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Pituitary adenylate cyclase‐activating peptide (PACAP) receptor (PAC1R) is a class B Gprotein‐coupled receptor (GPCR) that is widely expressed in the human body and is involved in neuronal differentiation. As class B GPCRs are known to form heterocomplexes with family members, we hypothesized that PAC1R mediates neuronal differentiation through interaction with a class B GPCR. We used the BRET assay to identify potential interactions between PAC1R and 11 class B GPCRs. Gastric inhibitory polypeptide receptor (GIPR) and secretin receptor were identified as putative binding partners of PAC1R. The effect of heterocomplex formation by PAC1R on receptor activation was evaluated with the cyclic (c)AMP, luciferase reporter, and calcium signaling assays; and the effects on receptor internalization and subcellular localization were examined by confocal microscopy. The results suggested he PAC1R/GIPR heterocomplex suppressed signaling events downstream of PAC1R, including cAMP production, serum response element and calcium signaling, and β‐arrestin recruitment. Protein–protein interaction was analyzed in silico, and induction of neuronal differentiation by the PAC1R heterocomplex was assessed in SH‐SY5Y neuronal cells by measure the morphological changes and marker genes expression by real‐time quantitative PCR and western blot. Over‐expression of GIPR suppressed PACAP/PAC1R‐mediated neuronal differentiation and the differentiation markers expression in SH‐SY5Y cells. GIPR regulates neuronal differentiation through heterocomplex formation with PAC1R. [ABSTRACT FROM AUTHOR]

Several hypotheses have been proposed regarding the origin and evolution of the secretin family of peptides and receptors. However, identification of homologous ligand–receptor pairs in invertebrates and vertebrates is difficult because of the low levels of sequence identity between orthologs of distant species. In this study, five receptors structurally related to the vertebrate class B1 G protein-coupled receptor (GPCR) family were characterized from amphioxus (Branchiostoma floridae). Phylogenetic analysis showed that they clustered with vertebrate parathyroid hormone receptors (PTHR) and pituitary adenylate cyclase-activating polypeptide (PACAP)/glucagon receptors. These PTHR-like receptors shared synteny with several PTH and PACAP/glucagon receptors identified in spotted gar, Xenopus, and human, indicating that amphioxus preserves the ancestral chordate genomic organization of these receptor subfamilies. According to recent data by Mirabeau and Joly, amphioxus also expresses putative peptide ligands including homologs of PTH (bfPTH1 and 2) and PACAP/GLUC-like peptides (bfPACAP/GLUCs) that may interact with these receptors. Functional analyses showed that bfPTH1 and bfPTH2 activated one of the amphioxus receptors (bf98C) whereas bfPACAP/GLUCs strongly interacted with bf95. In summary, our data confirm the presence of PTH and PACAP/GLUC ligand–receptor pairs in amphioxus, demonstrating that functional homologs of vertebrate PTH and PACAP/glucagon GPCR subfamilies arose before the cephalochordate divergence from the ancestor of tunicates and vertebrates. [ABSTRACT FROM AUTHOR]

In mammals, secretin is a 27-amino acid peptide that was first studied in 1902 by Bayliss and Starling from the extracts of the jejunal mucosa for its ability to stimulate pancreatic secretion. To date, secretin has only been identified in tetrapods, with the earliest diverged secretin found in frogs. Despite being the first hormone discovered, secretin's evolutionary origin remains enigmatic, it shows moderate sequence identity in nonmammalian tetrapods but is highly conserved in mammals. Current hypotheses suggest that although secretin has already emerged before the divergence of osteichthyans, it was lost in fish and retained only in land vertebrates. Nevertheless, the cognate receptor of secretin has been identified in both actinopterygian fish (zebrafish) and sarcopterygian fish (lungfish). However, the zebrafish secretin receptor was shown to be nonbioactive. Based on the present information that the earliest diverged bioactive secretin receptor was found in lungfish, and its ability to interact with both vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide potently suggested that secretin receptor was descended from a VPAC-like receptor gene before the Actinopterygii-Sarcopterygii split in the vertebrate lineage. Hence, secretin and secretin receptor have gone through independent evolutionary trajectories despite their concurrent emergence post-2R. A functional secretin-secretin receptor axis has probably emerged in the amphibians. Although the pleiotropic actions of secretin are well documented in the literature, only limited information of its physiological functions in nonmammalian tetrapods have been reported. To decipher the structural and functional divergence of secretin and secretin receptor, functional characterization of the ligand- receptor pair in nonmammals would be the next perspective for investigation. [ABSTRACT FROM AUTHOR]

Complexes of secretin (SecR) and angiotensin 1a (Atr1a) receptors have been proposed to be functionally important in osmoregulation, providing an explanation for overlapping and interdependent functions of hormones that bind and activate different classes of GPCRs. However, the nature of these crossclass complexes has not been well characterized and their signaling properties have not been systematically explored. We now use competitive inhibition of receptor bioluminescence resonance energy transfer and bimolecular fluorescence complementation to establish the dominant functionally important state as a symmetrical homodimeric form of SecR decorated by monomeric Atr1a, interacting through lipid-exposed faces of Atr1a TM1 and TM4. Conditions increasing prevalence of this complex exhibited negative allosteric modulatory impact on secretin-stimulated cAMP responses at SecR. In contrast, activating Atr1a with full agonist in such a complex exhibited a positive allosteric modulatory impact on the same signaling event. This modulation was functionally biased, with secretin-stimulated calcium responses unaffected, whereas angiotensin-stimulated calcium responses through the complex were reduced or absent. Further supporting this interpretation, Atr1a with mutations of lipid-exposed faces of TM1 and TM4 that did not affect its ability to bind or signal, could be expressed in the same cell as SecR, yet not exhibit either the negative or positive allosteric impact on cAMP observed with the inactive or activated states of wild type Atr1a on function, and not interfere with angiotensin-stimulated calcium responses like complexes with Atr1a. This may provide a more selective means of exploring the physiologic functional impact of this cross-class receptor complex without interfering with the function of either component receptor. [ABSTRACT FROM AUTHOR]

It is widely acknowledged that kisspeptin and its receptor Kiss1R play central regulatory roles in the hypothalamus-pituitary-gonad (HPG) axis and reproduction. Mutations of KISS1 and KISS1R lead to disorders associated with pubertal development, such as central precocious puberty (CPP) and idiopathic hypogonadotropic hypogonadism (IHH). This review focuses on KISS1 and KISS1R mutations found in CPP and IHH and its purposes are twofold: Firstly, based on the mutations found in KISS1 and KISS1R, this review provides insights into the precise mechanism of kisspeptin and the kisspeptin/Kiss1R pathway in the reproductive axis and in puberty. Secondly, G protein-coupled receptors (GPCRs) are known to share highly conserved structural motifs; therefore, knowledge of mutations found at different structural domains of Kiss1R in the diseased state, and how they affect Kiss1R function can be used to decipher GPCR domain function., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A growing body of evidence suggests that secretin (SCT) is an important element in the osmoregulatory pathway. It is interesting to note that both SCT and its receptor (SCTR) gene are activated upon hyperosmolality in the kidney. However, the precise molecular mechanisms underlying the induction of the SCTR gene expression in response to changes in osmolality have yet to be clarified. Detailed DNA sequence analysis of the promoter regions of the SCTR gene reveals the presence of multiple osmotic response elements (ORE). The ORE is the binding site of a key osmosensitive transactivator, namely, the nuclear factor of activated T-cells 5 (NFAT5). SCTR and NFAT5 are co-expressed in the kidney cortex and medulla collecting duct cells. We therefore hypothesize that NFAT5 is responsible for modulating SCTR expression in hypertonic environments. In this study, we found hypertonicity stimulates the promoter activities and endogenous gene expression of SCTR in mouse kidney cortex collecting duct cells (M1) and inner medulla collecting duct cells (mIMCD3). The overexpression and silencing of NFAT5 further confirmed it to be responsible for the up-regulation of the SCTR gene under hypertonic conditions. A significant increase in the interaction between NFAT5 and the SCTR promoter was also observed following chromatin immunoprecipitation assay. In vivo, osmotic stress up-regulates the SCTR gene in the kidney cortex and medulla of wild-type mice, but does not do so in NFAT5(+/-) animals. Hence, this study provides comprehensive information on how NFAT5 regulates SCTR expression in different osmotic environments., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In the present study, a functional neuron restrictive silencer element (NRSE) was initially identified in the 5' flanking region (-83 to -67, relative to ATG) of human secretin receptor (hSCTR) gene by promoter assays coupled with scanning mutation analyses. The interaction of neuron restrictive silencer factor (NRSF) with this motif was later indicated via gel mobility shift and ChIP assays. The silencing activity of NRSF was confirmed by over-expression and also by shRNA knock-down of endogenous NRSF. These studies showed an inverse relationship between the expression levels of NRSF and hSCTR in the cells. As hSCTR gene was previously shown to be controlled by two GC-boxes which are regulated by the ratio of Sp1 to Sp3, in the present study, the functional interactions of NRSF and Sp proteins to regulate hSCTR gene was investigated. By co-immunoprecipitation assays, we found that NRSF could be co-precipitated with Sp1 as well as Sp3 in PANC-1 cells. Interestingly, co-expressions of these factors showed that NRSF could suppress Sp1-mediated, but not Sp3-mediated, transactivation of hSCTR. Taken together, we propose here that the down-regulatory effects of NRSF on hSCTR gene expression are mediated via its suppression on Sp1-mediated transactivation., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Pituitary adenylate cyclase-activating polypeptide (PACAP)-related peptide (PRP) is a peptide encoded with PACAP in the same precursor protein. Non-mammalian PRPs were previously termed growth hormone-releasing hormone (GHRH)-like peptide, and was regarded as the mammalian GHRH homologue in non-mammalian vertebrates until the discovery of authentic GHRH genes in teleosts and amphibians. Although a highly specific receptor for PRP, which is lost in mammals, is present in non-mammals, a clear function of PRP in vertebrates remains unknown. Using goldfish as a model, here we show the expression of PRP and its cognate receptor in the brain-pituitary-gonadal (BPG) axis, thus suggesting a function of goldfish (gf) PRP in regulating reproduction. We found that gfPRP controls the expression of reproductive hormones in the brain, pituitary and ovary. Goldfish PRP exerts stimulatory effects on the expression of salmon gonadotropin-releasing hormone (sGnRH) in the brain, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in pituitary primary culture cells, but inhibits the expression of LH in the ovary. Using the same technique, we showed that gfPRP did not alter the mRNA level of growth hormone in the pituitary primary culture. In summary, we have discovered the first function of vertebrate PRP in regulating reproduction, which provides a new research direction in studying the neuroendocrine control of reproduction not only in teleosts, but also in other non-mammalian vertebrates., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Although recently discovered, orexins have been rapidly established as important neuropeptides in regulating physiological processes including food intake, sleep/wake cycles and reproduction through binding to two class B G protein-coupled receptors (OX1R and OX2R). To date, a handful of sequences for orexins and their receptors ranging from fish to mammalian species have been identified, allowing a glimpse into their evolution. Structurally, the genetic and molecular organization of the peptides and receptors amongst vertebrates are highly similar, underlining the strong evolutionary pressure that has been exerted to preserve structure and ultimately function. Furthermore, the absence of invertebrate orexin-like sequences suggests early vertebrates as the origin from which orexins evolved. With respect to the receptors, OX2R is probably evolutionary more ancient whilst OX1R is specific to mammalian species and evolved only during this later lineage. In common to all vertebrates studied, the hypothalamus remains to be the key brain region in which orexinergic neurons and fibers are localized in, establishing orexin to be an important player in regulating physiological processes especially those related to food intake and energy metabolism. To allow better understanding of the evolution of orexins and their receptors, this review will provide a comparative approach to their structures and functions in vertebrates., (Copyright © 2011 Elsevier Inc. All rights reserved.)

RASSF1A, which is frequently found inactivated in human cancers, is revealed as a tumor suppressor gene in nasopharyngeal carcinoma (NPC). Using RASSF1A-expressing (NP69 and HK-1) and non-RASSF1A-expressing (C666-1) cell models, the transcriptional regulation of RASSF1A was studied. By deletion analysis of 3.1 kb of 5' flanking region, the core promoter of RASSF1A was identified in the region between -431 and -1 upstream of the translation start site. Sequence analysis of this core promoter revealed several putative transcription factor binding sties. Using NP69 cells and by block replacement mutagenesis, the presence of three functional GC-boxes were identified, to which by competitive and supershift electrophoretic mobility shift assays (EMSA), the in vitro bindings of Sp1 and Sp3 were suggested. The in vivo functions of Sp-proteins in regulating RASSF1A gene were then investigated by overexpression studies; among the tested Sp-proteins, Sp1 or Sp3, but not Sp4, was able to augment promoter activities. More interestingly, co-expression of Sp1 and Sp3 could synergistically enhance RASSF1A promoter function. UV irradiation induces oxidation stresses and hence is routinely used to investigate expressions of oncogenes and tumor suppressors. In this report, upon UV irradiation, the RASSF1A promoter activity and endogenous transcript levels were found to be reduced. By chromatin immunoprecipitation (ChIP) and EMSA, we demonstrated that the binding of Sp1 and Sp3 onto -431 to -202 were significantly reduced after UV irradiation. This UV-mediated effect on RASSF1A promoter, as shown by specific inhibitors that interrupt cellular pathways, is MEK1-, but not JNK-dependent. In summary, our data provided a simple model to explain the potential development of NPC, via silencing of the tumor suppressor RASSF1A by reduced bindings of activators Sp1 and Sp3 onto the GC-boxes in the core promoter of the gene.

Small heterodimer partner (SHP) is an orphan nuclear receptor in which gene expression can be upregulated by bile acids. It regulates its target genes by repressing the transcriptional activities of other nuclear receptors including NeuroD, which has been shown to regulate secretin gene expression. Here, we evaluated the regulation on duodenal secretin gene expression by SHP and selected bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA). In vitro treatment of CDCA or fexaramine elevated the SHP transcript level and occupancy on secretin promoter. The increase in the SHP level, induced by bile acid treatment or overexpression, reduced secretin gene expression, whereas this gene inhibitory effect was reversed by silencing of endogenous SHP. In in vivo studies, double-immunofluorescence staining demonstrated the coexpression of secretin and SHP in mouse duodenum. Feeding mice with 1% CA-enriched rodent chow resulted in upregulation of SHP and a concomitant decrease in secretin transcript and protein levels in duodenum compared with the control group fed with normal chow. A diet enriched with 5% cholestyramine led to a decrease in SHP level and a corresponding increase in secretin expression. Overall, this study showed that bile acids via SHP inhibit duodenal secretin gene expression. Because secretin is a key hormone that stimulates bile flow in cholangiocytes, this pathway thus provides a novel means to modulate secretin-stimulated choleresis in response to intraduodenal bile acids.

PACAP-related peptide (PRP) and PACAP are structurally related peptides that are encoded in the same transcripts. In the past, it was believed that the mammalian PRPs are evolved from GHRHs in non-mammals. With the recent discovery of authentic GHRH and receptor genes in frog and fish, this review aims to (1) coin the name of all GHRH-like peptides in previous literature as PRPs and (2) provide the background for new research direction for PRP in vertebrates. As a goldfish receptor highly specific for PRP with distinct tissue distribution has previously been characterized, it is highly possible that PRP plays a physiological role in non-mammalian vertebrates and the function of PRP has somehow been lost in mammals as a consequence of the loss of its receptor in the genome. This information may provide clues to elucidate functions of PRP in the future.

Gonadotropin-releasing hormone (GnRH) I and II are hypothalamic decapeptides with pivotal roles in the development of reproductive competence and regulation of reproductive events. In this study, transcriptional regulation of the human GnRH II gene was investigated. By scanning mutation analysis coupled with transient promoter assays, the motif at -641/-636 (CATGCC, designated GII-Sil) was identified as a repressor element. Mutation of this motif led to full restoration of promoter activity in TE671 medulloblastoma and JEG-3 placenta choriocarcinoma cells. Supershift and chromatin immunoprecipitation assays showed in vitro and in vivo binding of NF-kappaB subunit p65 and the retinoic acid receptors, RARalpha and RXRalpha, to the promoter sequences. Over-expression of these protein factors indicated that p65 is a potent repressor, and the RARalpha/RXRalpha heterodimer is involved in the differential regulation of the GnRH II gene in neuronal and placental cells. This was confirmed by quantitative real-time PCR. Treatment of cells with the RARalpha/RXRalpha ligands, all-trans retinoic acid and 9-cis-retinoic acid, reduced and increased GnRH II gene expression in TE671 and JEG-3 cells, respectively. Taken together, these data demonstrate the differential roles of NF-kappaB p65 and RARalpha/RXRalpha, interacting with the same sequence in the promoter of the human GnRH II gene to influence gene expression in a cell-specific manner.

Previously, we found that secretin transcript levels were induced by all-trans retinoic acid (RA) in a neuroblastoma cell model, SH-SY5Y. In this article, this RA-dependent upregulation process was further investigated. In the cyclin-dependent kinase 1 (Cdk1) inhibitor-treated cells, the RA-dependent induction of secretin gene expression was inhibited. Together with our previous works, we propose here that the RA responsiveness of the secretin promotor is mediated by two different pathways. The first pathway is by changing the expression levels of NFI-C and Sp proteins while the second pathway is by modifying the phosphorylation status of both NFI-C and Sp proteins via Cdk1.

Previous studies have demonstrated the transcriptional repressive property of the atypical nuclear receptor, small heterodimer partner (SHP), on NeuroD. NeuroD is a basic helix-loop-helix transcription factor that has also been shown to be important in modulating secretin gene expression. The present study revealed the activation of the human secretin core promotor by overexpressing NeuroD, and the localization of SHP and secretin-producing cells in mouse duodenal epithelium by immunohistochemical stainings. These results indicated that SHP and secretin are potentially co-expressed and lead us to propose a novel regulatory pathway, in which SHP represses NeuroD's positive regulatory activity on secretin gene.

Repressor element 1 (RE-1) is a negative, cis-acting regulatory element that interacts with the transcription factor RE-1-silencing transcription factor (REST). REST represses gene expression by two repressor domains that recruit other factors including mSin3 and CoREST. RE-1 has been identified in an increasing number of neuronal-specific genes, and recently, functional REST sites have also been discovered in VIP and PACAP genes. In the present article, we demonstrated for the first time that RE-1 sites are present in the 5' flanking regions of several secretin/PACAP/VIP genes by in silico analysis. This observation suggests that RE-1/REST is a common negative regulatory pathway of this peptide family.