β‐arrestin1 phosphorylation by GRK5 regulates G protein‐independent 5‐HT4 receptor signalling

G protein‐coupled receptors (GPCRs) have been found to trigger G protein‐independent signalling. However, the regulation of G protein‐independent pathways, especially their desensitization, is poorly characterized. Here, we show that the G protein‐independent 5‐HT₄ receptor (5‐HT₄R)‐operated Src/ERK (extracellular signal‐regulated kinase) pathway, but not the G_s pathway, is inhibited by GPCR kinase 5 (GRK5), physically associated with the proximal region of receptor’ C‐terminus in both human embryonic kidney (HEK)‐293 cells and colliculi neurons. This inhibition required two sequences of events: the association of β–arrestin1 to a phosphorylated serine/threonine cluster located within the receptor C‐t domain and the phosphorylation, by GRK5, of β–arrestin1 (at Ser⁴¹²) bound to the receptor. Phosphorylated β‐arrestin1 in turn prevented activation of Src constitutively bound to 5‐HT₄Rs, a necessary step in receptor‐stimulated ERK signalling. This is the first demonstration that β‐arrestin1 phosphorylation by GRK5 regulates G protein‐independent signalling.     

The role of 5‐HT2A receptor and 5‐HT2A/5‐HT1A receptor interaction in the suppression of catalepsy

In the present study, the 5‐HT_2A and 5‐HT_1A receptors functional activity and 5‐HT_2A receptor gene expression were examined in the brain of ASC/Icg and congenic AKR.CBAD13Mit76C mouse strains (genetically predisposed to catalepsy) in comparison with the parental catalepsy‐resistant AKR/J and catalepsy‐prone CBA/Lac mouse strains. The significantly reduced 5‐HT_2A receptor functional activity along with decreased 5‐HT_2A receptor gene expression in the frontal cortex was found in all mice predisposed to catalepsy compared with catalepsy‐resistant AKR/J. 5‐HT_2A agonist DOI (0.5 and 1 mg/kg, i.p.) significantly reduced catalepsy in ASC/Icg and CBA/Lac, but not in AKR.CBAD13Mit76C mice. Essential increase in 5‐HT_1A receptor functional activity was shown in catalepsy‐prone mouse strains in comparison with catalepsy‐resistant AKR/J mice. However, in AKR.CBAD13Mit76C mice it was lower than in ASC/Icg and CBA/Lac mice. The inter‐relation between 5‐HT_2A and 5‐HT_1A receptors in the regulation of catalepsy was suggested. This suggestion was confirmed by prevention of DOI anticataleptic effect in ASC/Icg and CBA/Lac mice by pretreatment with 5‐HT_1A receptor antagonist p‐MPPI (3 mg/kg, i.p.). At the same time, the activation of 5‐HT_2A receptor led to the essential suppression of 5‐HT_1A receptor functional activity, indicating the opposite effect of 5‐HT_2A receptor on pre‐ and postsynaptic 5‐HT_1A receptors. Thus, 5‐HT_2A/5‐HT_1A receptor interaction in the mechanism of catalepsy suppression in mice was shown.     

Sequence complexity of the S receptor kinase gene family in Brassica

A genomic library from an S ₂₉/S ₂₉ self-incompatible genotype of Brassica oleracea was screened with a probe carrying part of the catalytic domain of a Brassica S-receptor kinase (SRK)-like gene. Six positive phage clones with varying hybridisation intensities (K1 to K6) were purified and characterised. A 650–700 by region corresponding to the probe was excised from each clone and sequenced. DNA and predicted protein sequence comparisons based on a multiple alignment identified K5 as a pseudogene, whereas the others could encode functional proteins. K3 was found to have lost an intron from its genomic sequence. The six genes display different degrees of sequence similarity and form two distinct clusters in a dendrogram. The 98% similarity between K4 and K6, which extends across intron sequences, suggests that these might be very recently diverged alleles or daughters of a duplication. In addition, K2 showed a comparably high similarity to the probe. Clones K1, K3 and K5 cross-hybridised with an SLG ₂₉ cDNA probe, indicating the presence of upstream receptor domains homologous to the Brassica SLG gene. This suggests that the previously reported S sequence complexity may be ascribed to a large receptor kinase gene family.     

Serotonergic activation of 5HT1A and 5HT2 receptors modulates sexually dimorphic communication signals in the weakly electric fish Apteronotus leptorhynchus

Serotonin modulates agonistic and reproductive behavior across vertebrate species. 5HT_1A and 5HT_1B receptors mediate many serotonergic effects on social behavior, but other receptors, including 5HT₂ receptors, may also contribute. We investigated serotonergic regulation of electrocommunication signals in the weakly electric fish Apteronotus leptorhynchus. During social interactions, these fish modulate their electric organ discharges (EODs) to produce signals known as chirps. Males chirp more than females and produce two chirp types. Males produce high-frequency chirps as courtship signals; whereas both sexes produce low-frequency chirps during same-sex interactions. Serotonergic innervation of the prepacemaker nucleus, which controls chirping, is more robust in females than males. Serotonin inhibits chirping and may contribute to sexual dimorphism and individual variation in chirping. We elicited chirps with EOD playbacks and pharmacologically manipulated serotonin receptors to determine which receptors regulated chirping. We also asked whether serotonin receptor activation generally modulated chirping or more specifically targeted particular chirp types. Agonists and antagonists of 5HT_1B/1D receptors (CP-94253 and GR-125743) did not affect chirping. The 5HT_1A receptor agonist 8OH-DPAT specifically increased production of high-frequency chirps. The 5HT₂ receptor agonist DOI decreased chirping. Receptor antagonists (WAY-100635 and MDL-11939) opposed the effects of their corresponding agonists. These results suggest that serotonergic inhibition of chirping may be mediated by 5HT₂ receptors, but that serotonergic activation of 5HT_1A receptors specifically increases the production of high-frequency chirps. The enhancement of chirping by 5HT_1A receptors may result from interactions with cortisol and/or arginine vasotocin, which similarly enhance chirping and are influenced by 5HT_1A activity in other systems.     

Transient expression of serotonin 5‐HT4 receptors in the mouse developing thalamocortical projections

The serotonin 5‐HT₄ receptor (5‐HT₄‐R) is an unusually complex G‐protein coupled receptor that is likely to play important roles in brain development and that may underlie the comorbidity of central and peripheral abnormalities in some developmental disorders. We studied the expression of 5‐HT₄‐Rs in the developing mouse forebrain at embryonic days 13, 15, 17, and at postnatal days 3 and 14 by using immunohistochemistry, tract tracing, and quantitative RT‐PCR. The developing thalamocortical projections transiently expressed 5‐HT₄‐Rs in the embryonic brain and the 5‐HT₄‐R expression in the forebrain changed from axonal to somatic around birth. From embryonic days 13–17, the forebrain mRNA levels of the 5‐HT_4(a)‐R and 5‐HT_4(b)‐R splice variants increased nine‐ and fivefold, respectively, whereas the levels of the 5‐HT_4(e)‐R and 5‐HT_4(f)‐R variants remained relatively low throughout the studied period of embryonic development. These results suggest that during development 5‐HT₄‐R expression undergoes a dynamic regulation and that this regulation may be important for the normal development of sensory and limbic processing. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2010.     

Larvae of the small white butterfly,Pieris rapae,express a novel serotonin receptor

The biogenic amine serotonin ( 5‐hydroxytryptamine, 5‐HT) is a neurotransmitter in vertebrates and invertebrates. It acts in regulation and modulation of many physiological and behavioral processes through G‐protein‐coupled receptors. Five 5‐HT receptor subtypes have been reported in Drosophila that share high similarity with mammalian 5‐HT_1A, 5‐HT_1B, 5‐HT_2A, 5‐HT_2B, and 5‐HT₇ receptors. We isolated a cDNA (Pr5‐HT₈) from larval Pieris rapae, which shares relatively low similarity to the known 5‐HT receptor classes. After heterologous expression in HEK293 cells, Pr5‐HT₈ mediated increased [Ca²⁺]_i in response to low concentrations (< 10 nM) of 5‐HT. The receptor did not affect [cAMP]_i even at high concentrations (> 10 μM) of 5‐HT. Dopamine, octopamine, and tyramine did not influence receptor signaling. Pr5‐HT₈ was also activated by various 5‐HT receptor agonists including 5‐methoxytryptamine, (±)‐8‐Hydroxy‐2‐(dipropylamino) tetralin, and 5‐carboxamidotryptamine. Methiothepin, a non‐selective 5‐HT receptor antagonist, activated Pr5‐HT₈. WAY 10635, a 5‐HT_1A antagonist, but not SB‐269970, SB‐216641, or RS‐127445, inhibited 5‐HT‐induced [Ca²⁺]_i increases. We infer that Pr5‐HT₈ represents the first recognized member of a novel 5‐HT receptor class with a unique pharmacological profile. We found orthologs of Pr5‐HT₈ in some insect pests and vectors such as beetles and mosquitoes, but not in the genomes of honeybee or parasitoid wasps. This is likely to be an invertebrate‐specific receptor because there were no similar receptors in mammals.

     

5‐HT2A Receptor Gene Promoter Polymorphism in Relation to Abdominal Obesity and Cortisol

Objective: There is considerable evidence that cortisol secretion is associated with obesity. The regulation of the 5‐hydroxytryptamine receptor 2A (5‐HT_2A) gene might play an essential role because it is involved in the control of cortisol secretion. Therefore, we examined the potential impact of the 5‐HT_2A ?1438G/A promoter polymorphism on obesity and estimates of insulin, glucose, and lipid metabolism as well as circulating hormones, including salivary cortisol, in 284 unrelated Swedish men born in 1944. Research Methods and Procedures: The subjects were genotyped by using polymerase chain reaction amplification of the promoter region of the gene for 5‐HT_2A followed by digestion of the reaction product with the restriction enzyme MspI. Results: The frequencies were 0.39 for allele ?1438A and 0.61 for allele ?1438G. Homozygotes for the ?1438G allele had, in comparison with ?1438A/A subjects, higher body mass index, waist‐to‐hip ratio, and abdominal sagittal diameter. Moreover, cortisol escape from 0.25‐mg dexamethasone suppression was found in subjects with the ?1438A/G genotype. Serum leptin, fasting insulin, and glucose, as well as serum lipids, were not different across the ?1438G/A genotype groups. Discussion: From these results, we suggest the possibility that an abnormal production rate of the 5‐HT_2A gene product might lead to the development of abdominal obesity. The pathophysiology could involve stress factors that destabilize the serotonin‐hypothalamic‐pituitary‐adrenal system in those with genetic vulnerability in the serotonin receptor gene.     

Antagonism of 5-HT1A receptors uncovers an excitatory effect of SSRIs on 5-HT neuronal activity, an action probably mediated by 5-HT7 receptors

Both microdialysis and electrophysiology were used to investigate whether another serotonin (5‐HT) receptor subtype next to the 5‐HT_1A autoreceptor is involved in the acute effects of a selective serotonin reuptake inhibitor on 5‐HT neuronal activity. On the basis of a previous study, we decided to investigate the involvement of the 5‐HT₇ receptors. Experiments were performed with the specific 5‐HT₇ antagonist SB 258741 and the putative 5‐HT₇ agonist AS19. In this study WAY 100.635 was used to block 5‐HT_1A receptors. Systemic administration of SB 258741 significantly reduced the effect of combined selective serotonin reuptake inhibitor and WAY 100.635 administration on extracellular 5‐HT in the ventral hippocampus as well as 5‐HT neuronal firing in the dorsal raphe nucleus. In the microdialysis study, co‐administration of AS19 and WAY 100.635 showed a biphasic effect on extracellular 5‐HT in ventral hippocampus, hinting at opposed 5‐HT₇ receptor mediated effects. In the electrophysiological experiments, systemic administration of AS19 alone displayed a bell‐shaped dose–effect curve: moderately increasing 5‐HT neuronal firing at lower doses while decreasing it at higher doses. SB 258741 was capable of blocking the effect of AS19 at a low dose. This is consistent with the pharmacological profile of AS19, displaying high affinity for 5‐HT₇ receptors and moderate affinity for 5‐HT_1A receptors. The data are in support of an excitatory effect of selective serotonin reuptake inhibitors on 5‐HT neuronal activity mediated by 5‐HT₇ receptors. It can be speculated, that the restoration of 5‐HT neuronal firing upon chronic antidepressant treatment, which is generally attributed to desensitization of 5‐HT_1A receptors alone, in fact results from a shift in balance between 5‐HT_1A and 5‐HT₇ receptor function.     

Cloning and sequencing of the gene for the DNA-binding 17 K protein of Escherichia coli

The skp gene encoding the 17 K protein, a basic DNA-binding nucleoid-associated protein of Escherichia coli, was cloned as part of a 2.3-kb genomic fragment. The gene was sequenced and a polypeptide of 161 amino acids (aa) was deduced from the nucleotide sequence. The primary translation product was processed by cutting off the N-terminal 20 aa residues, yielding a mature polypeptide of 141 aa. The M_r of the mature polypeptide was 15674. An E. coli transformant containing the skp gene on the plasmid pGAH317 was shown to overproduce the gene product some 20-fold.     

Characterisation of 5-HT3C, 5-HT3D and 5-HT3E receptor subunits: evolution, distribution and function

The 5‐HT₃ receptor is a member of the ‘Cys‐loop’ family of ligand‐gated ion channels that mediate fast excitatory and inhibitory transmission in the nervous system. Current evidence points towards native 5‐HT₃ receptors originating from homomeric assemblies of 5‐HT_3A or heteromeric assembly of 5‐HT_3A and 5‐HT_3B. Novel genes encoding 5‐HT_3C, 5‐HT_3D, and 5‐HT_3E have recently been described but the functional importance of these proteins is unknown. In the present study, in silico analysis (confirmed by partial cloning) indicated that 5‐HT_3C, 5‐HT_3D, and 5‐HT_3E are not human–specific as previously reported: they are conserved in multiple mammalian species but are absent in rodents. Expression profiles of the novel human genes indicated high levels in the gastrointestinal tract but also in the brain, Dorsal Root Ganglion (DRG) and other tissues. Following the demonstration that these subunits are expressed at the cell membrane, the functional properties of the recombinant human subunits were investigated using patch clamp electrophysiology. 5‐HT_3C, 5‐HT_3D, and 5‐HT_3E were all non‐functional when expressed alone. Co‐transfection studies to determine potential novel heteromeric receptor interactions with 5‐HT_3A demonstrated that the expression or function of the receptor was modified by 5‐HT_3C and 5‐HT_3E, but not 5‐HT_3D. The lack of distinct effects on current rectification, kinetics or pharmacology of 5‐HT_3A receptors does not however provide unequivocal evidence to support a direct contribution of 5‐HT_3C or 5‐HT_3E to the lining of the ion channel pore of novel heteromeric receptors. The functional and pharmacological contributions of these novel subunits to human biology and diseases such as irritable bowel syndrome for which 5‐HT₃ receptor antagonists have major clinical usage, therefore remains to be fully determined.     

Structural basis of ligand recognition in 5-HT3 receptors

The 5‐HT₃ receptor is a pentameric serotonin‐gated ion channel, which mediates rapid excitatory neurotransmission and is the target of a therapeutically important class of anti‐emetic drugs, such as granisetron. We report crystal structures of a binding protein engineered to recognize the agonist serotonin and the antagonist granisetron with affinities comparable to the 5‐HT₃ receptor. In the serotonin‐bound structure, we observe hydrophilic interactions with loop E‐binding site residues, which might enable transitions to channel opening. In the granisetron‐bound structure, we observe a critical cation–π interaction between the indazole moiety of the ligand and a cationic centre in loop D, which is uniquely present in the 5‐HT₃ receptor. We use a series of chemically tuned granisetron analogues to demonstrate the energetic contribution of this electrostatic interaction to high‐affinity ligand binding in the human 5‐HT₃ receptor. Our study offers the first structural perspective on recognition of serotonin and antagonism by anti‐emetics in the 5‐HT₃ receptor.     

Effects of 5‐HT1 and 5‐HT 2 Receptor Agonists on Electromagnetic Field‐Induced Analgesia in Rats

Much evidence demonstrates the antinociceptive effect of magnetic fields (MFs). However, the analgesic action mechanism of the electromagnetic field (EMF) is not exactly understood. The aim of the present study was to investigate the effects of 5‐HT₁ and 5‐HT₂ receptor agonists (serotonin HCl and 2,5‐dimethoxy‐4‐iodoamphetamine [DOI] hydrochloride) on EMF‐induced analgesia. In total, 66 adult male Wistar albino rats with an average body mass of 225 ± 13 g were used in this study. The animals were subjected to repeated exposures of alternating 50 Hz and 5 mT EMF for 2 h a day for 15 days. Prior to analgesia tests, serotonin HCl (5‐HT₁ agonist) 4 mg/kg, WAY 100635 (5‐HT₁ antagonist) 0.04 mg/kg, DOI hydrochloride (5‐HT₂ receptor agonist) 4 mg/kg, and SB 204741 (5‐HT₂ antagonist) 0.5 mg/kg doses were injected into rats. For statistical analysis of the data, analysis of variance was used and multiple comparisons were determined by Tukey’s test. Administration of serotonin HCl MF (5 mT)‐exposed rats produced a significant increase in percent maximal possible effect (% MPE) as compared with EMF group (P < 0.05). On the contrary, injection of WAY 100635 to MF‐exposed rats produced a significant decrease in analgesic activity (P < 0.05). Similarly, the administration of DOI hydrochloride significantly increased % MPE values as compared with the EMF group while SB 204741 reduced it (P < 0.05). In conclusion, our results suggested that serotonin 5‐HT₁ and 5‐HT₂ receptors play an important role in EMF‐induced analgesia; however, further research studies are necessary to understand the mechanism. Bioelectromagnetics. 2019;40:319–330. © 2019 Bioelectromagnetics Society.     

Sequence analysis of the 5′-untranslated region of the human H1 histamine receptor-encoding gene

A clone containing the H₁ histamine receptor (H₁HR)-encoding gene was isolated from a human genomic DNA library. The 5′-UTR of the H₁HR gene reported here differs upstream from bp −142 from that reported previously [Fukui et al., Biochem. Biophys. Res. Comm. 201 (1994) 894–901]. PCR amplification utilizing primer pairs derived from the 5′-UTR reported herein amplified a DNA fragment of the expected size from human genomic DNA whereas 5′-UTR primers derived from the Fukui et al. sequence did not yield a PCR product. The 5′-UTR of H₁HR contains potential TATA and CCAAT boxes, a CACCC sequence, potential GREs and other DNA-binding motifs.     

Complete cDNA Sequence, Genomic Structure, and Chromosomal Localization of the LPA Receptor Gene,lpA1/vzg-1/Gpcr26

Thelp_A1/Gpcr26locus encodes the first cloned and identified G-protein-coupled receptor that specifically interacts with lysophosphatidic acid. A murine full-length cDNA of size consistent with that seen on Northern blots (3.7 kb) was determined using 3′ rapid amplification of cDNA ends. Analysis of genomic clones revealed that the gene is divided into five exons, with one intron inserted in the coding region for transmembrane domain VI and one exon encoding the divergent 5′ sequence in another published cDNA clone variant (orphan receptor mrec1.3). This structure differs from the intronless coding region for a homologous receptor,Edg1,but is identical to another more similar orphan receptor (lp_A2) that has been deposited with GenBank. Using backcross analysis, both exons 1 and 4 mapped to a proximal region of murine Chromosome 4 indistinguishable from the vacillans gene. Exon 4 also mapped to a second locus on proximal Chromosome 6 inMus spretus,and this partial duplication was confirmed by Southern blot. The genomic structure indicates a distinct, divergent evolutionary lineage for thevzg-1/lp_A1subfamily of receptors compared to those of homologous orphan receptor genes.