Biochemical and Biophysical Research Communications
Regular ArticleCloning of a Splice Variant of the Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Type I Receptor
Abstract
The rat pancreatic acinar cell line AR 4-2J is endowed with numerous PACAP type I receptors (PACAPR1). The cDNA of this receptor was PCR amplified at low stringency using two degenerate primers based on conserved sequences in the TM2 and TM6 segments of secretin, parathormone and calcitonin receptors. One additional amplified band of 574 bp possessed an original 84 bp insert. This fragment, when used as probe for Northern blot analysis, revealed a high Mr (about 7.5 kb) transcript in AR 4-2J cells and also in rat brain. Screening a lambda Uni-Zap bacteriophage library of AR 4-2J cell cDNA yielded one hybridizing clone with an ORF of 1254 bp. The translated 418 amino acid peptide showed 206 identities with rat VIP receptors and 176 identities with secretin receptors. This sequence homology, together with the mRNA distribution, and the expression study of a similar cDNA published very recently (8) indicated that we had cloned PACAPR1 except for its 77 N-terminal amino acids. Its putative I3 loop contained a unique additional 28 amino acid sequence (with four hemi-cystines and several serine, threonine and basic residues). Using RT-PCR we then demonstrated the coexistence of a second form of mRNA, without this added insert, in DNAse-pretreated RNAs from both AR 4-2J cells and normal rat brain. This indicated that coninon alternative splicing provokes the diversification of PACAP type I receptors into PACAPR1A (unspliced) and PACAPR1B (spliced) variants.
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VPAC and PAC receptors: From ligands to function
2009, Pharmacology and TherapeuticsCitation Excerpt :Average EC50 values reported in these studies from the examination of cAMP production, were ~ 0.3 nM for helodermin and ~ 10 nM for VIP and the PACAPs. Finally, the rat PAC1R was simultaneously cloned by several groups within just a few weeks of each other (Hashimoto et al., 1993; Hosoya et al., 1993; Morrow et al., 1993; Pisegna & Wank, 1993; Spengler et al., 1993; Svoboda et al., 1993), either from rat brain cDNA libraries or the rat AR4-2J cell line. The human transcript was cloned in the same year, whereas the mouse variant was cloned three years later (Ogi et al., 1993; Hashimoto et al., 1996b).
Vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase activating polypeptides (PACAPs) share 68% identity at the amino acid level and belong to the secretin peptide family. Following the initial discovery of VIP almost four decades ago a substantial amount of knowledge has been presented describing the mechanisms of action, distribution and pleiotropic functions of these related peptides. It is now known that the physiological actions of these widely distributed peptides are produced through activation of three common G-protein coupled receptors (VPAC1, VPAC2 and PAC1R) which preferentially stimulate adenylate cyclase and increase intracellular cAMP, although stimulation of other intracellular messengers, including calcium and phospholipase D, has been reported. Using a range of in vitro and in vivo approaches, including cell-based functional assays, transgenic animals and rodent models of disease, VPAC/PAC receptor activation has been associated with numerous physiological processes (e.g. control of circadian rhythms) and clinical conditions (e.g. pulmonary hypertension), which underlies on-going research efforts and makes these peptides and their cognate receptors attractive targets for the pharmaceutical industry. However, despite the considerable interest in VPAC/PAC receptors and the processes which they mediate, there is still a paucity of selective and available, non-peptide ligands, which has hindered further advances in this field both at the basic research and clinical level. This review summarises the current knowledge of VIP/PACAP and the VPAC/PAC receptors with regard to their distribution, pharmacology, signalling pathways, splice variants and finally, the utility of animal models in exploring their physiological roles.
Kinins and Neuro-osteogenic Factors
2008, Principles of Bone Biology: Volume 1-2, Third EditionThis chapter summarizes the knowledge of the effects of kinins on bone and the local neuronal influence on bone tissue, as well as the interactions among kinins, neuroosteogenic factors, and cytokines on bone metabolism. Kinins are blood-derived short peptides released from kininogens as a result of the enzymatic action of kallikreins, proteolytic enzymes present in most tissues and body fluids. The biological effects of the kallikrein-kinin system are mainly exerted by bradykinin (BK) and kallidin (Lys-BK) acting on a variety of cells via cell surface receptors of the B2 subtype. This chapter briefly summarizes the kallikrein-kinin system. Following this, it discusses the effects of kinins on bone metabolism, highlighting that calcitonin, added simultaneously with BK, inhibits the bone-resorptive effect of BK. Prostaglandins also act as mediators of bone resorption induced by BK and kinin receptors are also present in bone cells. Studies have also indicated the possibility that kinins may interact with cytokines and activities of and interactions between different bone cells are regulated by a variety of systemic hormones. Finally, skeletal pain in patients with inflammatory and neoplastic disorders clearly suggests the existence of an extensive sensory nervous system in bone tissues and affirm that not only dramatic changes of the neuronal influence on the skeleton, but also more subtle fluctuations, may influence skeletal metabolism.
Cloning and characterization of a PAC1 receptor hop-1 splice variant in goldfish (Carassius auratus)
2006, General and Comparative EndocrinologyIn several vertebrates, it has been demonstrated that alternative splicing of PAC1 receptor (PAC1-R) transcripts can generate a number of functional receptor variants which utilize different signal transduction pathways to mediate their activities. As PACAP is a physiological growth hormone-releasing factor in fish, and PACAP and the PAC1-R are highly conserved in vertebrate evolution, it would be of interest to investigate the structure and cellular distribution, particularly in the pituitary, of PAC1-R splice variants in a fish model. Our laboratory has previously cloned a receptor cDNA corresponding to the goldfish PAC1-R-s (goldfish PAC1-R-short). In the present study, a goldfish PAC1-R-hop1 variant was characterized. Functional expression of goldfish PAC1-R-s and PAC1-R-hop1 in Chinese Hamster Ovary cells revealed that, upon stimulation by ovine PACAP38, these receptor variants exhibited similar EC50 values (8.7 ± 1.5 and 8.8 ± 1.9 nM, respectively) and maximal responses in activating intracellular cAMP production. The presence and expression levels of these transcripts were measured by quantitative real-time PCR in the brain, heart, pituitary and male gonad, and goldfish PAC1-R-s were found to be the predominant form. In situ hybridization of goldfish PAC1-R in the pituitary revealed its prevalent presence in the pars distalis. In summary, the present study provides information to confirm the role of PACAP in the pituitary and to elucidate the pleiotropic effects of PACAP in fish.
VIP as a trophic factor in the CNS and cancer cells
2003, PeptidesThe effects of vasoactive intestinal peptide (VIP) on the proliferation of central nervous system (CNS) and cancer cells were investigated. VIP has important actions during CNS development. During neurogenesis, VIP stimulates the proliferation and differentiation of brain neurons. Addition of VIP to embryonic mouse spinal cord cultures increases neuronal survival and activity dependent neurotrophic factor (ADNF) secretion from astroglial cells. VIP is an integrative regulator of brain growth and development during neurogenesis and embryogenesis. Also, VIP causes increased proliferation of human breast and lung cancer cells in vitro. VIP binds with high affinity to cancer cells, elevates the cAMP and increases gene expression of c-fos, c-jun, c-myc and vascular endothelial cell growth factor. The effects of VIP on cancer cells are reversed by VIPhybrid, a synthetic VPAC1 receptor antagonist. VIPhyb inhibits the basal growth of lung cancer cells in vitro and tumors in vivo and potentiates the ability of chemotherapeutic drugs to kill cancer cells. Due to the high density of VPAC1 receptors in cancer cells, VIP has been radiolabeled with , and to image tumors. It remains to be determined if radiolabeled VIP analogs will be useful agents for early detection of cancer in patients.
PAC1 and PACAP expression, signaling, and effect on the growth of HCT8, human colonic tumor cells
2002, Regulatory PeptidesThe pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1) is a heptahelical, G protein-coupled receptor that has been shown to be expressed by non-squamous lung cancer and breast cancer cell lines, and to be coupled to the growth of these tumors. We have previously shown that PACAP and its receptor, PAC1, are expressed in rat colonic tissue. In this study, we used polyclonal antibodies directed against the COOH terminal of PAC1, as well as fluorescently labeled PACAP, Fluor-PACAP, to demonstrate the expression of PAC1 on HCT8 human colonic tumor cells, using FACS analysis and confocal laser scanning microscopy. Similarly, anti-PACAP polyclonal antibodies were used to confirm the expression of PACAP hormone by this cell line. We then investigated the signal transduction properties of PAC1 in these tumor cells. PACAP-38 elevated intracellular cAMP levels in a dose-dependent manner, with a half-maximal (EC50) stimulation of approximately 3 nM. In addition, PACAP-38 stimulation caused an increase in cytosolic Ca2+ concentration [Ca2+]i, which was partially inhibited by the PACAP antagonist, PACAP-(6–38). Finally, we studied the potential role of PACAP upon the growth of these tumor cells. We found that PACAP-38, but not VIP, increased the number of viable HCT8 cells, as measured by MTT activity. We also demonstrated that HCT8 cells expressed the Fas receptor (Fas-R/CD95), which was subsequently down-regulated upon activation with PACAP-38, further suggesting a possible role for PACAP in the growth and survival of these tumor cells. These data indicate that HCT8 human colon tumor cells express PAC1 and produce PACAP hormone. Furthermore, PAC1 activation is coupled to adenylate cyclase, increase cytosolic [Ca2+]i, and cellular proliferation. Therefore, PACAP is capable of increasing the number of viable cells and regulating Fas-R expression in a human colonic cancer cell line, suggesting that PACAP might play a role in the regulation of colon cancer growth and modulation of T lymphocyte anti-tumoral response via the Fas-R/Fas-L apoptotic pathway.
Differential regulation of pituitary adenylate cyclase-activating peptide receptor variants in the rat suprachiasmatic nucleus
2002, NeurosciencePituitary adenylate cyclase-activating peptide is densely distributed in the suprachiasmatic nucleus, which functions as the circadian pacemaker. A receptor for pituitary adenylate cyclase-activating peptide, denoted as PAC1, exists in six variant forms. We used reverse transcriptase–polymerase chain reaction to identify the PAC1 variants that are expressed in the suprachiasmatic nucleus. Dominant variant forms of PAC1 in the suprachiasmatic nucleus were PAC1short, PAC1hip, and PAC1hop1. By in situ hybridization, we examined 24-h profiles of mRNAs for the identified receptor variants in the suprachiasmatic nucleus in constant darkness and during the light–dark cycle. In constant darkness there were clear circadian rhythms in PAC1short mRNA with a peak at circadian time 4 but no rhythmicity was observed in PAC1hip mRNA or PAC1hop1 mRNA. In light–dark cycles, on the other hand, PAC1hip mRNA displayed a bimodal rhythm with troughs at zeitgeber time 4 and 16 but PAC1hop1 mRNA stayed constant during the day.
These results suggest that PAC1 splice variants are differentially regulated in the rat suprachiasmatic nucleus.