Click
here to close Hello! We notice that
you are using Internet Explorer, which is not supported by Echinobase
and may cause the site to display incorrectly. We suggest using a
current version of Chrome,
FireFox,
or Safari.
???displayArticle.abstract??? Serotonin (5-HT) is an important neurotransmitter and neuromodulator that controls a variety of sensory and motor functions through 5-HT receptors (5-HTRs). The 5-HT4R subfamily is linked to Gs proteins, which activate adenylyl cyclases (ACs), and is involved in many responses in peripheral organs. In this study, the 5-HT4R from Apostichopus japonicus (Aj5-HT4R) was identified and characterised. The cloned full-length Aj5-HT4R cDNA is 1,544 bp long and contains an open reading frame 1,011 bp in length encoding 336 amino acid proteins. Bioinformatics analysis of the Aj5-HT4R protein indicated this receptor was a member of class A G protein coupled receptor (GPCR) family. Further experiments using Aj5-HT4R-transfected HEK293 cells demonstrated that treatment with 5-HT triggered a significant increase in intracellular cAMP level in a dose-dependent manner and induced a rapid internalisation of Aj5-HT4R fused with enhanced green fluorescent protein (Aj5-HT4R-EGFP) from the cell surface into the cytoplasm. In addition, the transcriptional profiles of Aj5-HT4R in aestivating A. japonicas and phosphofructokinase (AjPFK) in 5-HT administrated A. japonicus have been analysed by real-time PCR assays. Results have led to a basic understanding of Aj5-HT4R in A. japonicus, and provide a foundation for further exploration of the cell signaling and regulatory functions of this receptor.
Figure 1. Aj5-HT4R cDNA sequence and deduced amino acid sequence.The seven transmembrane domains (TM1-TM7) are noted by the black underline. The N-glycosylated sites are highlighted in gray. The phosphorylation sites are labeled in box with full lines. The initiation codon (ATG) and the termination codon (TGA) are shown in bold. The potential polyadenylation signal (AATAAA) is noted by the double underscore. The numbers on the left refer to the position of the nucleotides and the amino acids.
Figure 2. Alignment of the deduced Aj5-HT4R amino acid sequence with sequences from other species.Sequences of Strongylocentrotus purpuratus 5-HT4 receptor (Sp5-HT4R), Aplysia californica 5-HT4 receptor (Ac5-HT4R), Austrofundulus limnaeus 5-HT4 receptor (Al5-HT4R), Cavia porcellus 5-HT4 receptor (Cp5-HT4R) and Homo sapiens 5-HT4 receptor (Hs5-HT4R) were obtained from GenBank, along with a list of accession numbers (Suppl, Table S1). Alignment was generated using ClustalW and color align property was generated using Sequence Manipulation Suite online. The seven transmembrane domains (TM1–TM7) are marked with a black horizontal line above the sequence alignment. The three extracellular (EC) and three intracellular (IC) rings are noted above the sequence alignment. Black ↓ indicates the conserved cysteine residues. Percentage of sequences that must agree for identity or similarity coloring was set as 80%.
Figure 3. Predicted Aj5-HT4R protein structure and respective domain.(A) Predicted 3D structure of the Aj5-HT4R protein. Seven transmembrane domains (TM1-TM7), three extracellular (EC) rings and three intracellular (IC) rings are marked. The predicted 3D structure of the Aj5-HT4R protein was generated from SWISS-MODEL. (B) Aj5-HT4R protein binding domain and transmembrane region. The Dashboard overview was generated from PredictProtein.
Figure 4. Phylogenetic tree based on amino acid of 5-HTRs.The tree was constructed based neighbor-joining algorithms using MEGA 6.0. The topological stability of the NJ tree was achieved by running 1000 bootstrapping replications. Bootstrap values (%) are indicated by numbers at the nodes. The numerical number in parentheses showed the number of sequences used in each taxon. The GenBank accession numbers and identities are listed in Suppl. Table S2.
Figure 5. Confocal microscopy of HEK293 cells expressing the Aj5-HT4R-EGFP fusion protein.(A) Aj5-HT4R distribution in HEK293 cells. Cells were stained with a membrane plasma probe (DiI) and a nuclei probe (DAPI). Cells stably expressing Aj5-HT4R-EGFP were seeded on glass bottom six-well plates overnight, incubated with DiI (10 μM) and DAPI, and examined by confocal microscopy as described in the section Methods. (B) Internalisation of Aj5-HT4R in HEK293 cells. HEK293 cells transfected with Aj5-HT4R-EGFP were activated by treatment with 100 pM, 10 nM and 1 μM 5-HT during 60 min and detected by confocal microscopy. The “CTL†refers to control without 5-HT stimulation. All images represent at least three independent experiments.
Figure 6. 5-HT induced cAMP accumulation in HEK293 cells stably expressing Flag-Aj5-HT4R.(A) cAMP accumulation in HEK293 cells transientlyco-transfected with Flag-Aj5-HT4R and pCRE-Luc, and was determined in response to 5-HT treatment (1 μM). (B) cAMP accumulation in HEK293 cells stably expressing Flag-Aj5-HT4R/CRE-Luc was assayed in response to different doses of 5-HT. Data are expressed as the mean ± S.E. (n = 3).
Figure 7. Relative expression of Aj5-HT4R and AjPFK in sea cucumbers.(A) Relative expression of Aj5-HT4R in different tissues of active and aestivating sea cucumbers. Total RNA was isolated and purified from the respiratory tree (RT), intestine (IT) and muscle (MS). The expression value was normalised against the expression of the internal control gene (β-actin and β-tubulin). Each symbol and verticalbar represents mean ± SD (n = 6). Double asterisk above the bars indicates extremely significant differences (P < 0.01) between active and aestivation. (B) Tanscriptional variation of AjPFK in different tissues of 5-HT administrated sea cucumbers. Total RNA was isolated and purified from the respiratory tree (RT), intestine (IT) and muscle (MS). The expression value was normalised against the expression of the internal control gene (β-actin and β-tubulin). Each symbol and verticalbar represents mean ± SD (n = 6). Asterisk above the bars indicates extremely significant differences (P < 0.05) between control (PBS) and experimental (5-HT) groups.
Arnold,
The impact of glycosylation on the biological function and structure of human immunoglobulins.
2007, Pubmed
Arnold,
The impact of glycosylation on the biological function and structure of human immunoglobulins.
2007,
Pubmed
Arora,
Mutations of the conserved DRS motif in the second intracellular loop of the gonadotropin-releasing hormone receptor affect expression, activation, and internalization.
1997,
Pubmed
Barnes,
5-HT: the promiscuous and happy hormone!
2011,
Pubmed
Berger,
The expanded biology of serotonin.
2009,
Pubmed
Berumen,
Serotonin receptors in hippocampus.
2012,
Pubmed
Bielefeldt,
Disorders of gastrointestinal hypomotility.
2016,
Pubmed
Blenau,
Distribution of serotonin (5-HT) and its receptors in the insect brain with focus on the mushroom bodies: lessons from Drosophila melanogaster and Apis mellifera.
2011,
Pubmed
Blondel,
Cloning, expression, and pharmacology of four human 5-hydroxytryptamine 4 receptor isoforms produced by alternative splicing in the carboxyl terminus.
1998,
Pubmed
Bray,
Identification and functional characterization of the phosphorylation sites of the neuropeptide FF2 receptor.
2014,
Pubmed
Case,
Functional analysis of the murine cytomegalovirus chemokine receptor homologue M33: ablation of constitutive signaling is associated with an attenuated phenotype in vivo.
2008,
Pubmed
Chee,
Human cytomegalovirus encodes three G protein-coupled receptor homologues.
1990,
Pubmed
Chen,
Comparative phosphoproteomic analysis of intestinal phosphorylated proteins in active versus aestivating sea cucumbers.
2016,
Pubmed
,
Echinobase
Coelho,
Serotonin regulates 6-phosphofructo-1-kinase activity in a PLC-PKC-CaMK II- and Janus kinase-dependent signaling pathway.
2013,
Pubmed
Collingridge,
A nomenclature for ligand-gated ion channels.
2009,
Pubmed
De Maeyer,
Selective desensitization of the 5-HT4 receptor-mediated response in pig atrium but not in stomach.
2009,
Pubmed
De Maeyer,
5-HT4 receptor agonists: similar but not the same.
2008,
Pubmed
Dos Santos,
Distribution of serotonin 5-HT1A-binding sites in the brainstem and the hypothalamus, and their roles in 5-HT-induced sleep and ingestive behaviors in rock pigeons (Columba livia).
2015,
Pubmed
Du,
Transcriptome sequencing and characterization for the sea cucumber Apostichopus japonicus (Selenka, 1867).
2012,
Pubmed
,
Echinobase
Dumuis,
A nonclassical 5-hydroxytryptamine receptor positively coupled with adenylate cyclase in the central nervous system.
1988,
Pubmed
Dupont,
Investigation of 5-HT4 receptors in bronchial hyperresponsiveness in cigarette smoke-exposed mice.
2014,
Pubmed
Gershon,
The serotonin signaling system: from basic understanding to drug development for functional GI disorders.
2007,
Pubmed
Hannon,
Molecular biology of 5-HT receptors.
2008,
Pubmed
Hayes,
5-HT receptors and reward-related behaviour: a review.
2011,
Pubmed
Hoyer,
Molecular, pharmacological and functional diversity of 5-HT receptors.
2002,
Pubmed
Katow,
The 5-HT receptor cell is a new member of secondary mesenchyme cell descendants and forms a major blastocoelar network in sea urchin larvae.
2004,
Pubmed
,
Echinobase
Kaumann,
5-hydroxytryptamine receptors in the human cardiovascular system.
2006,
Pubmed
Kawahara,
Molecular cloning of a putative serotonin receptor gene from barnacle, Balanus amphitrite.
1997,
Pubmed
Komuniecki,
Biogenic amine receptors in parasitic nematodes: what can be learned from Caenorhabditis elegans?
2004,
Pubmed
Langlois,
5-HT4 receptor ligands: applications and new prospects.
2003,
Pubmed
Lee,
Identification of a serotonin receptor coupled to adenylyl cyclase involved in learning-related heterosynaptic facilitation in Aplysia.
2009,
Pubmed
Lefebvre,
Synergistic effect between 5-HT4 receptor agonist and phosphodiesterase 4-inhibitor in releasing acetylcholine in pig gastric circular muscle in vitro.
2016,
Pubmed
Liu,
Serotonin augments gut pacemaker activity via 5-HT3 receptors.
2011,
Pubmed
Livak,
Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.
2001,
Pubmed
Lu,
The role of the aspartate-arginine-tyrosine triad in the m1 muscarinic receptor: mutations of aspartate 122 and tyrosine 124 decrease receptor expression but do not abolish signaling.
1997,
Pubmed
Manabe,
New-generation 5-HT4 receptor agonists: potential for treatment of gastrointestinal motility disorders.
2010,
Pubmed
Meyer,
Effects of serotonin agonists and doxapram on respiratory depression and hypoxemia in etorphine-immobilized impala (Aepyceros melampus).
2010,
Pubmed
Morin,
The D136A mutation of the V2 vasopressin receptor induces a constitutive activity which permits discrimination between antagonists with partial agonist and inverse agonist activities.
1998,
Pubmed
Nagakura,
Regulation of protein kinase C Apl II by serotonin receptors in Aplysia.
2010,
Pubmed
Nirogi,
Synthesis and SAR of Imidazo[1,5-a]pyridine derivatives as 5-HT4 receptor partial agonists for the treatment of cognitive disorders associated with Alzheimer's disease.
2015,
Pubmed
Pérez-Maceira,
Food intake inhibition in rainbow trout induced by activation of serotonin 5-HT2C receptors is associated with increases in POMC, CART and CRF mRNA abundance in hypothalamus.
2016,
Pubmed
Priem,
The facilitating effect of prucalopride on cholinergic neurotransmission in pig gastric circular muscle is regulated by phosphodiesterase 4.
2012,
Pubmed
Pytliak,
Serotonin receptors - from molecular biology to clinical applications.
2011,
Pubmed
Qi,
Larvae of the small white butterfly, Pieris rapae, express a novel serotonin receptor.
2014,
Pubmed
RAPPORT,
Serum vasoconstrictor, serotonin; isolation and characterization.
1948,
Pubmed
Richter,
Serotonin receptors: guardians of stable breathing.
2003,
Pubmed
Rovati,
The highly conserved DRY motif of class A G protein-coupled receptors: beyond the ground state.
2007,
Pubmed
Scheer,
The activation process of the alpha1B-adrenergic receptor: potential role of protonation and hydrophobicity of a highly conserved aspartate.
1997,
Pubmed
Spitzer,
Conservation of structure, signaling and pharmacology between two serotonin receptor subtypes from decapod crustaceans, Panulirus interruptus and Procambarus clarkii.
2008,
Pubmed
Teran,
Serotonin neurons and central respiratory chemoreception: where are we now?
2014,
Pubmed
Thamm,
Function and distribution of 5-HT2 receptors in the honeybee (Apis mellifera).
2013,
Pubmed
Vandesompele,
Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.
2002,
Pubmed
Wirth,
How serotonin receptors regulate morphogenic signalling in neurons.
2017,
Pubmed
Yang,
Agonist-Activated Bombyx Corazonin Receptor Is Internalized via an Arrestin-Dependent and Clathrin-Independent Pathway.
2016,
Pubmed
Yao,
Tryptophan metabolism in animals: important roles in nutrition and health.
2011,
Pubmed
Yi,
Phosphofructokinase 1 glycosylation regulates cell growth and metabolism.
2012,
Pubmed
Zhang,
Identification of differential expressed proteins and characterization their mRNA expression in thermally stressed Apostichopus japonicus.
2013,
Pubmed
,
Echinobase
Zhao,
RNA-seq dependent transcriptional analysis unveils gene expression profile in the intestine of sea cucumber Apostichopus japonicus during aestivation.
2014,
Pubmed
,
Echinobase
Zhao,
DNA methylation levels analysis in four tissues of sea cucumber Apostichopus japonicus based on fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) during aestivation.
2015,
Pubmed
,
Echinobase
Zhao,
Differential gene expression in the respiratory tree of the sea cucumber Apostichopus japonicus during aestivation.
2014,
Pubmed
,
Echinobase
Zhu,
Gene structure, expression, and DNA methylation characteristics of sea cucumber cyclin B gene during aestivation.
2016,
Pubmed
,
Echinobase
Zhu,
Expression of serotonin receptors in the colonic tissue of chronic diarrhea rats.
2016,
Pubmed
