Kazuhiro Ogi

Conformation of a peptide ligand bound to its G-protein coupled receptor.

Many peptide hormones elicit a wide array of physiological effects by binding to G-protein coupled receptors. We have determined the conformation of pituitary adenylate cyclase activating polypeptide, PACAP(1–21)NH2, bound to a PACAP-specific receptor by NMR spectroscopy. Residues 3–7 form a unique β-coil structure that is preceded by an N-terminal extended tail. This β-coil creates a patch of hydrophobic residues that is important for receptor binding. In contrast, the C-terminal region (residues 8–21) forms an α-helix, similar to that in the micelle-bound PACAP. Thus, the conformational difference between PACAP in the receptor-bound and the micelle-bound states is limited to the N-terminal seven residues. This observation is consistent with the two-step ligand transportation model in which PACAP first binds to the membrane nonspecifically and then diffuses two-dimensionally in search of its receptor; a conformational change at the N-terminal region then allows specific interactions between the ligand and the receptor.

Free fatty acids regulate insulin secretion from pancreatic beta cells through GPR40.

Diabetes, a disease in which carbohydrate and lipid metabolism are regulated improperly by insulin, is a serious worldwide health issue. Insulin is secreted from pancreatic β cells in response to elevated plasma glucose, with various factors modifying its secretion. Free fatty acids (FFAs) provide an important energy source as nutrients, and they also act as signalling molecules in various cellular processes, including insulin secretion. Although FFAs are thought to promote insulin secretion in an acute phase, this mechanism is not clearly understood. Here we show that a G-protein-coupled receptor, GPR40, which is abundantly expressed in the pancreas, functions as a receptor for long-chain FFAs. Furthermore, we show that long-chain FFAs amplify glucose-stimulated insulin secretion from pancreatic β cells by activating GPR40. Our results indicate that GPR40 agonists and/or antagonists show potential for the development of new anti-diabetic drugs.

A novel peptide which stimulates adenylate cyclase: molecular cloning and characterization of the ovine and human cDNAs.

A novel neuropeptide which remarkably stimulates adenylate cyclase in rat anterior pituitary cell cultures has been recently isolated from ovine hypothalami by A. Arimura and his collaborators (Biochem.Biophys.Res.Commun.164, 567-574(1989)). This peptide was designated as PACAP38(Pituitary Adenylate Cyclase Activating Polypeptide with 38 residues). In an attempt to investigate physiological implications of PACAP38, we have succeeded in cloning the cDNAs encoding the precursor of PACAP38 from ovine hypothalamus and human testis. An ovine cDNA encodes a protein of 176 amino acids in which PACAP38 is proceeded by a putative signal peptide and a pro-region (107 amino acids), and followed by a Gly-Arg-Arg sequence for proteolytic processing and amidation. Deduced amino-acid sequence of human PACAP38 was completely identical to that of the ovine isolated peptide. Cloning of PACAP38 cDNAs confirms the expression of the corresponding mRNAs and the presence of this neuropeptide in ovine hypothalamus and also in human testis.

Isolation and cDNA Cloning of a Novel Galanin-like Peptide (GALP) from Porcine Hypothalamus

Galanin is a widely distributed neuropeptide with a variety of physiological functions. Three galanin receptor subtypes, GALR1, GALR2, and GALR3, have been reported. We isolated a novel galanin-like peptide (GALP) from porcine hypothalamus by observing its activity for increasing [35S]GTPγS binding to a membrane preparation of GALR2-transfected cells. The peptide had 60 amino acid residues and a non-amidated C terminus. The amino acid sequence of GALP-(9–21) was completely identical to that of galanin-(1–13). A cloned porcine GALP cDNA indicated that GALP was processed from a 120-amino acid GALP precursor protein. The structures of rat and human GALP-(1–60) were deduced from cloned cDNA, which indicated that the amino acid sequences 1–24 and 41–53 were highly conserved between humans, rats, and pigs. Receptor binding studies revealed that porcine GALP-(1–60) had a high affinity for the GALR2 receptor (IC50 = 0.24 nm) and a lower affinity for the GALR1 receptor (IC50 = 4.3 nm). In contrast, galanin showed high affinity for the GALR1 (IC50= 0.097 nm) and GALR2 receptors (IC50 = 0.48 nm). GALP is therefore an endogenous ligand that preferentially binds the GALR2 receptor, whereas galanin is relatively non-selective.

Structure of the human pituitary adenylate cyclase activating polypeptide (PACAP) gene

The human gene encoding pituitary adenylate cyclase activating polypeptide (PACAP) was isolated and its nucleotide sequence was determined. By comparison with a human PACAP cDNA, the exon/intron organization of PACAP gene was determined. The last exon encoded the longer form of PACAP, PACAP38 and 3-untranslated sequences, suggesting that the shorter form of PACAP, PACAP27 is not generated by alternative splicing mechanisms. The 5-flanking region of the PACAP gene contains several sequence motifs homologous to CRE, TRE, and GHF-1. On the basis of DNA isolated from mouse A9 microcell hybrid clone containing a single human chromosome, the PACAP gene was assigned to human chromosome 18. Furthermore, we determined the locus of the gene to be 18p11 by the chromosomal in situ hybridization technique.

Characterization of murine PACAP mRNA

A murine PACAP precursor cDNA was isolated by screening a brain cDNA library. The amino acid sequence of the precursor was highly similar (from 81% to 93% similarity) to its rat, human, and ovine counterparts. The primary structure of murine PACAP is identical with those from sheep, humans, and rats, indicating that the mature PACAP is well conserved among mammals. Northern blot analysis revealed that the approximately 2.4 kb transcript for the PACAP precursor is expressed in murine brain. The verification that murine PACAP is identical to its human counterpart provides a rationale for physiological and pathophysiological studies of PACAP in mice.

One of the endothelin gene family, endothelin 3 gene, is expressed in the placenta

A cDNA encoding human endothelin 3 (ET‐3) precursor was cloned from a cDNA library from the placenta, and its nucleotide and deduced amino acid sequences were determined. This ET‐3 cDNA was found to contain 2.3 kb pairs and encode prepro‐ET‐3 protein consisting of 224 amino acid residues. The putative big‐ET‐3 seems to consist of 42 amino acid residues. Two of the intron insertion sites were determined with information from nucleotide sequences of the cloned genomic ET‐3 gene. This is the first direct evidence that the ET‐3 gene is transcribed and expressed in the placenta.

Specific expression of human endothelin-2 (ET-2) gene in a renal adenocarcinoma cell line Molecular cloning of cDNA encoding the precursor of ET-2 and its characterization

Analysis of culture medium of human renal adenocarcinoma cells ACHN by RP‐HPLC suggested that the cells specifically secreted human endothelin‐2 (ET‐2). cDNAs encoding human ET‐2 precursor were cloned from a cDNA library constructed with mRNA derived from the ACHN cells, and the nucleotide and deduced amino acid sequences were determined. The ET‐2 cDNA was revealed to contain 1.3 kb and encode prepro‐ET‐2 protein consisting of 178 amino acid residues. The ET‐like sequence found in the prepro‐ET‐1 and ‐ET‐3 was conserved in this prepro‐ET‐2. The Northern blot analysis of mRNA suggested that the transcript of ET‐2 gene was 1.4 kb. This is the first direct evidence that human ET‐2 gene was expressed specifically in tumor cells.

Cloning and expression of a complementary DNA encoding the bovine receptor for pituitary adenylate cyclase-activating polypeptide (PACAP)

A cDNA encoding a pituitary adenylate cyclase-activating polypeptide (PACAP) receptor was cloned from a bovine brain cDNA library using a synthetic oligonucleotide probe corresponding to the partial N-terminal amino acid sequence of the PACAP receptor purified from the bovine brain. The cloned cDNA encoded a polypeptide of 513 amino acid residues with seven putative transmembrane domains. The deduced amino acid sequence exactly matched the N-terminal amino acid sequence of the purified PACAP receptor. It also shared an apparent similarity with the vasoactive intestinal peptide (VIP), secretin, growth hormone releasing hormone, calcitonin, and glucagon receptors, suggesting that the PACAP receptor is a member of the secretin receptor subfamily of the guanine nucleotide-binding regulatory protein-coupled receptor family. Northern blot analysis showed that the size of the major mRNA band which hybridized with the cDNA was about 7 kb in the bovine cerebral-cortex and hippocampus. An expression vector containing the cloned cDNA for the PACAP receptor was introduced into Chinese hamster ovary (CHO) cells. The affinity of PACAP receptors expressed on the transfected CHO cells was quite similar to that of natural PACAP receptors on the bovine brain membranes. Competitive binding experiments showed that PACAP38 displaced the binding of 125I-labeled PACAP27 to the receptors on the CHO cells more efficiently than PACAP27, while VIP was less effective. In addition, both of PACAP27 and PACAP38 elevated the levels of cAMP and inositol phosphates in the transformed CHO cells. These results indicate that the PACAP receptors encoded by the cloned cDNA are identical to the purified PACAP receptors, and that they can stimulate dual signaling cascades.

Distribution of thyrotropin-releasing hormone receptor mRNA in rat peripheral tissues

Since the thyrotropin-releasing hormone receptor (TRH-R) cDNA was isolated, the distribution of TRH-R mRNA has been investigated in the central nervous system (CNS) and the pituitary. However, there has been less genetical studies on the distribution of TRH-R mRNA in the peripheral tissues, although TRH exists not only in CNS but also in the peripheral tissues. In this study we investigated the distribution of TRH-R mRNA in rat peripheral tissues by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis. TRH-R mRNA was detected in almost all of the peripheral tissues tested, although the amount varied considerably depending on the tissues. In the uterus, thymus, ovary, and testis, TRH-R mRNA levels appeared to be relatively high. These results suggest that TRH and its receptor have specific functions in the peripheral tissues as well as in CNS and in the pituitary.