binding site with nonfluorescent ligand and use of a null-reactive CCK receptor construct. The measured distances between the fluorophore within the docked agonist and the sites within the first (residue 102) and third (residue 341) extracellular loops of the receptor were shorter than those directed to the second loop (residue 204) or to intramembranous helix two (residue 94). These distances were accommodated well within a refined molecular model of the CCK-occupied receptor that is fully consistent with all existing structure-activity and photoaffinitylabeling studies. This approach provides the initial insights into the conformation of extracellular loop regions of this receptor and establishes clear differences from analogous loops in the rhodopsin crystal structure.An understanding of the molecular details of agonist ligand binding to a receptor provides powerful insights into the tertiary structure of the receptor in its active conformation, which, in turn, can afford valuable information for possible structure-based drug design. In this work, we have applied fluorescence resonance energy transfer (FRET) to the determination of distances between a fixed position in an agonist ligand and a series of defined positions within its receptor. Experimentally derived distance constraints so determined can complement insights gained from ligand structure-activity series (Ding et al., 2002) and studies using receptor mutagenesis (Kennedy et al., 1997;Gigoux et al., 1999) and photoaffinity labeling (Ji et al., 1997;Hadac et al., 1998Hadac et al., , 1999Ding et al., 2001). These experimentally determined intermolecular distances can also be extremely useful as constraints for three-dimensional model construction and refinement for receptor-ligand complexes.The type A CCK receptor, a member of class I guanine nucleotide-binding protein (G protein)-coupled receptors, is normally activated by a linear peptide hormone. CCK is important for the regulation of nutritional homeostasis, playing roles in th...

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“…[8][9][10][11] FRET measurements have become widespread owing to the recent implementations for microscopic and flow cytometric setups. [12][13][14][15][16] Measurements usually involve detecting the quenching of donor fluorescence in the presence of acceptors [17,18] or scaling of sensitized emission of the acceptor to total unquenched donor intensity. [12,19] There are also several methods that measure the fluorescence lifetime of the donor [20,21] or changes in the bleaching kinetics of the donor fluorescent signal caused by FRET.…”

Section: Introductionmentioning

confidence: 99%

Strength in Numbers: Effects of Acceptor Abundance on FRET Efficiency

et al. 2010

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Fluorescence resonance energy transfer (FRET) is a strongly distance-dependent process between a donor and an acceptor molecule, which can be used for sensitive distance measurements and characterization of molecular interactions at the nanometer level. The original mathematical description of this process, however, is only valid for the interaction of one donor with one acceptor. This criterion is not always met, especially in biological systems, where multiple structures can interact simultaneously, often making distance estimations based on transfer efficiency values error-prone. Herein we investigate how the interaction of multiple acceptors and donors influences the transfer efficiency value in an intramolecular cellular FRET system by manipulating the fluorophore/protein ratio of the fluorophore-conjugated antibodies. We show that the labeling ratio of the acceptor has the largest influence on measured transfer efficiency and decreasing or increasing the acceptor labeling ratio can be utilized to manipulate the FRET response of the acceptor-donor pair and therefore is a tool for optimizing sensitivity of FRET measurements.

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Mapping the Antagonist Binding Site of the Serotonin Type 3 Receptor by Fluorescence Resonance Energy Transfer

Cited by 16 publications

References 23 publications

Characterization of the Ligand-binding Site of the Serotonin 5-HT3 Receptor

Characterization of the Ligand-binding Site of the Serotonin 5-HT3 Receptor

Measurement of Intermolecular Distances for the Natural Agonist Peptide Docked at the Cholecystokinin Receptor Expressed in Situ Using Fluorescence Resonance Energy Transfer

Strength in Numbers: Effects of Acceptor Abundance on FRET Efficiency

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