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PubMed2003

Prevalence and risk factors for LSD use among young women.

STUDY OBJECTIVE: To report the lifetime prevalence of lysergic acid diethylamide (LSD) and to identify unique correlates of using this substance in the last year among a large multiethnic sample of sexually active adolescent and young adult women aged 14 to 26 yrs. DESIGN, SETTING, PARTICIPANTS: A cross-sectional survey, administered at university-based ambulatory reproductive health clinics, was completed by 904 women between April and November of 1997 to identify risk factors associated with their use of LSD within the past 12 months. Subjects who reported lifetime, but not past 12 months', use of marijuana, LSD, or other illicit drugs were excluded, leaving a sample of 368 nonusers and 56 users of LSD. In addition, 231 young women who reported only using marijuana in the last year were used as a comparison group to identify unique factors associated with LSD use. RESULTS: Of the total sample (n=904), 13% (n=119) reported lifetime use of LSD, and 58% (n=536) reported lifetime use of marijuana. Logistic regression analyses controlling for age and race/ethnicity found distinct profiles for those who reported using LSD or only marijuana in the last year when compared to those who reported no substance use. Common to both groups was reporting being drunk at least 10 times during the last year, regular smoking of at least half a pack of cigarettes, and identification as a high-sexual-risk taker. However, LSD users as compared to nonusers were more likely to report white ethnicity (as compared to nonwhite), be less than or equal to 17 years of age (as compared to at least 18 years), report a history of physical abuse, and be categorized as having severe depressive symptomatology. In contrast, those who reported only using marijuana were more likely to report single marital status, young age at first intercourse, having half or more of their friends use marijuana, and poor grades. CONCLUSIONS: The female LSD user presents a distinct profile that might aid clinicians in identifying potential LSD use in this population as well as alerting clinicians to the relationship between LSD use and high-risk sexual behaviors.

Adolescent↗
PubMed1997

A naturally occurring amino acid substitution of the human serotonin 5-HT2A receptor influences amplitude and timing of intracellular calcium mobilization.

Recently, two naturally occurring amino acid substitutions were identified in the C-terminal region of the serotonin 5-HT2A receptor. One of these, His452Tyr, has a rarer allele Tyr frequency of 9%. If 452Tyr alters 5-HT2A function, it would thus be a candidate allele for human neurobehavioral variation. The present study was designed to evaluate the potential influence of the 452His and 452Tyr alleles on cellular 5-HT2A functions. Platelet 5-HT2A binding and 5-HT-induced Ca2+ response were compared in eight 452His/452His homozygous and eight 452His/452Tyr heterozygous individuals matched for sex, age, and diagnosis (all were patients with seasonal affective disorder). There was no difference in 5-HT2A binding measured using 125I-lysergic acid diethylamide. Nor were levels of G-protein subunits or PKC alpha, delta, epsilon, or zeta significantly altered. However, when Ca2+ response was stimulated by 2, 5, 10, or 25 microM 5-HT, significant differences were found. In 452His/452Tyr heterozygotes, 452Tyr was associated with both smaller peak amplitude in Ca2+ mobilization and a different time course of response, with slower peak latency and longer half-time in 452His/452Tyr heterozygotes compared with 452His/452His homozygotes. The overall difference in the response of the 5-HT2A receptor in individuals with 452Tyr was a blunting of the shape of the Ca2+ mobilization peak. The data reported here suggest that the primary sequence of this intracellular domain is important in function of the receptor and that the 452His and 452Tyr 5-HT2A alleles should be carefully evaluated for effects on human neurobehavioral variation.

Adult↗
PubMed1993

Molecular cloning and functional expression of 5-HT1E-like rat and human 5-hydroxytryptamine receptor genes.

Sequential polymerase chain reaction experiments were performed to amplify a unique sequence representing a guanine nucleotide-binding protein (G-protein)-coupled receptor from rat hypothalamic cDNA. Degenerate oligonucleotides corresponding to conserved amino acids from transmembrane domains III, V, and VI of known receptors [5-HT1A, 5-HT1C, and 5-HT2; 5-HT is serotonin (5-hydroxytryptamine)] were used as primers for the sequential reactions. The resulting product was subcloned and used to screen a rat genomic library to identify a full-length clone (MR77) containing an intronless open reading frame encoding a 366-amino acid seven-transmembrane domain protein. The human homolog was isolated, and its encoded protein had 93% overall amino acid identity with the rat sequence. Within the conserved transmembrane domains, the sequences exhibit approximately 52%, 59%, 65%, and 68% amino acid identity with the known rat 5-HT1A, rat 5-HT1B, rat 5-HT1D, and human 5-HT1E receptors, respectively. MR77 was subcloned into a eukaryotic expression vector system and expressed in CosM6 cells. Studies on broken cell preparations indicate that the expressed receptor exhibits 125I-labeled d-lysergic acid diethylamide (LSD) binding that can be displaced by serotonin but not by other biogenic amines. The specific binding is displaced by the selective 5-HT1D agonist sumatriptan but not by the mixed 5-HT1A/1D agonist 5-carboxyamidotryptamine. 125I-labeled LSD binding was competitively antagonized by the ergot alkaloids methysergide and ergotamine. HeLa cells transfected with the MR77 gene exhibited inhibition of adenylate cyclase in response to serotonin. MR77 is expressed at low levels throughout the brain, with the greatest expression in the cortex, hippocampus, and striatum. MR77 thus represents a 5-HT receptor of the 5-HT1 class, and we propose that, based on the pharmacological characterization, MR77 represents an additional 5-HT1E-like receptor.

Amino Acid Sequence↗
PubMed1985

Demonstration of an autoreceptor modulating the release of [3H]5-hydroxytryptamine from a synaptosomal-rich spinal cord tissue preparation.

A superfusion system employed to measure the K+-stimulated release of [3H]5-hydroxytryptamine [(3H]5-HT, [3H]serotonin) from a synaptosomal-rich spinal cord tissue preparation was carefully characterized, then used to examine the regulation of spinal 5-HT release. Spinal 5-HT release is apparently modulated by an autoreceptor. Exogenous 5-HT depressed, in a concentration-dependent manner, the K+-stimulated release of [3H]5-HT. Similarly, lysergic acid diethylamide (LSD) produced a concentration-dependent decrease in [3H]5-HT release. Methiothepin and quipazine blocked the inhibition of release induced by exogenous 5-HT. The 5-HT2 receptor antagonists spiperone and ketanserin failed to alter the action of 5-HT at the spinal 5-HT autoreceptor. Spiperone and ketanserin were shown, however, to alter the storage of [3H]5-HT. When used in concentrations greater than 10 nM, the drugs evoked increases in basal [3H]5-HT and [3H]5-hydroxyindoleacetic acid ( [3H]5-HIAA) effluxes which were independent of the presence of calcium ions. A good agreement existed between the potencies of drugs for modifying autoreceptor function and their abilities to compete for high-affinity [3H]5-HT binding in the spinal cord (designated 5-HT1). Furthermore quipazine, in concentrations that preferentially interact with the 5-HT1B subtype, antagonized the actions of exogenous 5-HT on K+-stimulated release. Spiperone, in a concentration that approximated the affinity constant of 5-HT1A sites for the drug, was ineffective in altering the ability of exogenous 5-HT to modulate K+-stimulated [3H]5-HT release. These results suggest that 5-HT1B sites are associated with serotonergic autoreceptor function in the spinal cord.

Animals↗
PubMed1990

Differential effect of gepirone on presynaptic and postsynaptic serotonin receptors: single-cell recording studies.

The sustained administration of the serotonin (5-hydroxytryptamine1A, 5-HT1A) agonist gepirone (15 mg/kg/day subcutaneously) in the rat produced an initial decrease of the firing activity of dorsal raphe 5-HT neurons, which was followed by a progressive recovery to normal after 14 days of treatment. At this point, the somatodendritic 5-HT1A autoreceptor had desensitized, as indicated by the reduced effectiveness of intravenous lysergic acid diethylamide (LSD) and of microiontophoretic applications of 5-HT, LSD, 8-hydroxy-2-(N,N-propylamino) tetralin (8-OH-DPAT), and gepirone, but not of gamma-aminobutyric acid in depressing the firing activity of 5-HT neurons. In contrast, the responsiveness of postsynaptic dorsal hippocampus pyramidal neurons to 5-HT, 8-OH-DPAT, and gepirone was not altered by the 14-day gepirone treatment. In an attempt to unravel the differential effect of sustained gepirone administration on presynaptic and postsynaptic 5-HT1A receptors, the properties of gepirone at these two receptors were assessed. The concurrent microiontophoretic application of gepirone readily blocked the effect of 5-HT on dorsal hippocampus pyramidal neurons, but not on dorsal raphe 5-HT neurons, thus indicating that gepirone is a partial agonist at postsynaptic 5-HT1A receptors and a full agonist at somatodendritic 5-HT1A receptors. It is proposed that gepirone, being a partial agonist at postsynaptic 5-HT1A receptors, fails to desensitize them; whereas, because of its full agonistic activity at the somatodendritic 5-HT1A receptor, it desensitizes this autoreceptor with long-term administration.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed2004

Capillary electrophoresis analysis of a wide variety of seized drugs using the same capillary with dynamic coatings.

Capillary electrophoresis methodology is presented for the routine analysis of a wide variety of seized drugs using the same capillary with dynamic coatings and multiple run buffers. The types of exhibits analyzed using diode array UV detection include phenethylamines, cocaine, oxycodone, heroin, lysergic acid diethylamide (LSD), opium, hallucinogenic mushrooms, and gamma-hydroxybutyrate-gamma-butyrolactone (GHB-GBL). Both qualitative and quantitative analyses are achieved using run buffers that contain additives that provide for secondary equilibrium and/or dynamic coating of the capillary. Dynamic coating of the capillary surface is accomplished by rapid flushes of 0.1 N sodium hydroxide, water, buffer containing polycation coating reagent, and a buffer containing a polyanionic coating reagent (with or without cyclodextrin(s)) or a micelle coating reagent. Dynamic coating with a polyanionic coating reagent is used for the analysis of moderately basic seized drugs and adulterants. The use of cyclodextrin in the run buffer not only allows for chiral analysis but also greatly enhances separation selectivity for achiral solutes. A capillary dynamically coated with a micelle allows for the analysis of neutral, acidic, and weakly basic drugs (GHB, GBL and neutral, acidic, and weakly basic adulterants). Dynamic coating, which gives rise to a relatively high and robust electroosmotic flow at pH < 7, allows for rapid, precise and reproducible separations. For a wide variety of drugs, excellent linearity and migration time precision and good peak area precision (external and internal standard) is obtained. Quantitative results for synthetic mixtures are in good agreement with actual values. Screening for adulterants is greatly enhanced by the use of automated library searches.

Alkaloids↗
PubMed1986

Solubilization and characterization of the serotonin 5-HT1c site from pig choroid plexus.

A novel type of serotonergic binding site, termed the 5-HT1c site, was recently identified on choroid plexus epithelial cells. In the present study, we describe the solubilization of pig choroid plexus 5-HT1c sites by the zwitterionic detergent 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate (CHAPS). High affinity labeling of both membrane-bound and solubilized 5-HT1c sites was obtained by use of the serotonergic radioligand N1-methyl-2-[125I]lysergic acid diethylamide (125I-MIL). In solubilized preparations, 125I-MIL exhibited a dissociation constant (Kd) of 2.0 nM and a 60-75% ratio of specific to total binding. Approximately 45% of the membrane binding sites were solubilized by CHAPS as measured by a postlabeling, polyethylene glycol precipitation method. The CHAPS-solubilized 5-HT1c site fulfilled the accepted criteria for receptor solubilization. The affinities of a series of serotonergic antagonists for the 5-HT1c site showed little or no change upon solubilization of the site. Serotonin, however, showed a 20-fold increase in affinity for the 5-HT1c site after solubilization, which may indicate the loss of a modulatory component during detergent treatment. Both gel filtration and equilibrium sedimentation experiments indicate that the CHAPS-solubilized site is a large molecular weight complex. These studies demonstrate that the pig choroid plexus 5-HT1c site can be solubilized with retention of its binding activity in a form suitable for further purification and characterization.

Animals↗
PubMed2004

Cloning and pharmacological characterisation of the guinea pig 5-ht5A receptor.

The guinea pig 5-hydroxytryptamine(5A) (gp5-ht(5A)) receptor was cloned from guinea pig brain using degenerate polymerase chain reaction (PCR) and shows 88%, 85% and 84% amino acid sequence identity versus the human, rat and mouse 5-ht(5A) receptors, respectively. The receptor was transiently expressed in human embryonic kidney (HEK) 293 cells. [(3)H]-Lysergic acid diethylamide (LSD) bound saturably to gp5-ht(5A)/HEK293 membranes with a K(d) of 2.3+/-0.1 nM and B(max) of 15.7+/-3.4 pmol/mg protein. The receptor binding profile, determined by competition with [(3)H]LSD, correlated well with that for the human 5-ht(5A) receptor. 5-HT stimulated [(35)S]GTPgammaS binding to gp5-ht(5A)/HEK293 membranes (pEC(50) 8.1+/-0.2), and the response was surmountably antagonised by methiothepin and ritanserin, giving apparent pK(B) values of 8.0 and 7.2, respectively. The 5-HT response was absent using membranes prepared from gp5-ht(5A)/HEK293 cells pretreated with pertussis toxin (PTX). These data suggest that the gp5-ht(5A) receptor couples to G(i)-proteins in this expression system and shows a similar pharmacological profile to that for the human 5-ht(5A) receptor.

Amino Acid Sequence↗
PubMed1993

Molecular cloning and expression of a 5-hydroxytryptamine7 serotonin receptor subtype.

We have utilized the polymerase chain reaction technique to selectively amplify a G protein-coupled receptor cDNA from rat kidney proximal convoluted tubule mRNA, which exhibits high homology with previously cloned serotonin receptors. Sequencing of a full-length clone isolated from a rat hippocampal cDNA library revealed an open reading frame of 1,212 base pairs encoding a 404-residue protein with seven hydrophobic regions predicted to represent transmembrane-spanning domains. Within the transmembrane regions, this receptor was found to be 44-50% identical with various members of the 5-HT1, 5-HT5, and 5-HT6 subfamilies with lower (37-40%) homology to the 5-HT2-like receptors. Northern blots revealed a approximately 3.6-kilobase transcript localized in various brain regions with the following rank order of abundance: hypothalamus > hippocampus = mesencephalon > cerebral cortex = olfactory bulb > olfactory tubercle. Expression of this clone in COS-7 cells resulted in the appearance of high affinity, saturable binding of [3H]lysergic acid diethylamide ([3H]LSD; KD = 5 nM) and [3H]serotonin ([3H]5-HT; KD = 1 nM). Among endogenous biogenic amines, only 5-HT completely inhibited radioligand binding. The inhibition of radioligand binding by other serotonergic agents revealed a pharmacological profile that does not correlate with any previously described serotonin receptor subtype. In addition, this receptor exhibits high affinity for a number of tricyclic antipsychotic and antidepressant drugs including clozapine, loxapine, and amitriptyline. In HEK-293 cells stably transfected with this receptor, serotonin elicits a potent stimulation of adenylylcyclase activity. The distinct structural and pharmacological properties of this receptor suggests that it represents a completely novel serotonin receptor subtype, which we propose to designate 5-HT7. Based on its pharmacology and its localization to limbic and cortical regions of the brain, it is likely that this receptor may play a role in several neuropsychiatric disorders that involve serotonergic systems.

Amino Acid Sequence↗
PubMed1998

Identification of residues in transmembrane regions III and VI that contribute to the ligand binding site of the serotonin 5-HT6 receptor.

We have examined the ligand binding site of the serotonin 5-HT6 receptor using site-directed mutagenesis. Replacing the highly conserved Asp106 in transmembrane region III by asparagine eliminated D-[3H]-lysergic acid diethylamide ([3H]LSD) binding to the mutant receptor transiently expressed in HEK293 cells. The potency of 5-HT and LSD to stimulate adenylyl cyclase was reduced by 3,600- and 500-fold, respectively, suggesting that an ionic interaction between the positively charged amino group of 5-HT and D106 is essential for high-affinity binding and important for receptor activation. In addition, basal cyclic AMP levels in cells expressing this mutant were increased. Mutation of a tryptophan residue one helix turn toward the extracellular side of transmembrane region III (Trp102) to phenylalanine produced significant changes in the binding affinity and potency of several ligands, consistent with a role of this residue in the formation of the ligand binding site. The exchange of two neighboring residues in the carboxy-terminal half of transmembrane region VI (Ala287 and Asn288) for leucine and serine resulted in a mutant receptor with increased affinities (seven- to 30-fold) for sumatriptan and several ergopeptine ligands. The identification of these interactions will help to improve models of the 5-HT6 receptor ligand binding site.

Adenylyl Cyclases↗
PubMed1974

Further studies on the mode of action of psychotomimetic drugs: antagonism of the excitatory actions of 5-hydroxytryptamine by methylated derivatives of tryptamine.

1 The actions of 5-methoxytryptamine (5-MeOT), N,N-dimethyltryptamine (DMT), 5-hydroxy-N,N-dimethyltryptamine (bufotenine, 5-HODMT) and 5-methoxy-N,N-dimethyltryptamine (5-MeODMT), and their interactions with 5-hydroxytryptamine (5-HT), acetylcholine, (-)-noradrenaline, and glutamate were studied by microiontophoresis on single neurones in the brain stem of rats anaesthetized with urethane or decerebrate cats.2 Like D-lysergic acid diethylamide (LSD 25) the three psychotomimetic derivatives (DMT, 5-HODMT, 5-MeODMT) specifically antagonized 5-HT excitations of single neurones, but the non-psychotomimetic 5-MeOT had no antagonistic effects.3 In contrast to LSD 25, the psychotomimetic tryptamines only rarely antagonized glutamate effects, indicating that the excitatory 5-HT receptors and the glutamate receptors on the same neurones may be closely related spatially, but are separate.4 The methylated tryptamine derivatives were able to mimic the actions of 5-HT on neurones. The non-psychotomimetic 5-MeOT was most potent in this respect, while the other three derivatives which are psychotomimetic, were less active.5 The 5-HT mimicking actions of 5-MeOT were the same in rats pretreated with p-chlorophenylalanine or reserpine as in untreated rats. It therefore seems that the 5-HT mimicking actions are unlikely to be due to release of 5-HT, but are due to direct actions on 5-HT receptors.6 The evidence presented supports the hypothesis that LSD-like psychotomimetics act by an antagonism of 5-HT in the lower brain stem, and is not compatible with the suggestion that the psychotomimetic action of these drugs is related to 5-HT receptor stimulation.

Acetylcholine↗
PubMed2004

Platelet serotonin 5-HT2A receptor binding in patients with carcinoid tumor.

BACKGROUND: As carcinoid tumors produce and secrete serotonin, various serotonin markers in blood, plasma and urine have been used as diagnostic tools, and quantification of the urinary excretion of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) is the method most frequently used. METHODS: [3H]lysergic acid diethylamide ([3H]LSD) binding to the platelet serotonin 5-HT2A receptor was investigated in nine patients with carcinoid tumors. The possible effect of serotonin-rich food on the receptor binding was also investigated. RESULTS: B(max) for [3H]LSD binding was significantly lower in the carcinoid group than in the control group (mean +/- SD: 17.6 +/- 1.3 vs. 23.9 +/- 5.2 fmol/mg protein; p = 0.007). Kd for [3H]LSD binding was significantly higher in the carcinoid group than in the control group (median: 1.14 vs. 0.71 nmol/L; p = 0.03). B(max) was inversely related to the urinary 5-HIAA excretion, but the correlation did not reach statistical significance (r(s) = -0.57; p = 0.14). Intake of five bananas per day for one week had no effect on B(max) or Kd in healthy volunteers. CONCLUSIONS: The results are consistent with a down-regulation of the 5-HT2A receptor as a response to the high serotonin levels found in patients with carcinoid tumors. Intake of serotonin-rich food does not affect the receptor characteristics. Further studies are needed to determine whether the platelet 5-HT2A receptor status can be used as a supplement to urinary 5-HIAA and other biochemical variables in carcinoid tumors.

Aged↗
PubMed1996

LSD and the phenethylamine hallucinogen DOI are potent partial agonists at 5-HT2A receptors on interneurons in rat piriform cortex.

Correlations between 5-hydroxytryptamine (5-HT) receptor binding affinities and human hallucinogenic potency have suggested that 5-HT2 receptors mediate the hallucinogenic effects of lysergic acid diethylamide (LSD) and phenethylamine hallucinogens. Electrophysiological studies have suggested that a subpopulation of gamma-aminobutyric acid (GABA)ergic interneurons in layer III of the rat piriform cortex are excited by serotonin (5-HT) via 5-HT2A receptors. These interneurons have inhibitory inputs on pyramidal cells in layer II. In the present study, we tested low concentrations of both LSD (3-100 nM) and the phenethylamine hallucinogen 1-(2,5-dimethoxy-4-iodophenyl-2-aminopropane (DOI; 0.3-10 microM) on rat piriform cortical interneurons that were excited by 5-HT. Both LSD (3-100 nM) and DOI (0.3-10 microM) excited almost every cell excited by 5-HT. The maximal excitation achieved with LSD and DOI was 39% and 55% of the effect of a near-maximal 5-HT concentration (100 microM). Consistent with a partial agonist action, LSD and DOI blocked the 5-HT excitation of piriform cortical interneurons only at the higher hallucinogen concentrations tested. A specific 5-HT2A receptor antagonist, MDL 100,907, blocked excitation of these interneurons by 5-HT, LSD and DOI, but not by norepinephrine or alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate. Again, consistent with a partial agonist action of the hallucinogens, intracellular experiments showed that a maximal concentration of DOI (10 microM) induced fewer postsynaptic inhibitory currents than did 5-HT (100 microM) in pyramidal neurons in layer II of the piriform cortex. Based on the present electrophysiological studies, we conclude that LSD and DOI, a phenethylamine hallucinogen, act as highly potent partial agonists at cortical 5-HT2A receptors.

Action Potentials↗
PubMed1997

Interaction of tryptamine and ergoline compounds with threonine 196 in the ligand binding site of the 5-hydroxytryptamine6 receptor.

We examined the ligand-binding site of the 5-hydroxytryptamine6 (5-HT6) receptor using site-directed mutagenesis. Interactions with residues in two characteristic positions of trans-membrane region V are important for ligand binding in several bioamine receptors. In the 5-HT6 receptor, one of these residues is a threonine (Thr196), whereas in most other mammalian 5-HT receptors, the corresponding residue is alanine. After transient expression in human embryonic kidney 293 cells, we determined the effects of the mutation T196A on [3H]d-lysergic acid diethylamide (LSD) binding and adenylyl cyclase stimulation. This mutation produced a receptor with a 10-fold reduced affinity for [3H]LSD and a 6-fold reduced affinity for 5-HT. The potency of both LSD and 5-HT for stimulation of adenylyl cyclase was also reduced by 18- and 7-fold, respectively. The affinity of other N1-unsubstituted ergolines (e.g., ergotamine, lisuride) was reduced 10-30 fold, whereas the affinity of N1-methylated ergolines (e.g., metergoline, methysergide, mesulergine) and other ligands, such as methiothepine, clozapine, ritanserin, amitriptyline, and mainserin, changed very little or increased. This indicates that in wild-type 5-HT6 receptor, Thr196 interacts with the N1 of N1-unsubstituted ergolines and tryptamines, probably forming a hydrogen bond. Based on molecular modeling, a serine residue in transmembrane region IV of the 5-HT2A receptor has previously been proposed to interact with the N1-position of 5-HT. When the corresponding residue of the 5-HT6 receptor (Ala154) was converted to serine, no change in the affinity of twelve 5-HT6 receptor ligands or in the potency of 5-HT and LSD could be detected, suggesting that this position does not contribute to the ligand binding site of the 5-HT6 receptor.

Amino Acid Sequence↗
PubMed1998

Comparative biochemical and pharmacological characterization of the mouse 5HT5A 5-hydroxytryptamine receptor and the human beta2-adrenergic receptor produced in the methylotrophic yeast Pichia pastoris.

Over the last few years, Pichia pastoris has been developed into a powerful expression system for a multitude of foreign genes. Here, we demonstrate that the P. pastoris expression system has similar power to the baculovirus expression system in high-level production of two G-protein-coupled receptors, the mouse 5HT5A 5-hydroxtryptamine receptor and the human beta2-adrenergic receptor. Different expression plasmids were constructed in which the cDNAs of the two receptors were cloned under the transcriptional control of the highly inducible promoter of the P. pastoris alcohol oxidase 1 (AOX1) gene. In three expression plasmids, the receptors were fused to the Saccharomyces cerevisiae alpha-factor prepropeptide and also to the c-myc tag or the FLAG tag to permit immunological detection of the receptors. After transformation into P. pastoris strains KM71 and SMD 1163, recombinant clones were selected and tested for the production of the 5HT5A receptor and the beta2-adrenergic receptor by radioligand binding using [N-methyl-3H]lysergic acid diethylamide and [5,7-3H](-)CGP-12177 respectively. The production level of the 5HT5A receptor was improved by a factor of three by fusion with the alpha-factor prepropeptide. Also, the higher gene dosage resulting from multiple insertions of the expression cassette led to an improvement in production by a factor of two for both receptors. The addition of the adrenergic antagonist alprenolol to the culture medium had a positive effect on the number of specific binding sites detectable in clones producing the beta2-adrenergic receptor. For the 5HT5A receptor the addition of yohimbine resulted in a similar but smaller effect. Binding assays revealed that approx. 25 pmol of beta2-adrenergic receptor and approx. 40 pmol of 5HT5A receptor per mg of membrane protein in crude membrane preparations were produced. The pharmacological profiles for the heterologously produced receptors, estimated by ligand-displacement analysis using certain adrenergic and serotoninergic agonists and antagonists, were comparable with those reported for the receptors expressed in mammalian systems. Immunoblot analysis of the 5HT5A receptor revealed an apparent molecular mass about 20 kDa higher than expected from the amino acid sequence. Here, the Kex2 endopeptidase failed to process the alpha-factor leader correctly. Blocking glycosylation in vivo by tunicamycin or in vitro deglycosylation of membranes by endoglycosidase H resulted in correct processing. In contrast, the beta2-adrenergic receptor fusion to the alpha-factor leader was correctly processed by the internal Kex2 endopeptidase. The Kex2-processed beta2-adrenergic receptor was not glycosylated. In conclusion, the high-level production of the two receptors in P. pastoris will allow their purification in quantities sufficient for subsequent biophysical and structural studies.

Adrenergic beta-Agonists↗
PubMed1993

Site-directed mutagenesis of the serotonin 5-hydroxytrypamine2 receptor: identification of amino acids necessary for ligand binding and receptor activation.

Serotonin 5-hydroxytryptamine (5-HT)2 receptors are implicated in the etiology of mental disease and depression. Drugs that interact with the 5-HT2 receptor are used therapeutically to treat such illnesses, and their mechanisms of action are of great interest. In this study 5-HT2 receptor-ligand interactions were examined by site-directed mutagenesis in which three aspartic acid to asparagine mutants (Asn-120, Asn-155, and Asn-172) were created and expressed in NIH3T3 cells. The Asp-120 to asparagine mutant exhibited the same affinity for 125I-lysergic acid diethylamide (125I-LSD) as did the wild-type receptor and showed a decreased and GTP-insensitive binding affinity for the agonists 5-HT and (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane (approximately 10-fold) and the antagonists ketanserin and mianserin (approximately 10-fold) but not spiperone. The mutation also abolished agonist-stimulated formation of [3H]polyphosphoinositides (PI). The Asn-155 mutant showed reduced binding affinity for 125I-LSD (Kd, 2.8 nM versus 0.6 nM for the wild-type receptor) and had reduced affinity for agonists (approximately 30-fold) and for antagonists (14-75-fold). However, the Asn-155 receptor retained GTP sensitivity and the ability to stimulate PI formation. The Asn-172 mutant retained the wild-type Kd value for 125I-LSD, exhibited only approximately 5-fold reduced affinity for 5-HT and (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane while retaining GTP-sensitive agonist binding showed no change in affinity for ketanserin, and had a small decrease in mianserin and spiperone binding (approximately 6-fold). The Asn-172 receptor also retained the ability to form PI. These results indicate that Asp-120 is necessary for allosteric activation of the guanine nucleotide-binding protein. Asp-155 is necessary for high affinity binding, probably by acting as a counterion for the amine group of the ligand. The different effects of the three mutations on ketanserin, mianserin, and spiperone binding affinity suggest that these antagonists may share overlapping but different binding domains. The information provided by this study may facilitate the design of therapeutic site-selective compound based on the structure of the 5-HT2 receptor.

3T3 Cells↗
PubMed2004

Tyramine receptor (SER-2) isoforms are involved in the regulation of pharyngeal pumping and foraging behavior in Caenorhabditis elegans.

Octopamine regulates essential processes in nematodes; however, little is known about the physiological role of its precursor, tyramine. In the present study, we have characterized alternatively spliced Caenorhabditis elegans tyramine receptor isoforms (SER-2 and SER-2A) that differ by 23 amino acids within the mid-region of the third intracellular loop. Membranes prepared from cells expressing either SER-2 or SER-2A bind [3H]lysergic acid diethylamide (LSD) in the low nanomolar range and exhibit highest affinity for tyramine. Similarly, both isoforms exhibit nearly identical Ki values for a number of antagonists. In contrast, SER-2A exhibits a significantly lower affinity than SER-2 for other physiologically relevant biogenic amines, including octopamine. Pertussis toxin treatment reduces affinity for both tyramine and octopamine, especially for octopamine in membranes from cells expressing SER-2, suggesting that the conformation of the mid-region of the third intracellular loop is dictated by G-protein interactions and is responsible for the differential tyramine/octopamine affinities of the two isoforms. Tyramine reduces forskolin-stimulated cAMP levels in HEK293 cells expressing either isoform with nearly identical IC50 values. Tyramine, but not octopamine, also elevates Ca2+ levels in cells expressing SER-2 and to a lesser extent SER-2A. Most importantly, ser-2 null mutants (pk1357) fail to suppress head movements while reversing in response to nose-touch, suggesting a role for SER-2 in the regulation of foraging behavior, and fail to respond to tyramine in assays measuring serotonin-dependent pharyngeal pumping. These are the first reported functions for SER-2. These results suggest that C. elegans contains tyramine receptors, that individual SER-2 isoforms may differ significantly in their sensitivity to other physiologically relevant biogenic amines, such as octopamine (OA), and that tyraminergic signaling may be important in the regulation of key processes in nematodes.

Adrenergic Uptake Inhibitors↗