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Previous studies from our laboratory have shown that the 5-HT2C serotonin receptor can be rendered constitutively active by changing amino acid 312 (third intracellular loop) from serine to lysine (S312K). In the present study, detailed radioligand binding analyses were performed to characterize the constitutively activated state of S312K mutant receptors. All agonists tested displayed high affinity for both [3H]5-HT and [3H]mesulergine binding to S312K receptors, but displayed low affinity for [3H]mesulergine binding to native 5-HT2C receptors. [3H]5-HT labeled the same total number of S312K binding sites as [3H]mesulergine. 5-HT2C antagonists inhibited S312K basal inositol phosphate production. These results suggest that S312K receptors mimic the active conformation of native 5-HT2C receptors and provide a good model system for evaluating drugs for inverse agonist activity. Also, S312K receptors may represent a new system for screening 5-HT2C agonist activity by comparing [3H]mesulergine binding to native and S312K mutant receptors.

We have identified a conserved threonine residue in the second intracellular (i2) loop of the 5-HT1A receptor that when mutated to alanine prevents coupling to G beta gamma-mediated signaling, while preserving G alpha i-induced actions. In this review, we investigate the characteristics and potential role of the i2 domain in the coupling of the 5-HT1A receptor and other receptors to G proteins. The i2 domain, as well as portions of the i3 domain, is predicted to form an amphipathic alpha-helix with a positively charged face and a hydrophobic face. Mutagenesis experiments support a model in which the hydrophobic faces of these alpha-helical domains form an intracellular binding "pocket" for interaction with G proteins. Embedded in the hydrophobic face, Thr 149 is crucial for signaling through G beta gamma subunits, perhaps via interaction with its hydroxyl side-chain. Mutation of other residues of the i2 domain of Gi-coupled receptors is required to substantiate the importance of the alpha-helical i2 domain in receptor-G beta gamma signaling. If confirmed in other receptors, these results support a general model in which activated receptor and G beta gamma subunits remain associated to interact with effectors in a receptor-specific manner.

The 5-hydroxytryptamine (5-HT)1B/1D receptor subtypes are involved in the regulation of 5-HT release and have gained particular interest because of their apparent role in migraine. Although selective antagonists for both receptor subtypes recently have been developed, the receptor domains involved in the pharmacological specificity of these antagonists are defined poorly. This was investigated with a chimeric 5-HT1B/1D receptor analysis and using ketanserin as a selective antagonist of h5-HT1D (h5-HT1D) Ki = 24-27 nM) as opposed to h5-HT1B (Ki = 2193-2902 nM) receptors. A domain of the h5-HT1D receptor encompassing the second extracellular loop and the fifth transmembrane domain is necessary and sufficient to promote higher affinity binding (Ki = 65-115 nM) for ketanserin to the h5-HT1B receptor. The same domain of the h5-HT1B receptor, when exchanged in the h5-HT1D receptor, abolished high affinity binding of ketanserin (Ki = 364-1265 nM). A similar observation was made with the antagonist ritanserin and seems specific because besides the unmodified binding affinities for 5-HT and zolmitriptan, only minor modifications (2-4-fold) were observed for the agonists L 694247 and sumatriptan and the antagonists GR 127935 and SB 224289. Generating point mutations of divergent amino acids compared with the h5-HT1B receptor did not demonstrate a smaller peptide region related to a significant modification of ketanserin binding. The antagonists ketanserin and ritanserin are likely to bind the h5-HT1D receptor by its second extracellular loop, near the exofacial surface of the fifth transmembrane domain, or both.

Dopaminergic and serotonergic ligands are widely applied in the therapy of some severe diseases in humans connected to the malfunctioning of the corresponding membrane receptors within the CNS. However, no pharmaceuticals of this type with an ideal therapeutic index have been synthesized so far and there is a constant need of producing new dopaminergic/serotonergic ligands with improved properties especially with regard to undesirable side effects expressed after a prolonged therapy. Dopaminergic/serotonergic ratio turned out to be important for a fine tuning of pharmacological profile of new ligands. Employing a bioisosteric approach, we have synthesized numerous quinoxalinediones, benztriazoles, benzimidazoles and 2-substituted benzimidazoles as potential dopaminergic and/or mixed dopaminergic/serotonergic compounds. With this purpose, benzimidazole and its derivatives were incorporated into phenylethylamine, 3- and 4-substituted phenylethylpiperidine, 1-substituted 4-arylpiperazine and semirigid 2-aminotetralin frame and the resulting ligands were checked for the binding affinity at the D1 and D2 dopamine and 5-HT1A serotonin receptors in radioligand binding assays in vitro. Synaptosomal membranes prepared from bovine caudate nuclei and hippocampi served as a source of the dopamine and serotonin receptors, respectively. [3H]SCH 23390 (D1 receptor-selective), [3H]spiperone (D2 receptor-selective) and 8 OH [3H]DPAT (5-HT1A receptor-selective) were employed as radioligands in competition binding assays. Properties of substituents introduced into position 2 of benzimidazole ring, as well as the nature of the frame into which benzimidazole pharmacophore was incorporated have been shown to determine ligand binding affinity, mode of action and receptor preference, i.e. dopaminergic/serotonergic affinity ratio. Benzimidazolyl-2-thione and benztriazole derivatives were the most potent dopaminergic/serotonergic ligands. Molecular ab initio calculations of the electronic properties of pharmacophoric entities of the new ligands revealed different electron density distribution around the benzene ring in the active and inactive ligands. It can be assumed that this difference influences the properties of pi-pi interactions in a receptor-ligand complex. The results are discussed in comparison with the data of other authors working on similar topics.

5-Hydroxytryptamine2 (serotonin2, 5-HT2)-family receptors are important for mediating many physiological functions, including vascular and nonvascular smooth muscle contraction, platelet aggregation, modulation of perception, mood, anxiety, and feeding behavior. A large number of psychopharmaceuticals, including atypical antipsychotic drugs, antidepressants, anxiolytics, and hallucinogens, mediate their actions, at least in part, via interactions with various 5-HT2-family receptors. This review article summarizes information about structure-function aspects of 5-HT2-family receptors. Evidence is presented that implies that conserved aromatic and charged residues are essential for ligand binding to 5-HT2A receptors. Additionally, findings are reviewed that are consistent with the hypothesis that residues located in intracellular loops 2 and 3 (i2 and i3) mediate coupling to specific G(alpha)-subunits such as G(alpha q). Studies are reviewed that suggest that 5-HT2-family receptors may be down-regulated by both agonists and antagonists, and usually this down-regulation is due to post-transcriptional mechanisms. Finally, a model for regulation of 5-HT2-family receptors by receptor-mediated endocytosis is advanced, and the particular structural features responsible for the various endocytotic pathways are emphasized. Taken together, these results suggest that discrete domains of the receptor structure are important for ligand binding, G-protein coupling, and internalization.

Transmembrane segment (TMS) 7 has been shown to play an important role in the signal transduction function of G-protein-coupled receptors (GPCRs). Although transmembrane segments are most likely to adopt a helical structure, results from a variety of experimental studies involving TMS 7 are inconsistent with it being an ideal alpha-helix. Using results from a search of the structure database and extensive simulated annealing Monte Carlo runs with the new Conformational Memories method, we have identified the conserved (N/D)PxxY region of TMS 7 as the major determinant for deviation of TMS 7 from ideal helicity. The perturbation consists of an Asx turn and a flexible "hinge" region. The Conformational Memories procedure yielded a model structure of TMS 7 which, unlike an ideal alpha-helix, is capable of accommodating all of the experimentally derived geometrical criteria for the interactions of TMS 7 in the transmembrane bundle of GPCRs. In the context of the entire structure of a transmembrane bundle model for the 5HT2a receptor, the specific perturbation of TMS 7 by the NP sequence suggests a structural hypothesis for the pattern of amino acid conservation observed in TMS 1, 2, and 7 of GPCRs. The structure resulting from the incorporation of the (N/D)P motif satisfies fully the H-bonding capabilities of the 100% conserved polar residues in these TMSs, in agreement with results from mutagenesis experiments. The flexibility introduced by the specific structural perturbation produced by the (NP/DP) motif in TMS 7 is proposed to have a significant role in receptor activation.

We screened the serotonin5A receptor gene coding region in 186 unrelated alcoholic patients and 187 controls. A relatively abundant amino acid substitution and two synonymous DNA substitutions were detected. Two synonymous variants, A12T and C789T, had rarer-allele frequencies of 23% and 1%, respectively. The Pro15Ser substitution is located in the amino terminal, extracellular domain of the receptor adjacent to a putative phosphorylation site. Pro15Ser had rarer-allele frequencies of 8.1% and 5.9% in Finnish alcoholic patients and controls, respectively (p=n.s.)., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

The biochemical characterization of a serotonin (5HT) receptor and the cloning of a dopamine (DA) receptor in the central nervous system (CNS) of the leech, Theromyzon tessulatum, is presented. Additionally, DA and 5HT binding sites were examined in the CNS by Scatchard analysis which showed a single, relatively high-affinity binding site with a Kd 1.1 nM and a Bmax 126+18 fmol/mg protein for [3H]DA and a Kd 2.1 nM and a Bmax 225 fmol/mg protein for [3H]5HT. The first 88 amino acids of the 5HT receptor, isolated by a 5HT-affinity column followed by anion exchange chromatography and C3 reverse-phase HPLC exhibited a 43% sequence homology with Lymnaea stagnalis 5HT-receptor. The isolated DA receptor revealed a single protein of 45 kDa with an anti-D1-R in Western blot. The first 80 N-terminal amino acid residues and a trypsin digested fragment of 31 residues were obtained, and based on these sequencing data, a molecular biology strategy using reverse transcriptase-polymerase chain reaction, was developed. An amplified 1-kb segment was obtained. The complete deduced sequence of 416 amino acid residues exhibited about 30.6% sequence homology with the vertebrate D1 receptor family. Moreover, we further demonstrate that the leech 5HT and DA receptors also exhibit 30% sequence identity with each other, explaining their pharmacological cross-reactivity. Finally, anti-D1-R immunocytochemistry revealed positive structures in the peripheral and central nervous system, e.g., neurons, sensory fibers and immune cells. This is the first biochemical and molecular characterization of a DA receptor in leeches., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

We have used a combination of sequence comparisons, computer-based modeling and site-directed mutagenesis to investigate the molecular interactions involved in ligand binding and signal transduction of the human 5-HT1B receptor. Two amino acid residues, S212 in transmembrane region (TM) V and F331 in TM VI, were replaced by alanines. These amino acids are conserved in many G protein-coupled receptors and therefore likely to be important for receptor function. The mutant receptors were expressed in Chinese hamster ovary cells. The 5-HT-like agonist 5-carboxamido-tryptamine (5-CT) bound with 15-fold lower affinity to the S212A mutant as compared to wild-type receptor and the antagonist methiothepin bound with 17-fold lower affinity to the F331A mutant. No reduction in the affinity of 5-HT was seen for the S212A mutant, although an equivalent mutation in the 5-HT1A receptor resulted in a 100-fold reduction of 5-HT binding. The inhibition of forskolin-stimulated cyclic AMP production by 5-HT was significantly reduced in cells expressing the F331A mutant, even though the endogenous ligand 5-HT bound with somewhat increased affinity. Methiothepin acted as an inverse agonist and increased the forskolin-stimulated cyclic AMP production at both the wild-type receptor and the mutants, and the effect was stronger on the F331A mutant. These results suggest that F331 is involved in the conformational changes necessary for signal transduction.

Single amino acid mutations in the third intracellular loop, as well as other domains of G protein-coupled receptors, have been shown to confer drastic changes in receptor properties and have been postulated to be responsible for various disease states. To determine whether an amino acid mutation can confer dramatic alterations in the 5-hydroxytryptamine2A (5-HT2A) receptor, we mutated amino acid 322 to lysine (C322K), glutamate (C322E) or arginine (C322R). Transient expression of the mutant receptors revealed properties associated with constitutive activity. Radioligand binding studies revealed an increase in 5-HT affinity from 293 nM (native) to 86 nM (C322E), 25 nM (C322K) and 11 nM (C322R). 5-HT potency for stimulation of inositol phosphate production increased from 152 nM (native) to 61 nM (C322E) and 25 nM (C322K). Basal inositol phosphate levels in COS-7 cells expressing C322K and C322E mutant receptors were 8-fold and 4-fold higher, respectively, than cells expressing native 5-HT2A receptors. Basal levels of inositol phosphate stimulated by C322K receptors represented 48% of total inositol phosphate production stimulated by native receptors in the presence of 10 microM 5-HT. Antipsychotic drugs (chlorpromazine, clozapine, haloperidol, loxapine and risperidone) displayed inverse agonist activity by inhibiting C322K constitutive activation of phosphatidylinositol hydrolysis. These data indicate that amino acid 322 in the 5-HT2A receptor plays an important role in maintaining the inactive conformation and provide further evidence that amino acid mutations can produce profound alterations in G protein-coupled receptor activity.

The 5-HT3 receptor complex is a ligand-gated ion channel, and is therefore likely to comprise multiple subunits in common with other members of this superfamily. To date, however, only one 5-HT3 receptor subunit, plus an alternatively spliced variant, have been identified. In this article, Stephanie Fletcher and Nicholas Barnes review some of the extensive data in the literature that suggest the presence of other 5-HT3 receptor subunits. This is particularly relevant given the recent demonstration that the 5-HT3 receptor purified from pig brain contains a non-5-HT3A-like protein(s).

Site-directed mutagenesis was used to investigate the molecular interactions involved in ligand binding to the human 5-HT1B receptor. Six mutants were constructed at four positions and expressed in Chinese hamster ovary cells. Substitution of the amino acid F185 in transmembrane region IV by an alanine increased the affinities of sumatriptan, methysergide and 8-hydroxy-2-(dipropylamino)tetralin (8-OH-DPAT) 3-4-fold and substitution by a methionine increased the affinities of methysergide and methiothepin 2- and 3-fold, respectively. Substitution of amino acid S334 in transmembrane region VI by an alanine increased the affinity of 8-OH-DPAT 5-fold. In accordance with this, the EC50 value of 8-OH-DPAT was decreased 7-fold. This suggests that the serine at position 334 causes steric hindrance for 8-OH-DPAT binding that is lost in the S334A mutant. Mutation of F354 in transmembrane region VII, which differs between receptor subtypes, increased the affinity of methiothepin 2-3-fold but the affinities of the other compounds tested were essentially unchanged.

The 5-HT3 receptor (5-HT3R) is part of a superfamily of ligand-gated ion channels which includes nicotinic acetylcholine receptors (nAChR). cRNA derived from the long isoform cloned mouse 5-HT3R was used to drive expression of 5-HT3Rs in Xenopus oocytes. 5-HT-induced currents were monitored using two-electrode voltage-clamp. Eight nicotinic agonists, including ACh and nicotine, but not alpha-anatoxin, were found to antagonize 5-HT-induced currents. With the exception of 3-(2,4)-dimethoxybenzylidene-anabaseine (DMXB-anabaseine; GTS-21) this antagonism appeared to be competitive since it could be overcome by increasing concentrations of 5-HT. Potency of 5-HT3 antagonism was comparable to reported values for nAChR alpha7 activation. These results confirm the notion of families of receptors and further indicate that strong similarities can exist in some critical binding domains.

The ability of ad hoc defined size and shape descriptors and theoretical descriptors derived on a single structure to give powerful interpretative and predictive QSAR models has been compared and evaluated with respect to the quality of the pharmacological data available for a series of structurally diverse 5-HT1A receptor antagonists, displaying selectivity towards the alpha 1-adrenergic receptor.

The screening of ligands for membrane receptor proteins is central to the discovery of new pharmaceutical drugs. We present a general method to reversibly attach receptor proteins via an affinity tag to a quartz surface and subsequently detect with high sensitivity the real-time binding of ligands by total internal reflection fluorescence. A serotonin-gated ion channel protein was immobilized, and the binding of a fluorescent ligand was investigated. The affinity and the kinetic parameters of binding were measured, and the effect of unlabeled compounds was determined by competition. The pharmacology of the immobilized receptor was identical to that of the native receptor. The affinity of unlabeled ligands was rapidly and effectively determined. The method described here is generally applicable for membrane proteins and opens new ways for the discovery of pharmacologically active compounds.

Using the yeast two-hybrid system we isolated a cDNA clone encoding a novel protein interacting with the C-terminal domain of the 5-HT2C receptor. The protein, named MUPP1 (multi-PDZ-domain protein), contains thirteen PDZ domains and no obvious catalytic domain; it is related to hINADL and a putative C. elegans polypeptide referred to as C52A11.4 containing six or ten PDZ domains, respectively. Domains highly similar to those of MUPP1 are arrayed in the same order in all three proteins. The MUPP1 gene is localized on human chromosome 9p24-p22. Transcripts encoding MUPP1 are abundant in the brain as well as in several peripheral organs.

In an effort to develop a quantitative ligand-binding model for 5-HT1A receptors, a pharmacophore mapping procedure, DIStance COmparison (DISCO) was used to identify structural features that are common in a novel set of pyridazinothiazepines and pyridazinooxazepines with moderate-to high affinity to 5-HT1A-receptors. The pharmacophore thus obtained provided a good starting point for a Comparative Molecular Field Analysis (CoMFA) study. The CoMFA gave acceptable statistical measure (R2CV = 0.52 by using six latent variables, whereas it afforded a non cross-validated R2 value of 1.00). Predictability of our model was tested by a separated prediction set of four compounds, for them the relative deviations between calculated and measured biological activity values did not exceed 10%.

Synthesis and pharmacological evaluation of a series of condensed quinoline and pyridine derivatives bearing a N-methylpiperazine moiety attached to the 2-position of the quinoline or pyridine nucleus are described. 5-HT receptor binding studies revealed subnanomolar affinity for the 5-HT3 receptor subtype in some of the compounds under study. The most active compound (5b) displayed a Ki value about 1 order of magnitude higher than that of quipazine along with a higher selectivity. The potential 5-HT3 agonist/antagonist activity of four selected compounds was assessed in vitro on 5-HT3 receptor-dependent [14C]guanidinium uptake in NG 108-15 cells. Compound 5j acted as a 5-HT3 agonist in this assay with an EC50 value close to that reported for quipazine, while 5b was a partial agonist with an EC50 value of about 0.25 nM, and compound 5c possessed antagonist properties with an IC50 value (approximately 8 nM) in the same range as those of previously characterized 5-HT3 receptor antagonists. Qualitative and quantitative structure-affinity relationship studies carried out by making use of theoretical molecular descriptors allowed to elucidate the role of the main pharmacophoric components and to develop a model for the interaction of the 5-HT3 ligands related to quipazine with their receptor.

A serotonin 5-HT3 receptor was functionally expressed to high levels and on a large scale in mammalian cells with the Semliki Forest virus system. Conditions were optimized to maximize detergent solubilization of the receptor, while preserving ligand binding activity. An efficient one-step purification yielding approximately 50% of the histidine-tagged 5-HT3 receptor was achieved with immobilized metal ion chromatography. The expressed receptor, in both membranes and purified preparations, exhibited wild-type ligand binding properties, characterized by one class of binding sites. The purity of the receptor was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, yielding a single band at 65 kDa, and was confirmed by the specific ligand binding activity of approximately 5 nmol/mg of protein. Deglycosylation of the receptor reduced the estimated relative molecular mass to 49 kDa. The apparent molecular mass of the functional receptor complex was determined by size exclusion chromatography to be 280 kDa, suggesting that the 5-HT3 receptor is a pentameric homooligomer. The secondary structure of the 5-HT3 receptor as determined by circular dichroism appeared to consist of mainly alpha-helices (50%) and beta-strands (24%), with minor contributions from nonregular structure (9%). The binding of either agonist or antagonist did not alter the secondary structure of the receptor.

1. Whole cell voltage clamp electrophysiology and radioligand binding were used to examine the agonist characteristics of the two splice variants of the 5-HT3 receptor which have been cloned from neuronal cell lines. Homo-oligomeric 5-HT3 receptors were examined in HEK 293 cells stably transfected with either long (5-HT3-L) or short (5-HT3-S) receptor subunit DNAs. 2. Functional homo-oligomeric receptors were formed from both subunits, and responses to 5-HT3 receptor agonists (5-hydroxytryptamine (5-HT), 2-methyl 5-HT and m-chlorophenylbiguanide) were qualitatively similar. 3. Maximum currents (Rmax) elicited by the 5-HT3 receptor agonists m-chlorophenylbiguanide (mCPBG) and 2-methyl-5-HT (2-Me-5-HT), as compared to 5-HT, differed in the two splice variants: Rmax mCPBG/Rmax 5-HT values were 0.68+/-0.04 and 0.91+/-0.01 in 5-HT3-L and 5-HT3-S receptors, respectively. Comparable values for 2-Me-5-HT were 0.30+/-0.02 and 0.23+/-0.02. 4. Radioligand binding data showed no difference in affinity of agonist or antagonist binding sites; thus the six amino acid deletion appears to cause differences in agonist efficacy. 5. The role of the 6 amino acid insertion was further investigated by use of site-directed mutagenesis to create two mutant receptors, one where serine 286 was replaced with alanine, and the second where all 6 amino acids were replaced with alanines. 6. Examination of the mutant receptors when stably expressed in HEK 293 cells revealed agonist properties resembling long and not short 5-HT3 receptors. Thus specific amino acids in this region are not responsible for the observed differences. 7. The data show intracellular structure can have significant effects on ligand-gated ion channel function, and suggest that minor changes in structure may be responsible for differences in function observed when ligand-gated ion channel proteins are modulated intracellularly.

A conformational analysis, performed on muscarinic M1 agonists, identified four structural features characteristic of the muscarinic M1 pharmacophore: (i) a protonable basic or quaternary nitrogen acting as a cationic head; (ii) an electronegative dipole usually part of a planar mesomeric ester, amide, or amidine function which can be replaced by an ether (muscarine) or a dioxolane (AF 30); (iii) an intercharge distance of 5 +/- 0.5 A between the cationic head and the electronegative atom of the dipole; (iv) an elevation of 0.5 +/- 0.03 A of the cationic head over the plane containing the electronegative dipole. During a reinvestigation of the conformational behavior of published structures of 5-HT3 antagonists, similar features were observed for the 5-HT3 pharmacophore. However many 5-HT3 antagonists possess additional aromatic planes not present in the muscarinic M1 agonists. These observations brought us to predict the chemical modifications that would change muscarinic M1 agonists into 5-HT3 antagonists. Four of the predicted aminopyridazines were actually synthesized and submitted to testing. The observed IC50 values for 5-HT3 receptor binding ([3H] BRL 43694) ranged from 10 to 425 nM, whereas the affinities for the muscarinic receptor preparations ([3H] pirenzepine) layed over 10,000 nM. In electrophysiological studies the two most active compounds 10 and 13 produced antagonist-like effects on the 5-HT receptor channel complexes responsible for the generation of the rapidly desensitizing ionic currents, and agonist-like effects on those responsible for the slowly desensitizing components.

With the use of the degenerated nucleotides that contain the conserved sequence of G protein-coupled receptor, we have identified a 648-bp clone (HDGRC02) from human genomic DNA with significant sequence homology to human neurotransmitter receptors. HDGRC02 was then used as a probe for the screening of full length gene. From human Lambda DASH II genomic library, a 1.6 Kb clone encoded a full length gene was isolated and named putative neurotransmitter receptor (PNR). PNR has a single open reading frame which predicts a 38.3 KD protein of 338 amino acids with seven transmembrane domain topography. The amino acid sequence of PNR exhibits considerable homology to the rat 5-HR1D receptor with 35% amino acid identity and 56% amino acid similarity. PNR also shows significant sequence homology to the 5-HT1D receptor from Japanese puffer fish fugu, to the 5-HT4L receptor from mouse, to the alpha-2 adrenergic receptor and to the D2 dopamine receptor. Northern blot analysis indicates that PNR is expressed in skeletal muscle and selected areas of the brain. A chromosome mapping study located the PNR gene with human chromosome band of 6q23. The findings in the present study demonstrate that PNR is a putative neurotransmitter receptor.

During a search for new G-protein-linked receptors for dopamine and serotonin, we found a serotonin-4 receptor-like pseudogene. This receptor-like pseudogene is intronless, contains an in-frame stop codon following transmembrane-3, and has two one-nucleotide insertions between transmembrane-5 and -6 regions which alter the reading frame. The predicted amino acid sequence of the human pseudogene is about 35% identical with that of the rat serotonin-4 receptor.

Serotonin 5-HT2A receptors are essential for a large number of physiological functions in the central nervous system and periphery. This review article summarizes our current knowledge of the molecular biology and mechanisms of regulation of 5-HT2A receptors. The mode of drug binding using data derived from molecular modeling and site-directed mutagenesis is described. The cellular and subcellular localization of 5-HT2A receptors is described, and the concentration of 5-HT2A receptors on apical dendrites of pyramidal neurons is emphasized. Various modes of regulation of 5-HT2A receptors are also summarized, including transcriptional, post-translational and mRNA editing processes. Finally, an integrated model of 5-HT2A receptor regulation that involves various protein kinases (protein kinase C, G-protein receptor kinases), arrestins, clathrin-coated vesicles, endosomes and lysosomes. The relevance of these pathways for antidepressant and antipsychotic drug actions is emphasized.

This paper describes a new 3D flexible searching system 3DFS which supports two types of query definitions: simple atom-based definition and generalized function-based definition. The simple and practical definitions of hydrogen bond acceptors/donors, charge centers, aromatic ring centers, and a rapid hydrophobe recognition algorithm are described in detail. 3DFS adopts a four-step searching strategy: a rapid ID screening, an exact 2D substructure searching using the GMA algorithm, a rigid 3D searching, and a conformationally flexible 3D searching using POWELL method. The utility of 3DFS is illustrated by several typical searching examples.

Molecular modeling techniques were used to build a three-dimensional model of the human 5-HT1B receptor. The receptor model was used to examine receptor interactions of 5-hydroxytryptamine (serotonin), (S)pindolol and of the tetrapeptide Leu-Ser-Ala-Leu (LSAL), which recently has been shown to interact specifically with the 5-HT1B receptor. We have assumed that the NH3(+)-LSAL-COO- form of the tetrapeptide is the biologically active, and propose that a negatively charged residue conserved among various species homologues of the 5-HT1B receptor may act as a counter-ion for the positively charged N-terminus of the tetrapeptide. The strongest LSAL-receptor interactions were obtained after molecular dynamics simulations that were started with the N-terminus of LSAL positioned close to Asp352 in transmembrane helix 7. The model suggests that Asp352 in transmembrane helix 7 may act as a counter-ion for the positively charged N-terminus, and that the side chains of Tyr109 (transmembrane helix 2) and Trp125 (transmembrane helix 3) may form hydrogen bonds with the negatively charged C-terminus of LSAL.

The present investigation utilised monoclonal antibodies directed against subunits of the nicotinic acetylcholine receptor in immunoblot and immunoprecipitation studies, which failed to demonstrate that the native 5-hydroxytryptamine3 (5-HT3) receptor complex purified from porcine brain contains the alpha1, alpha3, alpha4, alpha5, alpha7 or beta2 subunits of the nicotinic acetylcholine receptor.

15 amino acid peptide from the transmembrane 5-intracellular loop 3 region of the human 5HT1a receptor produced concentration-dependent decreases in agonist binding. This result is consistent with a competitive interaction between peptide, receptor, and G protein at the receptor-G protein interface. Bombesin and a 13 amino acid peptide from the carboxyl terminus region of the receptor were inactive. Additionally, the peptide decreased forskolin-mediated cAMP elevation. Overall, these results suggest that amino acid residues from this region of the receptor are involved in receptor-G protein coupling and that G protein is activated by the receptor.

Although the serotonin-7 receptor was cloned several years ago, its localization in brain tissues remains confusing because of the existence of a related expressed pseudogene, the sequence of which has not hitherto been reported. During the course of searching for related receptor genes, we also searched for this pseudogene to determine its sequence. Human genomic DNA was screened for dopamine and serotonin receptor-like genes, using the polymerase chain reaction method and degenerate oligonucleotide primers based on the similar sequences in the transmembrane-6 and -7 regions of the serotonin-5A, the serotonin-7, and the dopamine D2, D3 and D4 receptors. This resulted in one of the clones having a 115 bp fragment, of which 89% of the bases were identical to the transmembrane-6 and -7 regions of the serotonin-7 receptor sequence. The fragment was radiolabelled and used to screen a human fetal brain cDNA library. A novel cDNA clone of 1326 bp was isolated. Based on the nucleotide sequence, 88% of the bases in this sequence of the pseudogene are identical to the human serotonin-7 receptor coding sequence. However, compared to the serotonin-7 receptor DNA sequence, the pseudogene sequence has nucleotide deletions and insertions, resulting in frame-shifts and stop codons. It was concluded that this sequence represented a pseudogene related to the serotonin-7 receptor gene.

The cloning and pharmacological characterization of mouse 5-HT1D receptors as well as the comparative analysis of its embryonic expression vs that of 5-HT1B receptors are reported. High densities of both 5-HT1D receptors mRNA and specific 5-HT1D binding sites were detected at 8, 9.5, 10.5 and 13.5 days of prenatal development. In contrast, no specific 5-HT1B binding sites could be detected until 13.5 days of development, when they were present at lower levels than 5-HT1D receptors. This differs markedly from the situation in the adult brain, in which 5-HT1B receptors are present at a much higher density than the 5-HT1D subtype. These data suggest the involvement of 5-HT1D receptors in the mitogenic and proliferative effects of serotonin during early embryonic development.

Using a combination of library screening and nested PCR based on a partial human serotonin 5-HT4 receptor sequence, we have cloned the complete coding region for a human 5-HT4 receptor. The sequence shows extensive similarity to the published porcine 5-HT4A and rat 5-HT4L receptor cDNA; however, in comparison with the latter, we find an open reading frame corresponding to only 388 amino acids instead of 406 amino acids. This difference is due to a frame shift caused by an additional cytosine found in the human sequence after position 1,154. Moreover, we also found the same additional cytosine in the rat 5-HT4 sequence. We confirmed the occurrence of the sequence by examining this part of the sequence in genomic DNA of 10 human volunteers and in rat genomic DNA. Based on a part of the genomic 5-HT4 receptor sequence that was identified in the cloning process, there seem to be at least two possible splice sites in the coding region of the gene. The human 5-HT4 receptor, transiently expressed in COS-7 cells, showed radioligand binding properties similar to 5-HT4 receptors in guinea pig striatal tissue. [3H]GR 113808 revealed K(D) values of 0.15 +/- 0.01 nM for the human receptor and 0.3 +/- 0.1 nM in the guinea pig tissue. Binding constants were determined for four investigated 5-HT4 antagonists and three agonists, and appropriate binding inhibition constants were found in each case. Stimulation of transfected COS-7 cells with 5-HT4-specific agonists caused an increase in cyclic AMP levels.

We have characterized the alpha-bungarotoxin receptors (BgtRs) found on the cell surface of undifferentiated pheochromocytoma (PC12) cells. The PC12 cells express a homogeneous population of alpha7-containing receptors that bind alpha-Bgt with high affinity (Kd = 94 pM). The BgtRs mediate most of the response elicited by nicotine, because the BgtR-specific antagonists methyllycaconitine and alpha-Bgt block approximately 90% of the whole-cell current. The binding of nicotinic agonists to cell-surface BgtRs was highly cooperative with four different agonists showing Hill coefficients in the range of 2.3-2.4. A similar agonist binding cooperativity was observed for BgtR homomers formed from chimeric alpha7/5HT3 subunits expressed in tsA 201 cells. Two classes of agonist binding sites, in the ratio of 4:1 for PC12 cell BgtRs and 3:1 for alpha7/5HT3 BgtRs, were revealed by bromoacetylcholine alkylation of the reduced sites on both PC12 BgtRs and alpha7/5HT3 BgtRs. We conclude from this data that PC12 BgtRs and alpha7/5HT3 homomers contain at least three distinguishable agonist binding sites and thus are different from other nicotinic receptors.

Two mutations of the rat serotonin 5-HT2A receptor were made, expressed and examined for their ability to bind and be stimulated by certain tryptamines as well as their ability to bind antagonists. Mutation of Ser207 to an Ala (S207A) resulted in no substantial changes in binding of either 5-HT2A antagonists or agonists. In contrast, mutation of Ser239 to an Ala (S239A) resulted in significant changes in the 5-HT2A receptor with some but not all agonists and antagonists examined. Specifically, 5-HT had decreased affinity for the S239A mutated 5-HT2A receptor, showing over a 10-fold decrease in receptor-binding displacement, while still being capable of stimulating IP3 formation. However, the agonists tryptamine, 5-methoxytryptamine (5-MeOT), and N-1-isopropyl-5-methoxytryptamine; and the antagonists ketanserin, LY 86057, and LY 53857 were significantly less affected by a S239A mutation. These results suggest that while 5-HT might have a direct interaction with the Ser239 of the 5-HT2A receptor, tryptamine and 5-MeOT interact with this receptor in a different manner.

Efficacies of the 5-hydroxytryptamine (serotonin) 5-HT3 receptor (5-HT3R) agonists 2-methyl-5-HT, dopamine, and m-chlorophenylbiguanide on 5-HT3R native to N1E-115 cells and on homopentameric 5-HT3R expressed in Xenopus oocytes were determined relative to that of 5-HT. Efficacies of 2-methyl-5-HT and dopamine on 5-HT3R native to differentiated N1E-115 cells are high (54 and 36%) as compared with their efficacies on homopentameric 5-HT3R-A(L) and 5-HT3R-A(S) receptors expressed in oocytes (4-8%). m-Chlorophenylbiguanide does not distinguish between 5-HT3R in N1E-115 cells and in oocytes. The distinct pharmacological profile of 5-HT3R native to differentiated N1E-115 cells is conserved when poly(A)+ mRNA from these cells is expressed in oocytes. The results indicate that, apart from the known 5-HT3R subunits, N1E-115 cells express additional proteins involved in 5-HT3R function.

Site-directed mutagenesis was performed to create a mutant serotonin 5-HT2C receptor that would mimic the active conformation of the native receptor. Structural alteration of receptor conformation was achieved by changing amino acid no. 312 from serine to phenylalanine (S312F) or lysine (S312K). After expression in COS-7 cells, the binding affinity of 5-HT for [3H]mesulergine-labeled 5-HT2C receptors increased from 203 nM (native) to 76 nM for S312F and 6.6 nM for S312K mutant receptors. 5-HT potency for stimulation of phosphatidylinositol (PI) hydrolysis increased from 70 nM (native) to 28 nM for S312F and 2.7 nM for S312K mutant receptors. The mutant receptors were constitutively active, stimulating PI hydrolysis in the absence of agonist. S312F and S312K mutations resulted in twofold and five-fold increases, respectively, in basal levels of PI hydrolysis. Mianserin and mesulergine displayed inverse agonist activity by decreasing basal levels of PI hydrolysis stimulated by S312K mutant receptors. [3H]5-HT and [3H]mesulergine labeled the same number of S312K mutant receptors and 5'-guanylylimidodiphosphate had no effect on [3H]5-HT binding. These results indicate that serine --> lysine mutation at amino acid no. 312 produces an agonist high-affinity state of the 5-HT2C receptor that spontaneously couples to G proteins and stimulates PI hydrolysis in the absence of agonist.

Two splice variants of the 5-HT7 receptor were identified in human brain that differ in the lengths of their intracellular carboxy terminal tail. Identification of the variants of this receptor is of particular interest since the 5-HT7 receptor is known to have a high affinity for a number of antidepressants and is localized in brain regions thought to be implicated in depression. The two isoforms are expressed in roughly equal amounts in various regions of the human brain. When expressed in NIH-3T3 cells, both variants encode functional 5-HT7 receptors, positively coupled to adenylyl cyclase. We suggest that both variants are derived from a single gene by alternative mRNA splicing. Furthermore, our results from Southern blot analysis studies suggest that additional 5-HT7 receptor genes may exist in human.

5-Hydroxytryptamine (5-HT) has been shown to exert positive inotropic, chronotropic, and lusitropic effects and to stimulate the L-type calcium channel current (I(Ca)) in human atrial tissue through activation of the pharmacologically defined 5-HT4 receptor subtype. However, the molecular nature of the receptor(s) involved in these effects is still unknown. In the present study, we report the molecular nature of a 5-HT4 receptor cloned from human atrium, h5-HT4A. Sequence analysis reveals that h5-HT4A displays a 93% protein identity with the short form of the 5-HT4 receptor recently isolated from rat brain. h5-HT4A mRNA is expressed in human atrium but not ventricle, and is also found in brain and GI tract. h5-HT4A transiently expressed in COS-7 cells displays a classical 5-HT4 pharmacological profile. However, affinities of the h5-HT4A receptor for agonists such as ML10302, BIMU1, renzapride or zacopride were 4-10-fold lower than the ones found in brain. Moreover, the stimulatory patterns of cAMP formation by h5-HT4A in response to the 5-HT4 agonists ML10302 and renzapride were very similar to the patterns of stimulation of I(Ca) obtained in response to these compounds in human atrial myocytes. We conclude that h5-HT4A likely mediates the effects of 5-HT in human atrium and may differ from 5-HT4 receptor isoforms present in the brain and GI tract.

Several models of agonist binding to G protein-coupled 5-hydroxytryptamine [5-HT] (serotonin) receptors have highlighted the potential importance of highly conserved aromatic residues for ligand binding and agonist efficacy. In this study, we tested these models by constructing and characterizing a number of point mutations of conserved and nonconserved aromatic residues using the 5-HT2A receptor as a model system. Mutations of three highly conserved tryptophans (W200A, W336A, and W367A) proposed to reside near the binding pocket markedly reduced agonist affinity and efficacy at 5-HT2A receptors. Mutations of two other highly conserved aromatic residues postulated to be near the agonist binding site (F340L and Y370A) also had dramatic effects on agonist binding and efficacy. Point mutations of neighboring conserved phenylalanines (F339L and F365L) had minimal effects on agonist binding, although the F365L mutation diminished agonist efficacy. Finally, mutations of two nonconserved aromatic residues (F125L and F383A) not predicted to be near the binding pocket had no effects on agonist binding, potency, or efficacy. Our results are best explained by models that suggest that helices III, V, VI, and VII can form a unit of interacting helices in which highly conserved aromatic residues are oriented toward the center of the helical aggregate to form an aromatic pocket. In addition, our novel results identify a series of aromatic residues essential for agonist-induced second messenger production. These results demonstrate that highly conserved aromatic residues residing in neighboring helices provide the optimum environment for both agonist binding and activation of 5-HT2A receptors.

The mouse serotonin (5-HT) receptor subtype, 5-HT7, belongs to the family of seven transmembrane G-protein-coupled receptors. To identify the structural basis for the coupling of 5-HT7 receptor to G alpha(s) we constructed a number of receptor mutants in which amino acid residues were either substituted or deleted from the second and third intracellular loops. Wild-type and mutant 5-HT7 receptors were expressed in insect cells using the baculovirus vectors. Two mutant receptor species, 5-HT7(E325G) and 5-HT7(K327S), demonstrated markedly impaired abilities to stimulate adenylyl cyclase. The results suggest the importance of the C-terminal region of the third intracellular loop in receptor-G-protein interaction and that specific charged residues, E325 and K327, may play a critical role in this interaction.

Productive interaction between receptors and G proteins involves multiple intracellular receptor domains, but the role of individual receptor amino acids in directing the selection of specific signaling pathways has not yet been identified. Sequence alignment of several G protein-coupled receptors identified a highly conserved threonine residue in the i2 loop of the 5-hydroxytryptamine 1A (5-HT1A) receptor that is a putative protein kinase C phosphorylation consensus site and is located in a predicted amphipathic alpha-helical domain. To examine the role of this conserved threonine residue in 5-HT1A receptor coupling to Gi/Go proteins, this residue was mutated to alanine (T149A mutant). Wild-type and mutant 5-HT1A receptors were stably transfected into both Ltk- and GH4C1 cells to investigate receptor coupling to multiple signaling pathways. In both cell lines, the T149A mutant displayed similar agonist affinities as the wild-type receptor. In Ltk- cells, the T149A 5-HT1A receptor inhibited cAMP accumulation by 30% compared with wild-type (83%). A 2.6-fold increase in intracellular calcium (due to phospholipase C-mediated calcium mobilization) was observed for the wild-type receptor upon the addition of 100 nM 5-HT; whereas the T149A 5-HT1A receptor failed to mediate a calcium mobilization response at equivalent receptor levels to wild-type. When transfected in GH4C1 cells, the T149A receptor mutant fully inhibited basal cAMP and partially inhibited Gs-stimulated cAMP accumulation compared with wild-type receptor (57 +/- 14% versus 86 +/- 2%). In contrast, the T149A 5-HT1A receptor mutant failed to block the influx of calcium induced by calcium channel agonist (+/-)-Bay K8644, whereas the wild-type 5-HT1A receptor inhibited the calcium influx by 40%. Thus, the Thr149 residue is directly involved in G protein coupling to calcium mobilization (mediated by betagamma subunits of Gi2) and to inhibition of calcium channel activation (mediated by betagamma subunits of Go) but plays a minor role in coupling to alpha1-mediated inhibition of cAMP accumulation. The conserved i2 loop threonine may serve as a G protein contact site to direct the signaling specificity of multiple receptors.

Explicit molecular binding pockets were constructed and optimized around sets of superimposed ligands using the minireceptor concept. The resulting binding sites incorporate the properties of the different ligands and were shown to be suitable for the design of molecules presenting novel interaction patterns. Two applications of minireceptor construction and/or optimization, followed by molecular design are described. In the pursuit of new ligands mimicking the action of paclitaxel, a minireceptor was constructed using the primary amino acid sequence of the target protein as a guide. The active site extracted from a homology-based model of the serotonin 5-HT1A receptor was optimized around a set of three ligands using the same approach.

To characterize the structure of Fugu G-protein coupled receptor family and its evolutionary divergence, we have cloned and sequenced the Fugu 5-HT type 1 receptor genes by Polymerase Chain Reaction (PCR) with degenerate primers followed by phage library screening. The analysis of the deduced amino acid sequences showed that F1A alpha and F1A beta have the highest homology to the human 5-HT1A receptor (71.5% and 63.7%, respectively). Another clone, F1D, showed highest (70.5%) homology to the human type 1D receptor. The amino acid residues that are important for ligand binding have been conserved in these Fugu genes. The phylogenetic tree analysis suggests that the duplication event of the Fugu type 1A receptor may have occurred after the divergence of Fugu and the tetrapod lineage.

Fast synaptic neurotransmission is mediated by ligand-gated ion-channel (LGIC) receptors, which include receptors for acetylcholine, serotonin, GABA, glycine, and glutamate. LGICs are pentamers with extracellular ligand-binding domains and form integral membrane ion channels that are selective for cations (acetylcholine and serotonin 5HT3 receptors) or anions (GABAA and glycine receptors and the invertebrate glutamate-binding chloride channel). They form a protein superfamily with no sequence similarity to any protein of known structure. Using a 1D-3D structure mapping approach, we have modeled the extracellular ligand-binding domain based on a significant match with the SH2 and SH3 domains of the biotin repressor structure. Refinement of the model based on knowledge of the large family of SH2 and SH3 structures, sequence alignments, and use of structure templates for loop building, allows the prediction of both monomer and pentamer models. These are consistent with medium-resolution electron microscopy structures and with experimental structure/function data from ligand-binding, antibody-binding, mutagenesis, protein-labeling and subunit-linking studies, and glycosylation sites. Also, the predicted polarity of the channel pore calculated from electrostatic potential maps of pentamer models of superfamily members is consistent with known ion selectivities. Using the glycine receptor alpha 1 subunit, which forms homopentamers, the monomeric and pentameric models define the agonist and antagonist (strychnine) binding sites to a deep crevice formed by an extended loop, which includes the invariant disulfide bridge, between the SH2 and SH3 domains. A detailed binding site for strychnine is reported that is in strong agreement with known structure/function data. A site for interaction of the extracellular ligand-binding domain with the activation of the M2 transmembrane helix is also suggested.

Preparation and affinity to 5-HT1A and 5-HT2A receptors of new buspirone analogues 7-17 are reported. The compounds possess high to low affinity to 5-HT1A and moderate to low to 5-HT2A receptors. The crystal structures have been determined for compounds 11, 12, 13, and 14. For low affinity ligand (15) of 5-HT1A receptor conformational analysis was performed and compared with similar analyses performed for know high (buspirone 1) and very high (WY-48,723 2) affinity ligands of the receptor. Structure-activity relationship is discussed for the affinity to 5-HT1A receptor. A three-point pharmacophore explaining interactions of buspirone-like molecules with the receptor binding site is proposed.