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Backgrounds: Elucidation of peptide-receptor pairs is a prerequisite for many studies in the neuroendocrine, endocrine, and neuroscience fields. Recent omics analyses have provided vast amounts of peptide and G protein-coupled receptor (GPCR) sequence data. GPCRs for homologous peptides are easily characterized based on homology searching, and the relevant peptide-GPCR interactions are also detected by typical signaling assays. In contrast, conventional evaluation or prediction methods, including high-throughput reverse-pharmacological assays and tertiary structure-based computational analyses, are not useful for identifying interactions between novel and omics-derived peptides and GPCRs. Summary: Recently, an approach combining machine learning-based prediction of novel peptide-GPCR pairs and experimental validation of the predicted pairs have been shown to breakthrough this bottleneck. A machine learning method, logistic regression for human class A GPCRs and the multiple subsequent signaling assays led to the deorphanization of human class A orphan GPCRs, namely, the identification of 18 peptide-GPCR pairs. Furthermore, using another machine learning algorithm, the support vector machine (SVM), the peptide descriptor-incorporated SVM was originally developed and employed to predict GPCRs for novel peptides characterized from the closest relative of vertebrates, Ciona intestinalis Type A (Ciona robusta). Experimental validation of the predicted pairs eventually led to the identification of 11 novel peptide-GPCR pairs. Of particular interest is that these newly identified GPCRs displayed neither significant sequence similarity nor molecular phylogenetic relatedness to known GPCRs for peptides. Key Messages: These recent studies highlight the usefulness and versatility of machine learning for enabling the efficient, reliable, and systematic identification of novel peptide-GPCR interactions. [ABSTRACT FROM AUTHOR]

Omics studies contribute to the elucidation of genomes and profiles of gene expression. In the ascidian Ciona intestinalis Type A (Ciona robusta), mass spectrometry (MS)-based peptidomic studies have detected numerous Ciona -specific (nonhomologous) neuropeptides as well as Ciona homologs of typical vertebrate neuropeptides and hypothalamic peptide hormones. Candidates for cognate G protein-coupled receptors (GPCRs) for these peptides have been found in the Ciona transcriptome by two ways. First, Ciona homologous GPCRs of vertebrate counterparts have been detected by sequence homology searches of cognate transcriptomes. Second, the transcriptome-derived GPCR candidates have been used for machine learning-based systematic prediction of interactions not only between Ciona homologous peptides and GPCRs but also between novel Ciona peptides and GPCRs. These data have ultimately led to experimental evidence for various Ciona peptide-GPCR interactions. Comparative transcriptomics between the wildtype and Ciona vasopressin (CiVP) gene-edited Ciona provide clues to the biological functions of CiVP in ovarian follicular development and whole body growth. Furthermore, the transcriptomes of follicles treated with peptides, such as Ciona tachykinin and cionin (a Ciona cholecystokinin homolog), have revealed key regulatory genes for Ciona follicle growth, maturation, and ovulation, eventually leading to the verification of essential and novel molecular mechanisms underlying these biological events. These findings indicate that omics studies, combined with artificial intelligence and single-cell technologies, pave the way for investigating in greater details the nervous, neuroendocrine, and endocrine systems of ascidians and the molecular and functional evolution and diversity of peptidergic regulatory networks throughout chordates. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) have been found to form heterodimers and modulate or fine-tune the functions of GPCRs. However, the involvement of GPCR heterodimerization and its functional consequences in gonadal tissues, including granulosa cells, have been poorly investigated, mainly due to the lack of efficient method for identification of novel GPCR heterodimers. In this paper, we identified a novel GPCR heterodimer between prostaglandin E2 (PGE2) receptor 2 (EP2) and calcitonin (CT) receptor (CTR). High-resolution liquid chromatography (LC)-tandem mass spectrometry (MS/MS) of protease-digested EP2-coimmunoprecipitates detected protein fragments of CTR in an ovarian granulosa cell line, OV3121. Western blotting of EP2- and CTR-coimmunoprecipitates detected a specific band for EP2-CTR heterodimer. Specific heterodimerization between EP2 and CTR was also observed by fluorescence resonance energy transfer analysis in HEK293MSR cells expressing cyan- and yellow-fluorescent protein-fused EP2 and CTR, respectively. Collectively, these results provided evidence for heterodimerization between EP2 and CTR. Moreover, Ca2+ mobilization by CT was approximately 40% less potent in HEK293MSR cells expressing an EP2-CTR heterodimer, whereas cAMP production by EP2 or CT was not significantly altered compared with cells expressing EP2- or CTR alone. These functional analyses verified that CTR-mediated Ca2+ mobilization is specifically decreased via heterodimerization with EP2. Altogether, the present study suggests that a novel GPCR heterodimer, EP2-CTR, is involved in some functional regulation, and paves the way for investigation of novel biological roles of CTR and EP2 in various tissues. [ABSTRACT FROM AUTHOR]

• Lymnaea corazonin receptor (CRZR) was identified. • Phylogenetic analysis supports that GnRH and CRZ signaling systems are paralogous. • Lymnaea CRZ signaling involves Ca2+ mobilization but not cAMP production. • Wide distribution of the receptor confirms Lymnaea CRZ peptide as multifunctional. • Previously called molluscan GnRH signaling should be classified as CRZ signaling. In recent years, new concepts have emerged regarding the nomenclature, functions, and relationships of different peptide families of the gonadotropin-releasing hormone (GnRH) superfamily. One of the main driving forces for this originated from the emerging evidence that neuropeptides previously called molluscan GnRH are multifunctional and should be classified as corazonin (CRZ). However, research articles still appear that use incorrect nomenclature and attribute the same function to molluscan CRZs as vertebrate GnRHs. The aim of the present study was to further support the recent interpretation of the origin and function of the GnRH superfamily. Towards this goal, we report the characterization of CRZ signaling system in the molluscan model species, the great pond snail (Lymnaea stagnalis). We detected a CRZ-receptor-like sequence (Lym-CRZR) by homology-searching in the Lymnaea transcriptomes and the deduced amino acid sequence showed high sequence similarity to GnRH receptors and CRZ receptors. Molecular phylogenetic tree analysis demonstrated that Lym-CRZR is included in the cluster of molluscan CRZRs. Lym-CRZR transiently transfected into HEK293 cells was found to be localized at the plasma membrane, confirming that it functions as a membrane receptor, like other G protein-coupled receptors. The signaling assays revealed that the previously identified Lym-CRZ neuropeptide stimulated intracellular Ca2+ mobilization in a dose-dependent manner, but not cyclic AMP production, in HEK293 cells transfected with Lym-CRZR. Finally, we demonstrated a wide tissue distribution of Lym-CRZR. These results suggest that Lym-CRZ is a multifunctional peptide and provide further insights into the evolution of the GnRH neuropeptide superfamily. The present study also supports the notion that previously termed molluscan "GnRH" should be classified as "CRZ". [ABSTRACT FROM AUTHOR]