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Simple Summary: Apostichopus japonicus is a temperate marine invertebrate and an economically important aquatic echinoderm species in China. As a benthic organism, sea cucumbers feed on small benthic particulate matter and are easily affected by pollutants. Therefore, for conservation purposes, it is important to elucidate the effects of specific pollutants on these animals. Bisphenol A (BPA) has been identified as an endocrine disruptor that functions as an estrogen analog and typically causes reproductive toxicity by interfering with the endocrine system. We identified the G protein-coupled estrogen receptor 1 in A. japonicus and found that both BPA and estradiol could activate the estrogen-receptor-mediated mitogen-activated protein kinase signaling pathway. Using biological approaches, we have confirmed the estrogen-analog bioactivity of BPA on sea cucumbers by direct interaction with the estrogen receptor. We also determined that BPA affects the reproduction of sea cucumbers by metabolic disruption in ovary tissue. The sea cucumber, Apostichopus japonicus, is a marine benthic organism that feeds on small benthic particulate matter and is easily affected by pollutants. Bisphenol A (BPA, 4,4′-isopropylidenediphenol) has been identified as an endocrine disruptor. It is ubiquitously detectable in oceans and affects a variety of marine animals. It functions as an estrogen analog and typically causes reproductive toxicity by interfering with the endocrine system. To comparatively analyze the reproductive effects of estradiol (E2) and BPA on sea cucumbers, we identified a G protein-coupled estrogen receptor 1 (GPER1) in A. japonicus and investigated its effects on reproduction. The results showed that BPA and E2 exposure activated A. japonicus AjGPER1, thereby mediating the mitogen-activated protein kinase signaling pathways. High-level expression of AjGPER1 in the ovarian tissue was confirmed by qPCR. Furthermore, metabolic changes were induced by 100 nM (22.83 μg/L) BPA exposure in the ovarian tissue, leading to a notable increase in the activities of trehalase and phosphofructokinase. Overall, our findings suggest that AjGPER1 is directly activated by BPA and affects sea cucumber reproduction by disrupting ovarian tissue metabolism, suggesting that marine pollutants pose a threat to the conservation of sea cucumber resources. [ABSTRACT FROM AUTHOR]

The major effects of cannabinoids and endocannabinoids are mediated via two G protein-coupled receptors, CB1 and CB2, elucidation of the mechanism and structural determinants of the CB2 receptor coupling with G proteins will have a significant impact on drug discovery. In the present study, we systematically investigated the role of the intracellular loops in the interaction of the CB2 receptor with G proteins using chimeric receptors alongside the characterization of cAMP accumulation and ERK1/2 phosphorylation. We provided evidence that ICL2 was significantly involved in G protein coupling in coordination with the C-terminal end. Moreover, a single alanine substitution of the Pro-139 in the CB2 receptor that corresponds to Leu-222 in the CB1 receptor resulted in a moderate impairment in the inhibition of cAMP accumulation, whereas mutants P139F, P139M and P139L were able to couple to the Gs protein in a CRE-driven luciferase assay. With the ERK activation experiments, we further found that P139L has the ability to activate ERK through both Gi- and Gs-mediated pathways. Our findings defined an essential role of the second intracellular loop of the CB2 receptor in coordination with the C-terminal tail in G protein coupling and receptor activation. [ABSTRACT FROM AUTHOR]

Abstract: The cAMP response element binding protein, CREB, is a G protein-coupled receptor (GPCR) signal-activated transcription factor implicated in the control of many biological processes. In the current study, we constructed a cAMP response element (CRE)-driven luciferase assay system for GPCR characterization in insect cells. Our results indicated that Gs-coupled Bombyx adipokinetic hormone receptor (AKHR) and corazonin receptor could effectively initiate CRE-driven luciferase transcription, but forskolin, a reagent widely used to activate adenylyl cyclase in mammalian systems, failed to induce luciferase activity in insect cells co-transfected with a CRE-driven reporter construct upon agonist treatment. Further investigation revealed that the specific protein kinase C (PKC) inhibitors exhibited stimulatory effects on CRE-driven reporter transcription, and blockage of Ca2+ signals and inhibition of Ca2+-dependent calcineurin resulted in a significant decrease in the luciferase activity. Taken together, these results suggest that PKC likely acts as a negative regulator to modulate CREB activation; in contrast, Ca2+ signals and Ca2+-dependent calcineurin, in addition to PKA, essentially contribute to the positive regulation of CREB activity. This study presents evidence to elucidate the underlying molecular mechanism by which CREB activation is regulated in insects. [Copyright &y& Elsevier]

The sea cucumber Apostichopus japonicus is dioecious, with seasonal reproduction. G protein-coupled receptor (GPCR)-mediated signaling systems might play critical roles in the reproductive control of A. japonicus. Here, we classified GPCR from the genome in silico and used transcriptomic analyses to further mine those that function in gonadal-development control. Totally, 487 GPCRs were predicted from A. japonicus , and 183 of these were further annotated to molecular pathways. Transcriptome analysis revealed 327 GPCRs expressed in gonads, and these were classified into four families and 19 subfamilies. Three pathways were apparently associated with reproduction, including neuroactive ligand-receptor interaction, the mTOR and Wnt signaling pathways. Seven and eight ovary- and testis-specific GPCRs were filtered, and the gene expression profiles were determined in multiple tissues and gonads at different developmental stages by qPCR. These results provide new insights into the discovery of GPCR-mediated signaling control in sea cucumber reproduction, especially in gonadal development control. • 487 G protein-coupled receptors are predicted and classified from Apostichopus japonicus. • A variety of G protein-coupled receptors are detected to be expressed in the gonads of Apostichopus japonicus. • Gonad-specific G protein-coupled receptors have been screened out. [ABSTRACT FROM AUTHOR]

CCHamides are newly identified insect neuropeptides, which are widely occurring in most insects. However, our knowledge about their signaling characteristics and physiological roles is still limited. Here, we cloned two full-length cDNAs encoding putative CCHamide receptors, Bombyx neuropeptide GPCR A14 (BNGR-A14) and -A15 (BNGR-A15), from the brain of B. mori larvae. Characterization of signaling indicated that Bombyx CCHamide-1 and CCHamide-2 are specific endogenous ligands for BNGR-A15 and BNGR-A14, respectively. Further functional assays combined with specific inhibitors demonstrated that upon activation by CCHamide-2, BNGR-A14 elicited significant increases in CRE-driven luciferase activity, intracellular Ca2+ mobilization and ERK1/2 phosphorylation in a G q inhibitor-sensitive manner, while BNGR-A15 was activated by CCHamide-1, thus leading to intracellular accumulation of cAMP, Ca2+ mobilization, and ERK1/2 phosphorylation in a G s and G q inhibitor-sensitive manner. Based on these findings, we designated the receptors BNGR-A15 and -A14 as Bommo -CCHaR-1 and -2, respectively. In addition, our results showed that CCHamides are considered to require intrachain disulfide bonds to activate their respective receptor in the physiological concentration range. Moreover, quantitative RT-PCR analysis revealed that CCHamide-1 is more likely to serve as a brain-gut peptide to regulate feeding behavior and growth through BNGR-A15, whereas the CCHamide-2 signaling system might play an important role in the control of multiple physiological processes. Our findings provide in-depth information on CCHamide-1 and -2-mediated signaling, facilitating further elucidation of their endocrinological roles in the regulation of fundamental physiological processes. Image 1 • Bombyx CCHamide-1 and CCHamide-2 are specific endogenous ligands for BNGR-A15 and BNGR-A14, respectively. • BNGR-A15 dually couples to G s - and G q -dependent pathways, while BNGR-A14 activates G q -dependent cascades • A disulfide bond is critical for CCHamides to activate their respective receptor in the physiological concentration range. • BNGR-A14 might play multiple physiological roles, whereas BNGR-A15 is likely to function as a brain-gut peptide. [ABSTRACT FROM AUTHOR]

Neuropeptide Y (NPY) receptors are suggested to mediate the multi-physiological functions of NPY family peptides, such as food intake, in teleost fish. However, the structure and signaling of fish NPY receptors are yet to be fully elucidated. In this study, we report the cloning and characterization of two neuropeptide Y receptor subtypes, Y2 (NPY2R) and Y7 (NPY7R), in yellow croaker Larimichthys crocea (L. crocea) (Lc NPY2R, Lc NPY7R). The gene structure, pharmacological characterization, cell location, and tissue expression of these two receptors were explored. The phylogenetic results showed that Lc NPY2R and Lc NPY7R had typical G protein-coupled receptor profiles, associated with the Y2 subfamily, with coding sequences that are highly conserved in vertebrates. The expression of both Lc NPY2R and Lc NPY7R could be activated by Lc NPY in HEK293 cells. However, truncated Lc NPY 18 – 36 was only able to activate Lc NPY2R at the same level as full length Lc NPY. Expression analysis revealed that Lc NPY2R mRNA was predominantly expressed in the intestine and liver, whereas Lc NPY7R was expressed in the stomach, which indicated that both receptors were related to the digestive system. Overall, our data establishes a molecular basis to determine the actions of Lc NPY2R and Lc NPY7R, which could be used to elucidate the conserved roles of these receptor-ligand pairs in vertebrates. Unlabelled Image • The full length cDNA sequences of Larimichthys crocea NPY2R and NPY7R have been cloned and bioinformatically characterized. • Internalized receptors were demonstrated in HEK293 cells incubating of 1 μM L. crocea neuropeptide Y (Lc NPY). • Lc NPY activates both Lc NPY2R and Lc NPY7R, leading to the intracellular cAMP inhibition in a dose-dependent manner. • Transcriptional distribution of LcNPY2R/LcNPY7R has been revealed and physiological roles were suggested. [ABSTRACT FROM AUTHOR]