Mita M et al. (2020), A novel G protein-coupled receptor for starfish...

ECB-ART-49477

PLoS One 2020 Jan 01;1511:e0242877. doi: 10.1371/journal.pone.0242877.

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A novel G protein-coupled receptor for starfish gonadotropic hormone, relaxin-like gonad-stimulating peptide.

Mita M , Matsubara S , Osugi T , Shiraishi A , Wada A , Satake H .

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Gonadotropic hormones play important regulatory roles in reproduction. Relaxin-like gonad-stimulating peptide (RGP) is a gonadotropin-like hormone in starfish. However, a receptor for RGP remains to be identified. Here, we describe the identification of an authentic receptor for RGP (RGPR) in the starfish, Patiria pectinifera. A binding assay using radioiodinated P. pectinifera RGP (PpeRGP) revealed that RGPR was expressed in ovarian follicle cells. A RGPR candidate was identified by homology-searching of transcriptome data of P. pectinifera follicle cells. Based on the contig sequences, a putative 947-amino acid PpeRGPR was cloned from follicle cells. Like the vertebrate relaxin family peptide receptors (RXFP 1 and 2), PpeRGPR was a G protein-coupled receptor that harbored a low-density lipoprotein-receptor class A motif and leucine-rich repeat sequences in the extracellular domain of the N-terminal region. Sf9 cells transfected with Gαq16-fused PpeRGPR activated calcium ion mobilization in response to PpeRGP, but not to RGP of another starfish Asterias amurensis, in a dose-dependent fashion. These results confirmed the species-specific reactivity of RGP and the cognate receptor. Thus, the present study provides evidence that PpeRGPR is a specific receptor for PpeRGP. To the best of our knowledge, this is the first report on the identification of a receptor for echinoderm RGP.

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	Fig 1. Amino acid sequence of the starfish Patiria pectinifera RGP receptor (PpeRGPR).The signal peptide, low-density lipoprotein-receptor class A (LDLa), leucine-rich repeat (LRR), and transmembrane domains (TM) are indicated with gray, red, green and blue backgrounds, respectively. Yellow characters indicate the Ca2+-ligation motifs.
	Fig 2. Intracellular calcium mobilization in Sf9 cells.(A, B) Sf9 cells were transfected with GÎ±q16- fused PpeRGPR-recombinant baculovirus (A) and non-transfected Sf9 cells were used as wild type controls (B). Expression of PpeRGPR in Sf9 cells was examined by immunostaining using an anti-GÎ±q16 antibody. Immunoreactivity was observed using a fluorescence microscope after incubation with an Alexa Fluor 488-conjugated secondary antibody. Specific PpeRGPR immunoreactivity was localized at the cell membrane (A), whereas no signal was observed in the control cells (B). Scale bars represent 50 Î¼m. (C) Calcium mobilization dose-response curves for PpeRGP and AamRGP in Sf9 cells transfected with GÎ±q16- fused PpeRGPR-recombinant baculoviruses. The kinetics of real-time Fluo-8 fluorescence was observed at excitation/emission wavelengths of 490/514 nm in Sf9 cells exposed to various concentrations of PpeRGP (â) or AamRGP (â). PpeRGPR was specifically stimulated by PpeRGP in a dose-dependent fashion. Symbols and bars represent the mean Â± standard error of the mean from three to seven independent samples. Each dose-response curve was statistically evaluated by analysis of variance. The difference between PpeRGP and AamRGP was also examined by Student t test.
	Fig 3. Competitive inhibition curves of 125I-PpeRGP binding to Patiria pectinifera ovarian follicle cells in the presence of unlabeled PpeRGP or AamRGP.P. pectinifera ovarian follicle cell homogenates (25 Î¼g of protein) were incubated for 2 h at 20Â°C in assay buffer containing 125I-PpeRGP (about 100,000 cpm; 2.087 pg/100 ml; total volume 0.5 ml, final concentration 1.1 pM) in the presence of serially diluted unlabeled PpeRGP (â) or AamRGP (â). Specific binding was obtained by subtracting nonspecific binding from total binding. Data were obtained from four separate assays using follicle cells from ovaries of four different animals.
	Fig 4. Effect of RGP on 1-MeAde and cAMP production in Patiria pectinifera ovarian follicle cells.(A) Amino acid alignments of A- and B-chains in P. pectinifera (PpeRGP) and Asterias amurensis (AamRGP). To illustrate the conserved features, the amino acid types are color coded according to their properties, with basic residues in blue (Arg, Lys and His), acidic residues in red (Glu and Asp), hydrophobic residues in green (Ala, Val, Ile, Phe, Trp, Tyr, Pro and Met), hydrophilic in black (Ser, Thr, Asn and Gln) and glycine in light blue. The cysteine residues are highlighted in yellow and disulfide bonds are shown with solid dark red lines. (B) Effect of PpeRGP and AamRGP on 1-MeAde and cAMP production in follicle cells. Follicle cells were incubated in ASW in the presence of either PpeRGP (â) or AamRGP (â) for 2 h at 20Â°C. The amount of 1-MeAde released into the medium was estimated using a biological assay with an analytical curve generated with authentic 1-MeAde. Intracellular cAMP content was determined by EIA. Symbols and bars represent the mean of three independent samples and standard error of the mean (SEM), respectively.

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