Analysis of [^125-I]-ap-GnRH binding to S2 cells transfected with ap-GnRHR-L or ap-GnRHR-S.

<p>Binding to the radioligand was not displaced by increasing concentrations of unlabeled ap-AKH in S2 cells transfected with either ap-GnRHR-L or ap-GnRHR-S. Binding data are expressed as the percentage of [¹²⁵I]-ap-GnRH binding to the respective receptor in the absence of unlabeled hormone. Each data point represents N = 3 assays run in triplicates.</p

Expression of ap-GnRHR isoforms (top two panels) in different <i>A</i>. <i>californica</i> tissues examined by RT-PCR.

<p>Primers used for the top panel amplify only ap-GnRHR-L; primers used for the second panel amplify both ap-GnRHR-L and ap-GnRHR-S. Negative controls (no RT) were RNA samples that have not been reverse transcribed (third panel). ap-Actin was used as a control to ensure the quality of RNA samples (bottom panel). The sizes of the PCR products are shown on the right. BCN, bag cell neurons; Abg, abdominal ganglia; Ceb, cerebral ganglia; P/P, pedal/pleural ganglia; Buc, buccal ganglia; Tail, ventral tail tissue; Osp, osphradium; SHD, small hermaphroditic duct; Hrt, heart; Ovt, ovotestis;—Cont, negative water control.</p

Maximum likelihood analysis of the phylogeny of GnRHR-related receptors.

<p>The bootstrap values (in %) from 1000 replicas are indicated at each branch point. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 334 amino acid sequences and a total of 1171 positions in the final dataset. The invertebrate cardioacceleratory peptide receptors and vertebrate vasopressin receptor-like GPCRs were used as outgroups to root the tree. The detailed nomenclature and sequences are listed as data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160292#pone.0160292.s001" target="_blank">S1 Dataset</a>.</p

Analysis of [¹²⁵I]-ap-GnRH binding to S2 cells transfected with ap-GnRHR-L or ap-GnRHR-S.

Binding to the radioligand was displaced by increasing concentrations of unlabeled ap-GnRH in S2 cells transfected with ap-GnRHR-L (EC50 = 2.52 nM), but not ap-GnRHR-S. Binding data are expressed as the percentage of [125I]-ap-GnRH binding to the respective receptor in the absence of unlabeled hormone. Empty vector-transfected cells were used as a negative control. Each data point represents N = 3 assays run in triplicates.</p

ap-GnRH stimulated IP1 accumulation in S2 cells transfected with ap-GnRHR-L, but not ap-GnRHR-S.

Data are expressed as fold stimulation over IP1 levels in the untreated cells. Proctolin was used as a positive control in ap-GnRHR-L or ap-GnRHR-S-transfected cells, and empty vector-transfected cells were used as a negative control. N = 3 assays run in triplicates.</p

Functional Authentication of a Novel Gastropod Gonadotropin-Releasing Hormone Receptor Reveals Unusual Features and Evolutionary Insight

<div><p>A gonadotropin-releasing hormone (GnRH)-like molecule was previously identified in a gastropod, <i>Aplysia californica</i>, and named ap-GnRH. In this study, we cloned the full-length cDNA of a putative ap-GnRH receptor (ap-GnRHR) and functionally authenticated this receptor as a bona fide ap-GnRHR. This receptor contains two potential translation start sites, each accompanied by a Kozak sequence, suggesting the translation of a long and a short form of the receptor is possible. The putative ap-GnRHR maintains the conserved structural motifs of GnRHR-like receptors and shares 45% sequence identity with the octopus GnRHR. The expression of the putative ap-GnRHR short form is ubiquitous in all tissues examined, whereas the long form is only expressed in parts of the central nervous system, osphradium, small hermaphroditic duct, and ovotestis. The cDNA encoding the long or the short receptor was transfected into the <i>Drosophila</i> S2 cell line and subject to a radioreceptor assay using ¹²⁵I-labeled ap-GnRH as the radioligand. Further, the transfected cells were treated with various concentrations of ap-GnRH and measured for the accumulation of cAMP and inositol monophosphate (IP1). Radioreceptor assay revealed that only the long receptor bound specifically to the radioligand. Further, only the long receptor responded to ap-GnRH with an increased accumulation of IP1, but not cAMP. Our studies show that despite the more prevalent expression of the short receptor, only the long receptor is the functional ap-GnRHR. Importantly, this is only the second report on the authentication of a protostome GnRHR, and based on the function and the phylogenetic grouping of ap-GnRHR, we suggest that this receptor is more similar to protostome corazonin receptors than chordate GnRHRs.</p></div

Table_1_An Adipokinetic Hormone Acts as a Volume Regulator in the Intertidal Gastropod Mollusk, Aplysia californica.DOCX

Adipokinetic hormone (AKH) is a multifunctional neuropeptide in the gonadotropin-releasing hormone superfamily. In insects, AKH acts to mobilize energy stores during times of high energetic demand, but has been shown to have other effects. In lophotrochozoans, the presence and function of AKH are less characterized. We have previously identified an AKH in an intertidal gastropod mollusk, the California sea hare (Aplysia californica), and named it ac-AKH. Our previous data showed ac-AKH induced an acute weight loss, suggesting a role in volume regulation. The overarching goals of this study were to test the role of ac-AKH as a volume regulator and examine the mechanism by which ac-AKH induced the acute weight loss. Our results showed that ac-AKH reduced body mass, in part, through the reduction of hemolymph volume without altering hemolymph osmolality or specific osmolytes. The effect of ac-AKH on volume loss was accentuated under a hyposaline condition. We further showed that ac-akh expression was inhibited during a hyposaline challenge, and that the administration of ac-AKH partially reversed the increase in body mass, but not hemolymph osmolality change, caused by the hyposaline challenge. These data collectively show that ac-AKH is a proximate regulator controlling the fluid volume, but not osmolality, in A. californica. Importantly, our results highlight the functional divergence of this structurally conserved neuropeptide in the molluscan lineage.</p

Amino acid sequence alignment of ap-GnRHR with representative GnRHR-related receptors.

<p>Shaded amino acids indicate the transmembrane (TM) domains. The predicted intracellular loops (IL) and extracellular loops (EL) are numbered accordingly. The two putative start sites (M) are each denoted by a “+”. Ms-CRZR—corazonin receptor, <i>Manduca sexta</i> (AAR14318); Oct-GnRHR—Gonadotropin-releasing hormone receptor, <i>Octopus vulgaris</i> (GNRHR_OCTVU); Ci-GnRHRI—gonadotropin-releasing hormone receptor 1, <i>Ciona intestinalis</i> (NP_001028997); Ci-GnRHRII—gonadotropin-releasing hormone receptor 2, <i>Ciona intestinalis</i> (NP_001028996); Pm-GnRHRI—gonadotropin releasing-hormone receptor, <i>Petromyzon marinus</i> (AF439802_1); Mm-GnRH—gonadotropin-releasing hormone receptor isoform <i>Mus musculus</i> (NP_001297580). Identical, highly conserved, and less conserved amino acid residues are denoted by an asterisk (*), a colon (:), and a period (.), respectively. Ampersand (&) denotes cysteine residues involved in the disulfide bridge formation.</p

Nucleotide and deduced amino acid sequences of the full-length putative ap-GnRHR (Genbank Accession # AHE78444.1).

<p>The nucleotides (lower case letters) and amino acids (upper case letters) are numbered accordingly. The seven predicted transmembrane domains are underlined. The two putative translation start sites are highlighted in green, and the asterisk (*) denotes the stop codon. The nucleotides corresponding to the truncated polyadenylation signal (aata) are double-underlined.</p

ap-GnRH did not stimulate cAMP accumulation in S2 cells transfected with either ap-GnRHR-L or ap-GnRHR-S.

<p>Data are expressed as fold stimulation over cAMP levels in the untreated cells. Forskolin was used as a positive control. N = 3 assays run in triplicates.</p