Hideki Sugawara

Differential Intracellular Signaling through PAC1 Isoforms as a Result of Alternative Splicing in the First Extracellular Domain and the Third Intracellular Loop

Pituitary adenylate cyclase-activating polypeptide (PACAP), a pleiotropic neuropeptide, performs a variety of physiological functions. The PACAP-specific receptor PAC1 has several variants that result mainly from alternative splicing in the mRNA regions encoding the first extracellular (EC1) domain and the third intracellular cytoplasmic (IC3) loop. The effects on downstream signaling produced by combinations of alternative splicing events in the EC1 domain and IC3 loop have not yet been clarified. In this study, we have used semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to examine the tissue distributions of four PAC1 isoforms in mice. We then established cell lines constitutively expressing each of the PAC1 isoforms and characterized the binding properties of each isoform to PACAP-38, vasoactive intestinal polypeptide (VIP), and the PAC1-specific agonist maxadilan, as well as the resulting effects on two major intracellular signaling pathways: cAMP production and changes in the intracellular calcium concentration. The results demonstrate that the variants of the IC3 loop affect the binding affinity of the ligands for the receptor, whereas the variants of the EC1 domain primarily affect the intracellular signaling downstream of PAC1. Accordingly, this study indicates that the combination of alternative splicing events in the EC1 domain and the IC3 loop create a variety of PAC1 isoforms, which in turn may contribute to the functional pleiotropism of PACAP. This study not only contributes to the understanding of the multiple functions of PACAP but also helps to elucidate the relationship between the structures and functions of G-protein-coupled receptors.

Diverse effects of intrathecal pituitary adenylate cyclase-activating polypeptide on nociceptive transmission in mice spinal cord

Pituitary adenylate cyclase-activating polypeptide (PACAP) immunoreactive neural elements have been detected in the mouse spinal cord. The discrepancy of PACAP actions in the role of sensory transmission has been proposed to have potentiation and inhibition on nociceptive responses after intrathecal application of PACAP. The aim of the present study was to assess nociceptive transmission of PACAP in the mouse spinal cord by comparison with that of substance P (SP). The intrathecal injection of PACAP induced licking or scratching behavior similar to that of SP. These PACAP-induced aversive behaviors showed different manner from SP-induced responses in point of time course. SP-induced aversive responses quickly increased and suddenly disappeared almost within 1 min. Meanwhile, following a long latency after the injection, PACAP-induced aversive responses gradually appeared, and then persisted more than 60 min. In the early phase, PACAP produced an increase of tail flick latency. Pretreatment with 6-hydroxydopamine (6-OHDA) which destroys noradrenaline neuron of descending pain inhibitory systems in the spinal cord markedly abridged the latency and augmented the duration of PACAP-induced aversive responses. In this way, PACAP exhibits diverse effects on nociception, such as an analgesic role in early phase of the injection and subsequently lasting algesia. These results suggest that PACAP as a neurotransmitter or neuromodulator might have crucial role in nociceptive transmission system.

One‐year intranasal application of growth hormone releasing peptide‐2 improves body weight and hypoglycemia in a severely emaciated anorexia nervosa patient

In Japan, growth hormone releasing peptide‐2 (GHRP‐2) is clinically used as a diagnostic agent for growth hormone secretion deficiency, but the therapeutic application of GHRP‐2 has not been studied in anorexia nervosa. GHRP‐2 reportedly exhibits agonistic action for ghrelin receptor and increases food intake.

Implication of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) for Neuroprotection of Nicotinic Acetylcholine Receptor Signaling in PC12 Cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) functions as a neurotrophic factor through PAC1-R, PACAP-specific receptor, in the central nervous system. On the other hand, by interacting with nicotinic acetylcholine receptor (nAChR), nicotine exhibits several neuroprotective effects. Since the relevance of PACAP and nAChR signaling has not been reported so far, we attempted to investigate their relevance in terms of neuroprotection in PC12 cells. Regarding the effect of nicotine on PACAP gene expression, nicotine increased its mRNA level in time-dependent and dose-dependent manners in the PC12 cells differentiated with nerve growth factor (NGF). In addition, luciferase reporter assay demonstrated that nicotine treatment significantly augments the promoter activity of PACAP gene. Since PAC1-R mRNA expression was induced by NGF, PACAP exhibited neuroprotective effect against tunicamycin-induced cell death in the differentiated PC12 cells. Nicotine also exhibited the neuroprotective effect, which was significantly attenuated by Max.d.4 (PAC1-R specific antagonist). These results suggest that the increment of PACAP gene expression due to nicotine treatment might be involved in the neuroprotection by nicotine.

Characterization of the PAC1 Variants Expressed in the Mouse Heart

Abstract:  Pituitary adenylate cyclase‐activating polypeptide (PACAP), a pleiotropic neuropeptide, exerts a variety of physiological functions through three types of G protein–coupled receptors, PAC1, VPAC1, and VAPC2. Characterization of the molecular forms of PAC1 in mouse heart revealed the presence of four types of variant receptors harboring the N or S variant in the first extracellular domain (EC1 domain) with or without the HOP1 insert in the third intracellular cytoplasmic loop (IC3 loop). Then, we assessed the binding affinity and ability to stimulate adenylyl cyclase of the PCA1 variant‐expressing cells for PACAP. Adenylyl cyclase activation by PACAP was markedly influenced with the variant in the EC1 domain as well as that in the IC3 loop, in spite of a little difference in their binding properties. These data suggest that the combination of EC1 domain variants and IC3 loop variants might account for the diversity of intracellular signaling, which might contribute to multiple functions of PACAP including a role in the cardiovascular system.

Characterization of the testis-specific promoter region in the human pituitary adenylate cyclase-activating polypeptide (PACAP) gene

Pituitary adenylate cyclase‐activating polypeptide (PACAP) is a pleiotropic neuropeptide localized in the testis at concentration comparable to that found in the brain, suggesting involvement in spermatogenesis. In this study, we identified the human PACAP testis‐specific exon (TSE) 10.9 kb upstream from the translational start site and found that the testis‐specific transcript of the human PACAP gene was found to be spliced from the TSE into a region of intron 2 without a frameshift. The resulting PACAP precursor has no signal peptide, suggesting that PACAP functions physiologically in an intracrine manner in the testis. The 5′‐flanking region of the TSE contains an 80‐bp fragment with potent promoter activity in testicular F9 cell. Electrophoresis mobility shift assays showed that proteins from the F9 nuclear extract interacted specifically with the 80‐bp fragment. DNA affinity chromatography allowed isolation of the specific proteins bound to the 80‐bp fragment, two of which were identified as Poly (ADP‐ribose) polymerase‐1 (PARP‐1) and TIA‐1‐related protein (TIAR) by mass spectrometry. By using their siRNAs, the depletion of their proteins in F9 cells affected the potent promoter activity of the 80‐bp fragment, suggesting that they might be involved in the testis‐specific gene expression of PACAP.

Alternative Splicing of the Pituitary Adenylate Cyclase-activating Polypetide (PACAP) Receptor Contributes to Function of PACAP-27

Pituitary adenylate cyclase-activating polypeptide (PACAP)-27 and PACAP-38 are neuropeptides performing a variety of physiological functions. The PACAP-specific receptor PAC1 has several variants that result mainly from alternative splicing in the mRNA region encoding the first extracellular (EC1) domain and the third intracellular cytoplasmic (IC3) loop. To characterize the molecular forms of alternative splicing variants of PAC1, we examined the binding affinity and activation of two major second messenger pathways (cAMP production and changes in [Ca2+]i) by PACAP-27. Activation of cAMP was influenced by the variant in both of the EC1 domain and IC3 loops. In the N form in the EC1 domain, the suppressive effect of the HOP1 form in the IC3 loop was enhanced. Regarding the intracellular calcium mobilization assay, the rank order of the potency of PACAP-27 for the different PAC1 isoforms was S/HOP1 >> N/R ≅ S/R >> N/HOP1. In particular, PACAP-27 exhibited remarkable potency of calcium mobilization in the S/HOP1-expressing cells at sub-picomolar concentrations even though the affinities of PACAP-27 to the four PAC1 isoforms were not significantly different. This suggests the specific functions of PACAP-27 due to PACAP-27 preferring PAC1 activation, and leads in clarification of the pleiotoropic function of PACAP.