Rat Pituitary Adenylate Cyclase-activating Polypeptide Research Articles

Functional characterization of neural-restrictive silencer element in mouse pituitary adenylate cyclase-activating polypeptide (PACAP) gene expression.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is predominantly localized in the nervous system, but the underlying mechanism in its neuron-specific expression remains unclear. In addition to two neural-restrictive silencer-like element (NRSLE1 and 2), as reported previously, we have identified the third element in -1,601 to -1,581bp from the translational initiation site of mouse PACAP gene and termed it as NRSLE3, of which, the sequence and location were highly conserved among mouse, rat, and human PACAP genes. In luciferase reporter assay, the deletion or site-directed mutagenesis of NRSLE3 in the reporter gene construct, driven by heterologous SV40 promoter, cancelled the repression of luciferase activity in non-neuronal Swiss-3T3 cells. Furthermore, its promoter activity was significantly repressed in Swiss-3T3 cells, but not in neuronal-differentiated PC12 cells. The electrophoretic mobility shift assay (EMSA) with nuclear extracts of Swiss-3T3 cells demonstrated a specific complex with NRSLE3 probe that exhibited the same migration with the neural-restrictive silencer element (NRSE) probe of rat type II sodium channel gene. During neuronal differentiation of PC12 cells, the increment of PACAP mRNA exhibited the correlation with that of REST4 mRNA, which is a neuron-specific variant form of neural-restrictive silencer factor (NRSF). In undifferentiated PC12 cells, trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, which indirectly inhibits NRSF-mediated gene silencing, increased PACAP mRNA level and attenuated the repression of promoter activity of 5' flanking region of mouse PACAP gene containing NRSLEs. These suggest that the NRSE-NRSF system implicates in the regulatory mechanism of neuron-specific expression of PACAP gene.

Androgen Receptor Drives Transcription of Rat PACAP in Gonadotrope Cells

Gonadotropin expression is precisely regulated within the hypothalamic-pituitary-gonadal axis through the complex interaction of neuropeptides, gonadal steroids. and both gonadal- and pituitary-derived peptides. In the anterior pituitary gland, the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) modulates gonadotropin biosynthesis and secretion, acting both alone and in conjunction with GnRH. Steroid hormone feedback also influences gonadotropin expression via both direct and indirect mechanisms. Evidence from nonpituitary tissues suggests that PACAP may be a target for gonadal steroid regulation. In the present study, we show that androgen markedly stimulates rat (r) PACAP promoter-reporter activity in the LβT2 mature mouse gonadotrope cell line. 5'-Serial deletion analysis of reporter constructs identifies 2 regions of androgen responsiveness located at (-915 to -818) and (-308 to -242) of the rPACAP promoter. Androgen receptor (AR) binds directly to DNA cis-elements in each of these regions in vitro. Site-directed mutagenesis of 3 conserved hormone response element half-sites straddling the (-308 to -242) region dramatically blunts androgen-dependent PACAP promoter activity and prevents AR binding at the mutated promoter element. Chromatin immunoprecipitation demonstrates that endogenous AR binds the homologous region on mouse chromatin in LβT2 cells in both the presence and absence of androgen. These data demonstrate that androgen stimulates PACAP gene expression in the pituitary gonadotrope via direct binding of AR to a specific cluster of evolutionarily conserved hormone response elements in the proximal rPACAP gene promoter. Thus, androgen regulation of pituitary PACAP expression may provide an additional layer of control over gonadotropin expression within the hypothalamic-pituitary-gonadal axis.

GnRH Stimulates Expression of PACAP in the Pituitary Gonadotropes via Both the PKA and PKC Signaling Systems

Recent studies have demonstrated a clear role for pituitary adenylate cyclase-activating polypeptide (PACAP) in the regulation of gonadotropin biosynthesis and secretion, both alone and in conjunction with GnRH. First defined as a hypothalamic releasing factor, PACAP subsequently has been identified in the gonadotrope subpopulation of the anterior pituitary gland, suggesting that PACAP may act as an autocrine-paracrine factor in this tissue. In initial studies, we determined that GnRH markedly stimulated endogenous PACAP mRNA levels and promoter-reporter activity in the mature gonadotrope cell line, LbetaT2. GnRH-stimulated rat PACAP promoter activity was blunted with deletion from position -915 to -402 and eliminated with further truncation to position -77 relative to the transcriptional start site. Site-directed mutagenesis demonstrated a functional requirement for a cAMP response element (CRE)-like site at position -205 and an activating protein-1 (AP-1)-like site at position -275, both of which bound CRE binding protein and AP-1 family members on EMSA. Treatment with pharmacological activators or inhibitors of second messenger signaling pathways implicated the protein kinase A, protein kinase C, and MAPK pathways in the GnRH response. In support of these in vitro data, we demonstrate that JunB binds to the rat PACAP gene promoter by chromatin immunoprecipitation assay and that small interfering RNA knockdown of JunB, cFos, and CRE binding protein factors blunts PACAP expression. In summary, these results further elucidate the complex functional interactions between PACAP and GnRH in the anterior pituitary. Specifically, these studies demonstrate that GnRH-stimulated PACAP gene expression is mediated via multiple signaling pathways acting on CRE/AP-1 sites in the proximal gene promoter. Because both PACAP and GnRH regulate gonadotropin biosynthesis and secretion, these results provide important insight into the critical fine tuning of gonadotrope function and, thereby, the maintenance of normal reproductive function.

PACAP/VIP receptors in pancreatic beta-cells: their roles in insulin secretion.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide belonging to the vasoactive intestinal polypeptide (VIP)/glucagon/secretin family. We have isolated a third PACAP receptor subtype, designated PACAPR-3, by molecular cloning. The cDNA encoding PACAPR-3 has been isolated from a mouse insulin-secreting beta-cell line MIN6 cDNA library. Mouse PACAPR-3 is a protein of 437 amino acids that has 50% and 51% identity with rat PACAP type I and type II receptors, respectively. We have expressed PACAPR-3 in mammalian cells and Xenopus oocytes. PACAPR-3 binds to VIP as well as PACAP-38 and -27, with a slightly higher affinity for PACAP-38, and is positively coupled to adenylate cyclase. PACAP-38, -27, and VIP evoked Ca2+ activated-Cl- currents in Xenopus oocytes. RNA blotting studies reveal that PACAPR-3 mRNA is expressed widely in tissues and cell lines, including pancreatic islets, insulin-secreting cell lines (MIN6, HIT-T15, and RINm5F), lung, brain, stomach, colon, and heart. Furthermore, insulin secretion from the MIN6 cells is stimulated significantly by PACAP-38 and VIP. The possible mechanisms of insulin secretion by PACAP and VIP are also discussed.

Neural-restrictive silencers in the regulatory mechanism of pituitary adenylate cyclase-activating polypeptide gene expression

Pituitary adenylate cyclase-activating polypeptide (PACAP) is known as a pleiotropic neuropeptide and is present abundantly in central nervous system. During a detailed analysis of the 5′-flanking region of the mouse PACAP gene, we found and characterized two negative regulatory elements, which are homologous to the neural-restrictive silencer element, and are termed neural-restrictive silencer-like elements 1 and 2 (NRSLE1 and NRSLE2). Their sequence and position were significantly conserved among mouse, human, and rat PACAP genes. In the electrophoretic mobility shift assay (EMSA) with nuclear extracts of Swiss-3T3 cells and individual oligonucleotide probes for NRSLE1 and NRSLE2, a specific complex was observed to have the same migration as compared with the NRSE probe of rat type II sodium channel gene (NaII). Furthermore, these complexes were efficiently competed by the unlabeled NaII probe. In the luciferase reporter assay, the reporter gene constructs containing NRSLEs, driven by heterologous SV40 promoter, exhibited repression of luciferase activity almost equal to basal level in Swiss-3T3 cells. In contrast, the repression was not observed in differentiated PC12 cells with NGF. These results suggested that the neural-restrictive silencer system might be involved in the regulatory mechanism of neuron-specific PACAP gene expression.

The regulatory mechanism for neuron specific expression of PACAP gene

Pituitary adenylate cyclase-activating polypeptide (PACAP), a pleiotropic neuropeptide, is present abundantly in the central nervous system. In the 5'-flanking region of the PACAP gene, we found and characterized two negative regulatory elements, which are homologous to the neural-restrictive silencer element (NRSE). Their sequence and position were significantly conserved among mouse, human, and rat PACAP genes. NRSE is a crucial negative-acting DNA regulatory element for neuron-specific gene expression. NRSE acts through the transcription factor known as neural-restrictive silencer factor (NRSF). In non-neuronal cells, NRSF suppresses the expression of neuron-specific genes. On the other hand, in neuronal cells, NRnV, a NRSF truncated form, repress their expressions in a dominant negative manner. The electrophoretic mobility shift assay with 3T3 cells extract demonstrated the identical complexes among NRSLE-1, NRSLE2, and the NRSE of rat type II sodium channel gene. In the luciferase reporter assay, NRSLEs suppressed SV40 promoter activity in 3T3 cells, but not in PC12 cells. RT-PCR analysis revealed that PACAP and NRnV mRNAs are expressed in neuronal cells (differentiated PC12), but not in non-neuronal cells (3T3 or C6). These results suggested that the NRSE-NRSF system might be involved in the regulatory mechanism of neuron-specific expression of the PACAP gene.

The effect of pituitary adenylate cyclase activating polypeptide on cultured rat cardiocytes as a cardioprotective factor.

In the cardiovascular system, pituitary adenylate cyclase activating polypeptide (PACAP) exhibits not only vasodilation but also positive inotropic action by increasing cardiac output. Then the effect of PACAP in cultured cardiovascular cells was examined. In neonatal rat myocytes, PACAP evoked concentration-dependent increase in intracellular cyclic AMP content more potently than vasoactive intestinal polypeptide (VIP). However, in neonatal rat nonmyocytes, PACAP and VIP showed equal potency. The characterization of the subtype of PACAP/VIP receptors by RT-PCR analysis revealed that PAC1 receptor mRNA is dominantly present in the myocytes, but VPAC2 receptor mRNA is abundant in the nonmyocytes. In the myocytes, PACAP did not change the protein synthesis stimulated by endothelin or by itself. However, PACAP moderately stimulated the secretion of atrial natriuretic polypeptide (ANP). On the other hand, PACAP inhibited the protein synthesis and DNA synthesis of the nonmyocytes. These indicate that PACAP might be involved in the regulation of cardiac hypertrophy and fibrosis as a cardioprotective factor.

A mutation in the second intracellular loop of the pituitary adenylate cyclase activating polypeptide type I receptor confers constitutive receptor activation

The pituitary adenylate cyclase activating polypeptide (PACAP) type I receptor belongs to the glucagon/secretin/vasoactive intestinal polypeptide (VIP) receptor family. We mutated and deleted an amino acid residue (E261) which is located within the second intracellular loop of the rat PACAP type I receptor and which is highly conserved among the receptor family. The wild-type receptor and the mutant receptors were efficiently expressed at the surface of COS-7 cells at nearly the same level and revealed the same high affinity for the agonist PACAP-27. The cAMP contents of COS cells transfected with the E261A, E261Q, and the deletion mutant receptor were 4.6-, 5.7-, and 6.7-fold higher as compared with COS cells transfected with the wild-type receptor. Thus, all the mutant PACAP receptors were constitutively active. The data suggest that the glutamic acid in the second intracellular loop of the PACAP receptor may be a key residue to constrain the receptor in the inactive conformation with respect to its coupling to G s proteins.

Specific antibody recognition of rat pituitary adenylate cyclase activating polypeptide receptors.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a new member of the secretin/VIP family of peptides. The specific receptor for PACAP has been cloned in rat, human, and bovine tissues. The distribution of the transcripts of PACAP receptor genes has been studied in various tissues using in situ hybridization. However, the unavailability of a specific antibody against the PACAP receptor has hampered further study of the expression of receptor proteins. In the present study, rabbit antisera were generated against a synthetic 25-residue peptide corresponding to the C-terminal intracellular domain of the rat PACAP receptor. To validate the specificity of the antisera, CHO cells and cells stably transfected with rat PACAP receptor cDNA were prepared. Using one of these antisera, the membrane and soluble fractions of the transformants were examined by Western blot analysis. Three bands were observed in subcellular fractions from the transfected CHO cells, but no bands were found in similar preparations from the nontransfected cells. A distinct 57-kDa band, which corresponds to the size of cloned rat PACAP receptor, was detected. In addition, a less intense band, larger than 57 kDa, and a very weakly stained band, smaller than 57 kDa, were demonstrated. All of these bands disappeared or were considerably diminished when the antiserum was preabsorbed with the synthetic immunogen peptide. This suggests that these bands are PACAP receptor-related proteins. The membranes from the transfected CHO cells bound to [125I]PACAP27. The size of the ligand/protein crosslinked product approximated 60 kDa, corresponding to the combined size of the PACAP receptor and PACAP27. No additional bands were observed, indicating that the immunopositive proteins larger or smaller than 57 kDa do not bind to the ligand and are not functional. Unlabeled PACAP27 and PACAP38, but not VIP, displaced the binding, suggesting that the receptors expressed in CHO cells are specific for PACAP. Solubilized membrane fractions prepared from rat brains were used for an immunoprecipitation study with [125I]PACAP27 and [125I]VIP. The PACAP receptor antiserum recognized [125I]PACAP-, but not [125I]VIP-bound proteins in the solubilized brain membrane fractions. Immunohistochemistry using this antiserum showed a distribution of PACAP receptor-like immunoreactivities similar to the distribution of the mRNA of PACAP receptor in the rat brain. Thus, the PACAP receptor antiserum is sufficiently specific to be used as a tool for studying the expression of PACAP receptors and related proteins.

Vasoactive intestinal polypeptide and pituitary adenylate cyclase-activating polypeptide receptor chimeras reveal domains that determine specificity of vasoactive intestinal polypeptide binding and activation.

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) receptors are closely related G protein-coupled receptors with seven-transmembrane domains. The VIP receptor can bind both VIP and PACAP with high affinity, whereas the PACAP receptor binds only PACAP with high affinity. To elucidate the structural domains involved in a selectivity for VIP binding and the subsequent receptor activation, a series of chimeric receptors between the VIP and PACAP receptors was constructed, expressed in COS-7 cells, and analyzed for ligand binding and cAMP generation. All chimeric constructs bound PACAP with high affinity and subsequently activated cAMP generation similarly to the wild-type receptors. In contrast, profound differences were observed in the potencies of VIP for competition of 125I-labeled PACAP binding to both wild-type receptors and the chimeric receptors. The cAMP responses of these receptors generally correlated with the ability of VIP to compete for PACAP radioligand binding with the exceptions for some particular chimeras. In this report we demonstrate that several domains, including the amino-terminal extracellular domain, the transmembrane domains I and II, and the first extracellular loop of the VIP receptor, are important for the selectivity for VIP binding and responsiveness to VIP. We further show that the third extracellular loop and its proximal domains of the VIP receptor appear to be involved in the VIP recognition, especially the receptor activation process. On the other hand, the direct binding experiments of the VIP radioligand demonstrated that both wild-type receptors and all chimeric receptors have a high affinity binding site for VIP, although this high affinity VIP binding resulted in a biological response only in the VIP receptor or VIP receptor-like chimeras. This suggests that there is a nonbiologically relevant high affinity VIP-binding site within the rat PACAP receptor.

The Third Intracellular Domain of the Platelet-activating Factor Receptor Is a Critical Determinant in Receptor Coupling to Phosphoinositide Phospholipase C-activating G Proteins: STUDIES USING INTRACELLULAR DOMAIN MINIGENES AND RECEPTOR CHIMERAS

Platelet activating factor (PAF) is a potent phospholipid mediator which elicits a diverse array of biological actions by interacting with G protein-coupled PAF receptors (PAFR). Binding of PAF to PAFRs leads to activation of G protein(s) that stimulate phosphoinositide phospholipase C and subsequent intracellular signaling responses. To identify the potential role of intracellular domains of the rat PAFR (rPAFR) in signaling, we examined effects of transfecting minigenes encompassing rPAFR intracellular domains 1 (1i), 2 (2i), and 3 (3i) on inositol phosphate (IP) production mediated by the co-transfected rPAFR cDNA. Although transfection of the rPAFR1i and rPAFR2i minigenes had no effects on PAF-stimulated signaling, transfection of the rPAFR3i minigene inhibited PAF-stimulated IP production by approximately 50% compared to controls. The rPAFR3i domain did not inhibit IP production mediated by the multifunctional rat pituitary adenylate cyclase-activating polypeptide receptor (rPACAPR), demonstrating the specificity of the competition by the rPAFR3i domain. In further experiments, the rPAFR3i domain was engineered onto the homologous domain of a monofunctional transmembrane variant of the rPACAPR (rPACAPR2) that activates only adenylyl cyclase. The rPACAPR2/rPAFR3i chimera responded to PACAP with increases in IP production which were attenuated nearly completely in cells cotransfected with the rPAFR3i domain. In contrast, PACAP had no effects on IP production in a receptor chimera expressing a mutated form of the rPAFR3i domain (rPACAPR2/rPAFR3imut). These results demonstrate the ability of the rPAFR3i domain to confer a phospholipase C-signaling phenotype to a receptor deficient in this activity and show that this activity is specific for the engineered rPAFR3i domain. These results suggest that the third intracellular loop of the rPAFR is a primary determinant in its coupling to phosphoinositide phospholipase C-activating G proteins, providing the first insight into the molecular basis of interaction of PAFRs with signal-transducing G proteins.

Cloning and molecular characterization of complementary deoxyribonucleic acid corresponding to a novel form of pituitary adenylate cyclase-activating polypeptide messenger ribonucleic acid in the rat testis.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide that greatly stimulates adenylyl cyclase activity in cultured anterior pituitary cells, was isolated from ovine hypothalamus in 1989. Investigation of the distribution of PACAP messenger RNA (mRNA) in rat tissues by Northern blot analysis revealed an anomalous signal in the testis. In this study we have isolated and characterized this unusual mRNA, which is approximately 800 bases long (approximately 1.5 kilobases shorter than that reported in the rat hypothalamus). Cloning and sequencing of the complementary DNA corresponding to this message revealed that the sequences are identical except for 126 bases at the 5'-end of the 5'-untranslated region of the smaller transcript. This region has no homology to either the published hypothalamic sequence or any other known sequence. Northern blot analysis of total RNA from various species showed that a smaller form of PACAP mRNA is also present in human, murine, and bovine testis, although in these species the message is slightly smaller. In addition, Northern blot analysis of these tissues using a probe directed to the 126-base 5'-region, revealed conservation of this sequence between species. Although the structure of the rat PACAP gene is unknown, preliminary investigations into the origins of the two mRNA species by PCR of genomic DNA suggests that they are transcribed from separate genes and not the product of alternate splicing.

Cloning and functional characterization of a third pituitary adenylate cyclase-activating polypeptide receptor subtype expressed in insulin-secreting cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide belonging to the vasoactive intestinal polypeptide/glucagon/secretin family. It is widely distributed in the body, and a variety of biological actions have been reported. PACAP exerts its biological effects by binding to specific receptors that are coupled to GTP-binding proteins. Recent studies have shown that there is a family of PACAP receptors (PACAPRs), and two members of this family have been identified. We report here the cloning, functional expression, and tissue distribution of a third PACAPR subtype, designated PACAPR-3. The cDNA encoding PACAPR-3 has been isolated from a mouse insulin-secreting beta-cell line MIN6 cDNA library. Mouse PACAPR-3 is a protein of 437 amino acids that has 50% and 51% identity with rat PACAP type I and type II receptors, respectively. Expression of recombinant mouse PACAPR-3 in mammalian cells shows that it binds to vasoactive intestinal polypeptide as well as PACAP-38 and -27, with a slightly higher affinity for PACAP-38, and is positively coupled to adenylate cyclase. The expression of PACAPR-3 in Xenopus oocytes indicates that calcium-activated chloride currents are evoked by PACAP and vasoactive intestinal polypeptide, suggesting that PACAPR-3 can also be coupled to phospholipase C. RNA blot analysis studies reveal that PACAPR-3 mRNA is expressed at high levels in MIN6, at moderate levels in pancreatic islets and other insulin-secreting cell lines, HIT-T15 and RINm5F, as well as in the lung, brain, stomach, and colon, and at low levels in the heart. Furthermore, insulin secretion from MIN6 cells is significantly stimulated by PACAP-38. These results suggest that the diverse biological effects of PACAP are mediated by a family of structurally related proteins and that PACAPR-3 participates in the regulation of insulin secretion.

Molecular Cloning and Functional Expression of a cDNA Encoding a Human Pituitary Adenylate Cyclase Activating Polypeptide Receptor

A functional cDNA clone for a human pituitary adenylate cyclase activating polypeptide (PACAP) receptor was isolated from a human pituitary cDNA library. The cDNA encoded a polypeptide consisting of 525 amino acids with putative seven hydrophobic domains. Chinese hamster ovary (CHO)-K1 cells transfected with the cDNA specifically bound PACAP and mediated PACAP-triggered intracellular accumulation of cAMP, indicating that this cDNA encoded a functional human PACAP Type I receptor. This receptor was structurally related to the vasoactive intestinal peptide (VIP), secretin, calcitonin and parathyroid hormone receptors and is much more homologous to a rat PACAP receptor. Northern blot analysis revealed that PACAP receptor mRNAs were expressed mainly in the brain and widely distributed in the central nervous system.

Molecular cloning and tissue distribution of a receptor for pituitary adenylate cyclase-activating polypeptide

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a polypeptide hormone related to vasoactive intestinal polypeptide (VIP). Rat PACAP receptor cDNA was isolated from a brain cDNA library by cross-hybridization with rat VIP receptor cDNA. The recombinant PACAP receptor expressed in COS cells bound PACAP with about 1000 times higher affinity than VIP, and PACAP stimulated adenylate cyclase through the cloned PACAP receptor. The rat PACAP receptor consists of 495 amino acids, contains seven transmembrane segments, and has a significant similarity with other G s-coupled receptors, such as VIP, glucagon, and secretin receptors. PACAP receptor mRNA was abundantly expressed in the brain, but not in the peripheral tissues except for the adrenal gland. In situ hybridization revealed a high level of expression of PACAP receptor mRNA in the hippocampal dentate gyrus, olfactory bulb, and cerebellar cortex.

Molecular Cloning and Functional Expression of Rat cDNAs Encoding the Receptor for Pituitary Adenylate Cyclase Activating Polypeptide (PACAP)

Two types of cDNA encoding PACAP receptors were isolated from the rat brain cDNA library by homology screening with a cDNA probe for VIP receptor. Nucleotide sequence analysis indicated that these two types of receptor mRNA were generated by alternative splicing mechanisms. These two cloned cDNAs were introduced into CHO cells respectively. Resultant transformants showed specific binding to [ 125I]PACAP27 which was displaced by unlabeled PACAP27 but not by VIP. Thus, these receptors are two subtypes of Type I PACAP receptor (Type I-A and Type I-B). The amino acid sequences of rat PACAP receptors deduced by the cDNAs showed a remarkable similarity with rat receptors for VIP, secretin, glucagon, and GHRH. A 6.5 kb significant hybridizing signal of the PACAP receptor mRNA was detected in the rat brain, and slight signal was also detected in the lung and the liver.

Molecular cloning and characterization of cDNA for the precursor of rat pituitary adenylate cyclase activating polypeptide (PACAP)

We have isolated and cloned a cDNA for rat pituitary adenylate cyclase activating polypeptide (PACAP) precursor protein from the brain. The nucleotide and the deduced amino acid sequences revealed that the rat PACAP cDNA encoded a 175 amino acid protein highly homologous to the ovine and human PACAP precursors. The rat PACAP precursor protein included a putative signal peptide at the amino-terminus, a PACAP-related peptide (PRP) of 29 amino acid residues and the rat PACAP of 38 amino acid residues flanked by basic processing sites, and a carboxy-teminus amidation signal for PACAP. Furthermore, the amino acid sequence of the rat PACAP was completely identical to that of the ovine and human PACAP. Northern blot analysis of rat brain RNA revealed an approximately 3.0 kb transcript for the PACAP precursor in rat brain.