Yuan J , Yang J , Xu X , Wang Z , Jiang Z , Ye Z , Ren Y , Wang Q , Wang T .

32072952 and 42076130 National Natural Science Foundation of China, 2022C02040 "Pioneer" and "Leading Goose" R&D Program of Zhejiang

	Figure 1. Coding sequence (CDS) and amino acid sequence of AjGPER1. Red underlines represent the seven transmembrane helices (7TMs).
	Figure 2. Structural characteristics and multiple sequence alignment of AjGPER1 amino acid sequences. (A) The 2D structure of the AjGPER1 amino acid sequences was predicated by Protter 1.0. N-glycosylation motifs were labeled with green diamonds, and tyrosine, threonine, and serine phosphorylation sites are indicated by yellow, purple, and blue circles. (B) The 3D structure of AjGPER1 protein was predicated using Robetta. (C) Multiple sequence alignment of GPER1s from Apostichopus japonicus and other vertebrate species. A black background represents the same amino acid residue. Sequences with a similarity greater than 80% are represented by a dark-gray background, and a light-gray background represents a similarity greater than 60%. The 7TMs are represented by the blue underlines. The GenBank accession numbers of Callorhinchus milii GPER1 (CmGPER1), Monopterus albus GPER1 (MaGPER1), Hippocampus comes GPER1 (HcGPER1), Larimichthys crocea GPER1 (LcGPER1), Xenopus laevis GPER1 (XlGPER1), Danio rerio GPER1 (DrGPER1), Gallus gallus GPER1 (GgGPER1), Mus musculus GPER1 (MmGPER1), and Homo sapiens GPER1 (HsGPER1) are presented in Supplementary Table S1.
	Figure 3. Phylogenetic analysis of GPER1 protein sequences. The phylogenetic tree was guided 1000 times with MEGA 5.0. The AjGPER1 was highlighted in red and the orange, pink, sky blue, or light green backgrounds represented the GPER1, ERα, ERβ, or KISS1R families, respectively. Sequence ID numbers of indicated receptors used in the phylogenetic analysis were elucidated in Supplementary Table S1.
	Figure 4. Localization of AjGPER1-EGFP fusion protein in HEK293 cells. Confocal microscopy of the HEK293 cells expressing AjGPER1-EGFP fusion protein. The nuclei and cell membrane were visualized with the nuclear probe (DAPI) and the cell membrane probe (DiI). The endoplasmic reticulum resident protein CALR and red-fluorescent protein fusion expression protein (CALR-RFP) or the Golgi body resident protein TGN38 and red-fluorescent protein fusion expression protein (TGN38-RFP) was coexpressed with AjGPER1-EGFP (scale = 10 μm). All images are representative of at least three independent experiments.
	Figure 5. ERK1/2 phosphorylation in AjGPER1-EGFP-expressing HEK293 cells activated by estradiol (E2). (A) Blank (nontransfected) cells, control (transfected with empty vector pEGFP-N1) cells, and AjGPER1-EGFP-expressing cells were treated with DMSO or E2 (100 nM) for 10 min. (B) Time course of E2 (100 nM) treatment induced ERK1/2 phosphorylation in AjGPER1-EGFP-expressing HEK293 cells. p-ERK1/2 was normalized to the control (t-ERK1/2). Values are presented as means ± standard error of the mean (SEM) (n = 3). Data were analyzed using Student’s t-test (ns p > 0.05; **** p < 0.0001). All images and data shown are representative of at least three independent experiments. (See Figures S2–S5 for the original Western blot images.)
	Figure 6. AjGPER1 activation-mediated intracellular signaling pathway. (A) AjGPER1 was activated by E2 (100 nM) to stimulate the phosphorylation of ERK1/2 in HEK293 cells. (B–D) Activation of ERK1/2 phosphorylation mediated by AjGPER1 by E2 was reduced or blocked by PKA, PKC, or Gαq protein inhibitors. Serum-starved AjGPER1-expressing cells were pretreated with DMSO, PKA inhibitor (H89, 10 μM), PKC inhibitor (Go 6983, 1 μM), or Gαq protein inhibitor (FR900359, 1 μM) for 1 h before E2 (100 nM) administration. (E) Statistical summary of the gray-scale analysis of ERK1/2 phosphorylation inhibitory effects by H89, Go 6983, or FR900359 in E2 stimulated AjGPER1-expressing cells. Values are presented as means ± SEM. Data were analyzed using one-way ANOVA followed by Tukey’s multiple comparison tests (** p < 0.01; *** p < 0.001; **** p < 0.0001). (F) Schematic representation of the AjGPER1-mediated cell signaling pathway. E2: estradiol; PLC: phospholipase C; PIP2: phosphatidylinositol (4,5) bisphosphate; DAG: diacylglycerol; IP3: inositol triphosphate; IP3R: inositol triphosphate receptor; ER: endoplasmic reticulum; Ca2+: calcium ion; PKC: protein kinase C; PKA: protein kinase A; ERK1/2: extracellular regulated kinase 1/2; P: phosphorylation. All pictures and data shown are representative of at least three independent experiments. (See Figures S6–S10 for the original Western blot images.)
	Figure 7. Activation of ERK1/2 in AjGPER1-expressing HEK293 cells by Bisphenol A (BPA). (A) Blank (nontransfected) cells, control (transfected with empty vector pEGFP-N1) cells, and AjGPER1-EGFP-expressing cells were stimulated with DMSO or BPA (10 μM) for 10 min. (B) Time course of BPA (100 nM)-induced ERK1/2 phosphorylation in AjGPER1-EGFP-expressing cells. The p-ERK1/2 was normalized to the loading control (t-ERK1/2). Values are represented as means ± SEM (n = 3). Data were analyzed using Student’s t-test (ns p > 0.05; * p < 0.05). All pictures and data shown are representative of at least three independent experiments. (See Figures S11–S14 for the original Western blot images.)
	Figure 8. High expression of AjGPER1 and physiological functions activated by E2 and BPA in sea cucumber ovarian tissues. (A) Tissue-specific expression profile of AjGPER1 in multiple tissues. The relative expression level of AjGPER1 mRNA in nerve ring, intestine, muscle, tentacle, respiratory tree, and ovary. Data were presented as mean ± SEM (n = 4), and data were analyzed using an independent-sample t-test, and different lowercase letters indicated significant differences (p < 0.05) between tissues. (B) Activation of ERK1/2 in A. japonicus respiratory tree and ovary tissues by E2 and BPA. Tissues were stimulated with different concentrations of E2 or BPA for 30 min, and the p-ERK1/2 was normalized to the loading control (t-ERK1/2). Concentrations of E2 Log[M] −11: 2.7238 ng/L; −9: 272.382 ng/L; −7: 22.8286 μg/L and of BPA Log[M] −11: 2.2829 ng/L; −9: 228.286 ng/L; −7: 22.8286 μg/L. (C–F) Effects of 4 h exposure to E2 or BPA on enzyme activities in the respiratory tree and ovary tissue of A. japonicus. Values shown are multiples of the lowest activity of the control group expressed as means ± SEM (n = 3). Different color bars represent different tissues. Data were analyzed using one-way ANOVA followed by Tukey’s multiple comparison tests (ns p > 0.05), and different lowercase letters indicated significant differences (p < 0.05). All data shown are representative of at least three independent experiments. (See Figures S15–S17 for the original Western blot images.)

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