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Sci Rep
2017 Jan 06;7:40247. doi: 10.1038/srep40247.
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Identification and functional characterisation of 5-HT4 receptor in sea cucumber Apostichopus japonicus (Selenka).
Wang T
,
Yang Z
,
Zhou N
,
Sun L
,
Lv Z
,
Wu C
.
???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.
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