Consequences of DJ-1 upregulation following p53 loss and cell transformation.

p53 is a tumor suppressor that responds to various stress signals by initiating cell-cycle arrest, senescence and apoptosis. Mutations of the p53 gene are found in over 50% of human tumors, highlighting the importance of p53 in tumor suppression. Numerous studies have reported on the interactions between p53, IGF-1-AKT and mTOR pathways as potentially explaining some of the tumor suppressive activities of p53. To further understand the basis of these interactions, we analyzed the involvement of DJ-1, an oncogene known to drive AKT-mediated cell survival, in the p53-AKT axis. In this study, we show that DJ-1 and p53 are tightly 'linked': p53 prevents the accumulation of DJ-1 protein, whereas loss of p53 leads to stabilization and enhancement of DJ-1 expression. Interestingly, this increase in DJ-1 level is only observed when p53 loss is accompanied by transformation of cells. Moreover, DJ-1 seems to be required for the enhanced activation of AKT observed in p53-deficient cells. Such observation confers a new property to DJ-1 associated to transforming-process to its oncogenic ability to drive AKT activation. We also show that DJ-1 is necessary for p53 activation following oxidative stress, suggesting the existence of a finely regulated loop between these two proteins in transformed cells. Finally, we demonstrate that in the absence of p53, DJ-1 is stabilized by ROS accumulation, and surprisingly seems to be required for this high intracellular ROS production. These data offer new insights into the regulation of DJ-1 and suggest that DJ-1 is a target of p53. Importantly, our study highlights that during transformation, DJ-1 is having a key role in the p53-regulated AKT pathway and p53-driven oxidative-stress response.

Therapeutic implications of constitutive activity of receptors: the example of the histamine H3 receptor.

Some G-protein-coupled receptors display constitutive activity, that is spontaneous activity in the absence of agonist: a proportion of the receptor population adopts a conformation that can bind and activate G proteins. Whereas this was mainly shown to occur with recombinant or pathologically mutated receptors, the physiological relevance of the process has remained debated. We have adressed this question in the case of the histamine H3 receptor, a presynaptic inhibitory receptor regulating histamine release in brain. Having identified a neutral antagonist and inverse agonists with variable intrinsic activity, we show that the native H3 receptor in brain displays high constitutive activity in vitro and, in vivo, controls the release of endogenous histamine. This implies that inverse agonists with high intrinsic activity should be preferred for therapeutic application as "cognitive enhancers" in several psychiatric disorders.

A detailed mapping of the histamine H(3) receptor and its gene transcripts in rat brain.

The detailed distribution of histamine H(3) receptor mRNAs in rat brain was analyzed by in situ hybridization using a 33P-labelled riboprobe and was combined for the first time with the detailed autoradiographic distribution of the receptor determined in the same animals with [(125)I]iodoproxyfan, a selective radioligand. The signals generated on adjacent brain sections by each probe were quantified and/or rated and were compared in order to identify neuronal populations expressing the receptor. In addition, the cellular localization of the transcripts within various brain structures was analyzed in sections dipped in a photographic emulsion. In the cerebral cortex, the strong mRNA expression in intermediate and deep layers indicates the presence of H(3) receptors on several types of neurons. The binding is dense except in layer V, suggesting that H(3) receptors are located on granule cells and apical dendrites of pyramidal cells. In addition to their localization on monoaminergic afferents, the dense binding in layer IV and strong mRNA expression in thalamic nuclei suggest the presence of heteroreceptors on thalamocortical projections. In the hippocampus, the strong mRNA expression but low binding in pyramidal layers of the CA1 and ventral CA3 fields suggest that H(3) receptors are abundant on efferent projections of pyramidal cells. In the dentate gyrus, some binding sites in the molecular layer may correspond to H(3) receptors synthesized in granule cells and coexpressed with H(1) and H(2) receptors in their dendrites. In the basal ganglia, H(3) receptors are highly expressed in the striatal complex and olfactory tubercles but not in islands of Calleja. Some of the striatal binding sites may correspond to presynaptic receptors present on afferents. The mRNAs in cortical layer V may encode for heteroreceptors on corticostriatal neurons. The presence of mRNAs in the substantia nigra pars compacta suggests that H(3) receptors are located upon nigrostriatal afferents. However, the absence of any signal in the ventral tegmental area indicates that some but not all dopaminergic neurons express H(3) receptors. In addition, the homogeneous mRNA expression within the caudate putamen and nucleus accumbens suggests that many striatal H(3) receptors are present on medium-sized, spiny projection neurons of both the direct and indirect movement pathways. In agreement, a dense binding, but low mRNA expression, is observed in external and internal pallidum and in substantia nigra pars reticulata. In the amygdala, the dense binding and mRNA expression indicate the presence of receptors on both afferents and projections. In the thalamus, the binding in some association nuclei may correspond to receptors present on neurons emanating from the deep cortical layers that strongly express the mRNAs, as well as receptors on the visual systems. However, the low binding and high mRNA expression in most nuclei indicate that many receptors are present upon thalamic projections. In the hypothalamus, the mRNA expression parallels the density of binding sites and is the highest in the tuberomammillary nucleus. Further investigation is needed to know if the dense binding and mRNA expression observed in other nuclei such as the paraventricular, ventromedial and medial tuberal nuclei correspond to pre- and/or postsynaptic receptors. mRNAs are also observed in several areas projecting to the tuberomammillary nucleus, such as the ventrolateral preoptic nucleus. In the lower brainstem, the high mRNA expression and very low binding in the locus coeruleus and raphe nuclei indicate that presynaptic rather than somatodendritic receptors regulate noradrenaline and serotonin release, respectively. A similar pattern in vestibular nuclei suggests that receptors located on projections account for the anti-vertigo properties of H(3) receptor antagonists. In the cerebellum, binding is hardly detectable but a strong mRNA expression is found in most, if not all, Purkinje cells as well as in several central cerebellar nuclei, suggesting the presence of H(3) receptors on efferent projections. The present study reports the first detailed quantification and/or rating of H(3) receptor mRNAs in the brain. The comparison, performed in the same animals, with the distribution of the H(3) receptor protein provides evidence for the presence of H(3) receptors on many neuronal perikarya, dendrites and projections. Although some localizations, mainly as auto- or heteroreceptors, are consistent with previous functional studies, the physiological role, if any, of most of these presynaptic or postsynaptic receptors remains to be established.

Histamine H3-receptor-mediated [35S]GTP gamma[S] binding: evidence for constitutive activity of the recombinant and native rat and human H3 receptors.

Constitutive activity of the recombinant and native rat and human H(3) receptors (H(3)Rs) was studied using H(3)R-mediated [(35)S]GTPgamma[S] binding and [(3)H]-arachidonic acid release. Ciproxifan, an inverse agonist at the rat H(3)R (rH(3)R), decreased [(3)H]arachidonic acid release from CHO cells expressing moderate densities (approximately 200 - 300 fmol mg(-1) protein) of the human H(3)R (hH(3)R). This effect occurred with the same magnitude than at the rH(3)R. The expression of the hH(3)R was associated with an increase in [(35)S]GTPgamma[S] binding to membranes of CHO cells. Ciproxifan decreased [(35)S]GTPgamma[S] binding to membranes of CHO (hH(3)R) cells. Both effects were correlated to receptor density and revealed that constitutive activity of the hH(3)R, although lower than that of the rH(3)R in this assay, was again observed at physiological densities (<500 fmol mg(-1) protein). Ciproxifan was less potent at the human than the rat receptor, not only as an antagonist (K(i)=45 nM), but also as an inverse agonist (EC(50)=15 nM). Constitutive activity of the hH(3)R was also evidenced using inhibition of [(35)S]GTPgamma[S] binding by unlabelled GTPgammaS. The expression of the hH(3)R generated a high affinity binding for GTPgammaS which was increased by imetit, but partially decreased by ciproxifan, therefore acting as a partial inverse agonist. [(35)S]GTPgamma[S] binding to rat brain membranes was decreased in several regions by thioperamide, ciproxifan and FUB 465, three inverse agonists at the H(3)R, whose effects were blocked by proxyfan, a neutral antagonist. [(35)S]GTPgamma[S] binding was also decreased by an A(1)-adenosine receptor inverse agonist, but remained unchanged in the presence of inverse agonists at D(2)/D(3) dopamine, H(1) and H(2) histamine, alpha(2)-adrenergic and delta opioid receptors. In conclusion, the present study shows that the recombinant rat and human H(3) receptors expressed at physiological densities display constitutive activity and suggests that constitutive activity of native H(3)Rs is one of the highest among G-protein-coupled receptors present in rat brain.

Acute and chronic effects of methamphetamine on tele-methylhistamine levels in mouse brain: selective involvement of the D(2) and not D(3) receptor.

We have explored the role of endogenous dopamine in the control of histaminergic neuron activity in mouse brain regions evaluated by changes in tele-methylhistamine (t-MeHA) levels. In vitro, methamphetamine released [(3)H]noradrenaline but failed to release [(3)H]histamine from synaptosomes. In vivo, methamphetamine enhanced t-MeHA levels by about 2-fold with ED(50) values of approximately 1 mg/kg in caudate putamen, nucleus accumbens, cerebral cortex, and hypothalamus. This response selectively involved the D(2) and not the D(3) receptor as indicated by its blockade by haloperidol and by its persistence after administration of nafadotride, a D(3) receptor preferential ligand, or in (-/-) D(3) receptor-deficient mice. The t-MeHA response to methamphetamine was delayed compared with the locomotor-activating effect of this drug, suggesting that it is of compensatory nature. In agreement, ciproxifan, an inverse agonist known to enhance histamine neuron activity, decreased the hyperlocomotion induced by methamphetamine. Repeated methamphetamine administration resulted in the expected sensitization to the hyperlocomotor effect of the drug but did not modify either the ED(50) or the E(max) regarding t-MeHA levels. However, it resulted in an enhanced basal t-MeHA level (+30-40%), which was sustained for at least 11 days after withdrawal in hypothalamus, striatum, and cerebral cortex and suppressed by haloperidol. Hence, both the acute and chronic administration of methamphetamine enhance histamine neuron activity, presumably in a compensatory manner. Repeated methamphetamine administration also resulted in a modified balance in the opposite influences of dopamine and serotonin on histaminergic neurons as revealed by the enhanced response to haloperidol and abolished response to ketanserin, respectively.

Application of genomics to drug design: the example of the histamine H3 receptor.

The histamine H(3) receptor was characterized in the 1980s as an autoreceptor regulating histamine release in brain. Since then, selective drugs have been designed, many of them displaying a high potency in vivo, and used in many studies to delineate the implications of cerebral histaminergic systems in physiological functions such as arousal or cognitive functions. The recent cloning of the H(3) receptor, more than 15 years later, has allowed to start molecular studies that led to important findings for optimization of drug design. In agreement some ligands display distinct affinities for the recombinant rat and human H(3) receptors, a difference that we assign to two amino acids in the third transmembrane domain. In addition, H(3) autoreceptors present in the brain display high constitutive activity including in vivo. As a consequence, inverse agonists enhance histamine neuron activity and constitute a novel potential therapeutic approach to schizophrenia and Alzheimer's disease.

The rat H3 receptor: gene organization and multiple isoforms.

Genomic DNA analysis revealed that the coding region of the rat histamine H3 receptor comprises three exons interrupted by two introns of approximately 1 kb each. Several H3 receptor mRNA variants were identified by PCR and cDNA cloning and sequencing. Four variants generated by pseudo-intron retention/deletion at the level of the third intracellular loop were designated H3(445), H3(413), H3(410), and H3(397), according to the length of their deduced amino acid sequence and display differential tissue expression. When expressed in CHO-K1 or Cos-1 cells, the H3(445), H3(413), and H3(397) were found to generate specific 125I iodoproxyfan binding of similar pharmacological profile. In addition, we identified two short variants, termed H3(nf1) and H3(nf2), which correspond to frame shift and stop codon interposition, respectively, and are presumably nonfunctional, among which H3(nf2) displays brain expression similar to that of the longer isoforms.

Chromosomal mapping and organization of the human histamine H3 receptor gene.

The histamine H3 receptor (H3R) was recently cloned, and two isoforms, termed H3L and H3S, differing in the third intracytosolic loop, were isolated but the chromosomal mapping and organization of its gene remained unknown. PCR analysis of a human x rodent cell hybrid panel indicated that the H3R gene is located in the telomeric region of chromosome 20q. Alignment of human H3R cDNA sequences with DNA sequences of this chromosome revealed that its coding region comprises three exons interrupted by two introns located in the second transmembrane domain (TM2) and second intracytosolic loop, respectively. Thus the organization of the H3R gene indicates that the H3L and H3S isoforms, that we characterized not only in rodents but also in humans, are generated by retention and deletion, respectively, of a pseudo-intron located in the third intracytosolic loop.

High constitutive activity of native H3 receptors regulates histamine neurons in brain.

Some G-protein-coupled receptors display 'constitutive activity', that is, spontaneous activity in the absence of agonist. This means that a proportion of the receptor population spontaneously undergoes an allosteric transition, leading to a conformation that can bind G proteins. The process has been shown to occur with recombinant receptors expressed at high density, and/or mutated, but also non-mutated recombinant receptors expressed at physiological concentrations. Transgenic mice that express a constitutively active mutant of the beta2-adrenergic receptor display cardiac anomalies; and spontaneous receptor mutations leading to constitutive activity are at the origin of some human diseases. Nevertheless, this process has not previously been found to occur in animals expressing normal levels of receptor. Here we show that two isoforms of the recombinant rat H3 receptor display high constitutive activity. Using drugs that abrogate this activity ('inverse agonists') and a drug that opposes both agonists and inverse agonists ('neutral antagonist'), we show that constitutive activity of native H3 receptors is present in rodent brain and that it controls histaminergic neuron activity in vivo. Inverse agonists may therefore find therapeutic applications, even in the case of diseases involving non-mutated receptors expressed at normal levels.

Distinct pharmacology of rat and human histamine H(3) receptors: role of two amino acids in the third transmembrane domain.

Starting from the sequence of the human histamine H(3) receptor (hH(3)R) cDNA, we have cloned the corresponding rat cDNA. Whereas the two deduced proteins show 93.5% overall homology and differ only by five amino acid residues at the level of the transmembrane domains (TMs), some ligands displayed distinct affinities. Thioperamide and ciproxifan were about 10 fold more potent at the rat than at the human receptor, whereas FUB 349 displayed a reverse preference. Histamine, (R)alpha-methylhistamine, proxyfan or clobenpropit were nearly equipotent at H(3) receptors of both species. The inverse discrimination patterns of ciproxifan and FUB 349 were partially changed by mutation of one amino acid (V122A), and fully abolished by mutation of two amino acids (A119T and V122A), in TM3 of the rH(3)R located in the vicinity of Asp(114) purported to salt-link the ammonium group of histamine. Therefore, these two residues appear to be responsible for the distinct pharmacology of the H(3)R in the two species.

Cloning and cerebral expression of the guinea pig histamine H3 receptor: evidence for two isoforms.

We cloned the full length guinea pig H3 receptor cDNA using RT-PCR amplification with primers from the human receptor and templates from brain areas. Evidence was obtained for two isoforms, designated H3L and H3S, differing by a 30 amino acid stretch within the third cytosolic loop, presumably generated by alternative splicing. In situ hybridization using a selective cRNA probe showed the gene transcripts to be highly expressed in discrete neuronal populations, e.g. pyramidal cells in the cerebral cortex or cerebellar Purkinje cells, in some instances already known to express other histamine receptor subtypes.

Atypical neuroleptics enhance histamine turnover in brain via 5-Hydroxytryptamine2A receptor blockade.

Clozapine and olanzapine behave as weak H3-receptor antagonists in vitro with Ki values around 1 and 50 microM, respectively. Despite these modest apparent affinities, both compounds given orally to mice, nearly doubled steady-state tele-methylhistamine levels in brain, with ED50 values as low as 1 and 3 mg/kg, respectively, an effect comparable to those of potent H3-receptor antagonists. This effect corresponded to an enhancement of histamine turnover rate from 45 to 73 ng/g/h as measured in the case of olanzapine using the pargyline test. Other antipsychotics displaying, such as clozapine and olanzapine, high 5-hydroxytryptamine (5-HT)2A receptor antagonist potency, i.e., risperidone, thioridazine, seroquel, and iloperidone, also enhanced markedly tele-methylhistamine levels. This effect was 1) additive with that of a pure H3-receptor antagonist, ciproxifan, 2) mimicked by a 5-HT2A receptor antagonist, ketanserin, 3) reversed by a 5-HT2A receptor agonist, DOI, 4) not shared by antipsychotics with low affinity for the 5-HT2A receptor, i.e., haloperidol, sulpiride, raclopride, or remoxipride that, on the contrary, tended to reduce tele-methylhistamine levels. We conclude that in contrast to "typical" antipsychotics, "atypical" antipsychotics stimulate histamine neuron activity via blockade of the 5-HT2A receptor in vivo. This effect does not appear to account for their reduced extrapyramidal side-effects but may underlie their pro-cognitive properties.

Detailed mapping of the histamine H2 receptor and its gene transcripts in guinea-pig brain.

Autoradiographic studies of the distribution of the histamine H2 receptor and its messenger RNAs were performed on serial frontal and a few sagittal sections of guinea-pig brain using [(125)I]iodoaminopotentidine for radioligand binding and a 33P-labelled complementary RNA probe for in situ hybridization, respectively. Both probes were validated by assessing non-specific labelling using non-radioactive competing H2 receptor ligands and a sense probe for binding sites and gene transcripts, respectively. In some areas, e.g., cerebral cortex, hippocampal complex or cerebellum, such studies were completed by identification of neurons expressing the H2 receptor messenger RNAs on emulsion-dipped sections. Nissl-stained sections from comparable levels were used to localize brain structures. In many brain areas, the distribution of the H2 receptor and its messenger RNAs appeared to parallel that known for histaminergic axons. For instance. high levels of both H2 receptor markers were detected in striatal and limbic areas known to receive abundant histaminergic projections. In contrast, in septum, hypothalamic, pontine and several thalamic nuclei, a comparatively low density of both H2 receptor markers was detected, suggesting that histamine actions in these areas are mediated by H1 and/or H3 receptors. Generally, the distribution of H2 receptor messenger RNA correlates well with that of [(125)I]iodoaminopotentidine binding sites, although some differences were observed. In a few regions (e.g., substantia nigra, locus coeruleus) high or moderate densities of binding sites were accompanied by a much more restricted expression of H2 receptor transcripts. Conversely, the mammillary region and the pontine nucleus exhibited higher levels of hybridization than of binding sites. In hippocampus, cerebral and cerebellar cortex there was a selective localization of the H2 receptor messenger RNA in the granule cells of dentate gyrus, pyramidal cells of the Ammon's horn and cerebral cortex, and Purkinje cells of cerebellum, whereas [(125)I]iodoaminopotentidine binding sites were located in layers where the dendritic trees of these messenger RNA-expressing neurons extend. The same discrepancy between messenger RNAs and binding sites suggests that striatonigral endings are endowed with the H2 receptor. The histamine H1 and H2 receptors both appear to be present in several brain areas, in some cases in a way suggesting their potential co-expression by the same neuronal populations, e.g., in granule and pyramidal cells in the hippocampal formation. This co-expression accounts for synergic responses, e.g., on cAMP generation, previously observed upon co-stimulation of both receptor subtypes. The widespread distribution of the H2 receptor, namely in thalamic nuclei or in telencephalic areas such as most layers of the cerebral cortex, together with its excitatory role previously established in electrophysiological studies, support its alleged function in mediating the histamine-driven control of arousal mechanisms. In addition, the detection of H2 receptor expression in brainstem areas from which other monoaminergic pathways involved in the control of states of sleep and wakefulness emanate, e.g., several raphe nuclei, locus coeruleus or substantia innominata, suggests possible interrelationships between all of these systems with highly divergent projections to the thalamus and telencephalon. The present mapping of the H2 receptor and its gene transcripts should facilitate neurochemical, neurophysiological and behavioural studies aimed at clarifying the role of histaminergic systems in brain.

The guinea pig histamine H2 receptor: gene cloning, tissue expression and chromosomal localization of its human counterpart.

The guinea pig is the prototypic animal species for the histamine H2 receptor. Using a strategy based upon nucleotide sequence homology and starting from the sequence of the rat histamine H2 receptor (Ruat et al., Biochem. Biophys. Res. Commun. 1991, 179: 1470-78), we have cloned an intronless highly homologous DNA very likely encoding the guinea pig H2 receptor. The encoded 359 amino acid protein displays 83 to 86% identity with the rat-, human- or dog-H2 receptors. Northern blot analysis identified a single transcript of 4.6 kb in peripheral tissues and brain areas in which the presence of the H2 receptor had been revealed previously by either photoaffinity labeling or binding studies. In brain, the distribution of transcripts, established by either Northern blots or in situ hybridization studies, was consistent with the localization of the H2-receptor. In addition, using Southern analysis of a chromosome mapping panel constructed from human x hamster hybridomas, we assigned the H2 receptor gene to human chromosome 5.

Guinea pig histamine H1 receptor. I. Gene cloning, characterization, and tissue expression revealed by in situ hybridization.

An intronless DNA encoding the guinea pig H1 receptor was cloned from a genomic library using probes derived from the bovine H1 receptor. It encodes a protein of 488 amino acids with a calculated molecular mass of 55,619 daltons compared with a size of 56-68 kDa for the photoaffinity-labeled receptor as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The protein displays a 66% homology with the bovine receptor. Stable expression of the H1 receptor, characterized by the appearance of [3H]mepyramine binding sites with a pharmacology similar to that of the native H1 receptor, was obtained following transfection of Chinese hamster ovary cells. Southern blot analysis, using a variety of restriction enzymes, did not provide any evidence of multiple H1 isoreceptors. Northern blot analysis of a variety of guinea pig peripheral or cerebral tissues identified, in most cases, a single transcript of 3.3 kb, but also, in some tissues, a second transcript of 3.7 kb, possibly generated by the use of different promoter or polyadenylation sites or corresponding to a transcript from a distinct gene. In situ hybridization studies showed the highly contrasted cerebral expression of H1-receptor gene transcripts, which was compared with autoradiographic receptor localization. This allowed the identification of some major cell populations expressing the H1 receptor, e.g., Purkinje cells in cerebellum or pyramidal cells in the hippocampal complex.

Guinea pig histamine H1 receptor. II. Stable expression in Chinese hamster ovary cells reveals the interaction with three major signal transduction pathways.

A cDNA encoding a guinea pig histamine H1 receptor was stably expressed in Chinese hamster ovary (CHO) cells. In one resulting clone, named CHO(H1), the H1 receptor was found to be coupled to several major signal transduction pathways. In each case the involvement of a Gi/Go protein with pertussis toxin (PTX) was assessed, as well as the influence of extracellular Ca2+ and of protein kinase C activation by phorbol 12-myristate 13-acetate (PMA). Histamine induced, in a PTX- and PMA-insensitive manner, a biphasic increase in the intracellular Ca2+ level of which only the second sustained phase was dependent on the extracellular Ca2+ level. Histamine also caused a threefold elevation of inositol phosphate production, which was PTX-insensitive, but slightly inhibited by PMA and reduced by 75% in the absence of extracellular Ca2+. Histamine also caused a massive release of arachidonic acid, which occurred in a Ca(2+)- and PMA-sensitive manner, probably through the activation of a cytosolic phospholipase A2, which partly involves coupling to a PTX-sensitive G protein. In comparison, in HeLa cells endowed with a native H1 receptor, the histamine-induced arachidonic acid release was also Ca(2+)- and PMA-sensitive, but totally PTX-insensitive. Finally, in CHO(H1) cells, histamine in very low concentrations potentiated the cyclic AMP accumulation induced by forskolin. This response appeared to be insensitive to PTX, extracellular Ca2+, and PMA.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning, characterization, and localization of a high-affinity serotonin receptor (5-HT7) activating cAMP formation.

By using a strategy based on nucleotide sequence homology, we have cloned a cDNA encoding a functional serotonin (5-HT) receptor. The deduced amino acid sequence of the 5-HT7 receptor displays limited homology with that of other 5-HT receptors. In addition to the seven stretches of hydrophobic amino acids that characterize the superfamily of receptors interacting with guanine nucleotide-binding proteins, the 448-aa sequence of the 5-HT7 receptor contains a hydrophobic domain located at its N-terminal end. Genomic analysis indicated the presence of introns interrupting the coding sequence. The 5-HT7 receptor, stably expressed in transfected CHO cells, bound [3H]5-HT with high affinity (Kd = 1 nM), like receptors of the 5-HT1 subfamily from which, however, it was clearly distinguished by its pharmacology. 5-HT in nanomolar concentrations stimulated cAMP accumulation in these CHO cells by approximately 10-fold, whereas lysergic acid diethylamide displayed low intrinsic agonist activity. These various properties differentiate the 5-HT7 receptor from the four other subfamilies of mammalian 5-HT receptors (i.e., the 5-HT1-, 5-HT2-, 5-HT3-, and 5-HT4-like subfamilies) and, therefore, appear to define another receptor subfamily. Northern blot and in situ hybridization analyses showed the 5-HT7 transcripts to be expressed in discrete areas of the limbic brain (e.g., pyramidal hippocampus cells, tenia tecta, amygdaloid, or mammillary nuclei), suggesting that the receptor mediates serotoninergic controls in functions like mood, learning, or neuroendocrine and vegetative behaviors.

A novel rat serotonin (5-HT6) receptor: molecular cloning, localization and stimulation of cAMP accumulation.

Using a strategy based upon nucleotide sequence homology and starting from the sequence of the rat histamine H2 receptor (Ruat et al., Biochem. Biophys. Res. Commun. 1991, 179, 1470-1478), we have cloned a rat cDNA encoding a functional serotonin receptor (5-HT6). Its coding sequence corresponds to a glycoprotein of 436 amino acids displaying significant homology with other cloned monoaminergic receptors, e.g., various serotonin receptors. Genomic analysis of its gene indicated the presence of at least one intron. The major transcript of the 5-HT6 receptor gene has a size of approximately 4.1 kb but another minor 3.2 kb transcript was also evidenced. The highest expression, detected by Northern blot analysis as well as by in situ hybridization occurs in various serotoninergic areas of rat or guinea pig brain such as striatum, olfactory tubercle, nucleus accumbens and hippocampus, but a faint expression is also detectable in rat stomach. When transiently expressed in transfected COS-7 cells the 5-HT6 receptor appears to be positively coupled to cyclic AMP production.

Hormone-associated variation of the glycan microheterogeneity pattern of human sex steroid-binding protein (hSBP).

hSBP is a steroid-binding protein (human) whose serum concentration is increased by estrogens and decreased by androgens. This regulation is independent of a direct effect on the hSBP gene transcription. The purpose of this work was to study the glycan microheterogeneous composition of the mature protein under physiological estrogen stimulation, by means of crossed affinoimmunoelectrophoresis using concanavalin-A. In men hSBP always divided into 2 fractions, both retarded. In women hSBP showed two other components, still more retarded. An explanation for these differences is given and the role of the glycan moiety of hSBP is discussed.