Insulin-like growth factor II precursor gene organization in relation to insulin gene family

The insulin gene family, comprised of insulin, relaxin, insulin-like growth factors I and II (IGF-I and IGF-II) and possibly the beta-subunit of 7S nerve growth factor, represents a group of structurally related polypeptides whose biological functions have diverged. The IGFs, or somatomedins, constitute a class of polypeptides that have a key role in pre-adolescent mammalian growth (see ref. 4 for review). IGF-I expression is regulated by growth hormone and mediates postnatal growth, while IGF-II appears to be induced by placental lactogen during prenatal development. The primary structures of both human IGFs have been determined and are closely related. A polypeptide highly homologous to human IGF-II is secreted by the rat liver cell line, BRL-3A. As this polypeptide, termed multiplication stimulating activity (MSA), differs from human IGF-II by only five amino acid residues, MSA probably represents the rat IGF-II protein. Using molecular cloning techniques, we have isolated cDNA and chromosomal genes coding for the MSA and human IGF-II precursors, respectively. Our data, presented here, indicate that both MSA and human IGF-II are synthesized initially as larger precursor molecules. The deduced preprohormones both have molecular weights (MWs) of 20,100 and contain C-terminal propeptides of 89 amino acid residues, which we have named E-peptides. The organization of the IGF-II precursor gene is discussed in relation to that of other insulin gene family members.     

Sequence of cDNA encoding human insulin-like growth factor I precursor

Somatomedins (SM) or insulin-like growth factors (IGF) constitute a heterogeneous group of peptides with important growth-promoting effects in vitro as well as in vivo. Amino acid sequences have been determined for only two of them, IGF-I and IGF-II, which are highly homologous. IGF-I, which is identical with SM-C, is composed of 70 amino acid residues and IGF-II contains 73 amino acids and may be identical with SM-A. Other peptides with different charge properties but with similar SM-like or insulin-like behaviour in biological and receptor assays, have been described but have not yet been fully characterized. The liver is known to be a major site of production of these peptides, but many other tissues--especially in the fetus--may synthesize them as well. We report here the nucleotide sequence of a human liver cDNA encoding the complete amino acid sequence of IGF-I. The IGF-I coding region is flanked by sequences encoding an amino-terminal peptide of at least 25 amino acid residues and a carboxyl-terminal peptide of 35 amino acids. This provides evidence that IGF-I is synthesized as a precursor protein and that formation of IGF-I from this precursor requires proteolytic processing at both ends.     

Localization of insulin-like growth factor genes to human chromosomes 11 and 12

The insulin-like growth factors IGF-I and IGF-II are required for growth and development. Both are single-chain proteins (of 70 and 67 amino acids respectively) derived from precursors by proteolytic processing. IGF-I may be particularly important in promoting normal stature and IGF-II may be a fetal growth hormone. The IGF proteins are probably synthesized by many normal tissues and by some tumours. The secretion of growth factors by tumours and tumour-derived cell lines suggests that they may act as autocrine regulators of cell proliferation. Because of the possible role of these proteins in growth disorders and cancer, and their sequence homology with insulin, we have determined their chromosomal localization. Using somatic cell hybrids and cloned cDNA probes for these proteins, we have assigned the genes for IGF-I and IGF-II to human chromosomes 12 and 11, respectively. We present evidence that the IGF-II gene is located on the short arm of chromosome 11 with a ras proto-oncogene and the insulin structural gene, and also suggest the existence of a fragment length polymorphism using the IGF-I probe.     

Structural identification of beta- and gamma-human atrial natriuretic polypeptides

Atrial natriuretic polypeptides (ANPs) of varying chain length have been identified recently in human and rat atrial tissue. Their potent natriuretic-diuretic activities indicate their key role in the regulation of extracellular fluid volume and electrolyte balance. Furthermore, human and rat cDNAs encoding their precursor have been cloned and identified. Natriuretic-diuretic activity in human atrial extract comprises three distinct components (alpha, relative molecular mass (Mr) approximately 3,000; beta, Mr approximately 6,000; gamma, Mr approximately 13,000). However, only the 3,000-Mr peptide, alpha-human atrial polypeptide (alpha-hANP), comprising 28 amino acids, has so far been identified. We report here the purification and sequence analysis of two novel hANPs of higher Mr, beta- and gamma-hANP, both of which exhibit natriuretic and hypotensive activity. gamma-hANP, composed of 126 amino acids, carries the alpha-hANP sequence at its carboxy terminus. The identification of gamma-hANP reveals that the peptide, being the largest form of hANP, is processed directly from a 151-residue precursor by removal of a 26-residue signal peptide. In contrast, beta-hANP (56 residues) comprises an anti-parallel dimer of alpha-hANP; such a dimeric peptide possessing bioactivity has never been found in the tissue as an endogenous entity.     

Sequence of a cDNA clone encoding human preproinsulin-like growth factor II

The insulin-like growth factors (IGF) I and II are single-chain serum proteins of 70 and 67 amino acids, respectively, which are synthesized by the liver and possibly other tissues. They are probably required for normal fetal and postnatal growth and development. They also stimulate the growth of cultured cells, possibly by controlling the progression through the G1 phase of the cell cycle. In contrast to IGF-II whose concentration does not vary during postnatal development, the serum levels of IGF-I increase several-fold to adult levels during puberty. The serum concentration of IGF-I is a sensitive monitor of growth hormone levels and is decreased in individuals with growth hormone deficiency and elevated in those with growth hormone-secreting tumours. As a first step in studying the biosynthesis of these proteins and elucidating their role(s) in normal development and in tumorigenesis, we have isolated and sequenced cDNAs prepared from adult human liver mRNA which encode the precursors to IGF-I and -II. We report here the sequence of a cDNA encoding a 180-amino acid protein which is the precursor to IGF-II.     

Human chromosomal mapping of genes for insulin-like growth factors I and II and epidermal growth factor

Many of the actions previously attributed to pituitary-derived growth hormone are mediated by polypeptide growth factors. These include the insulin-like growth factors I and II (IGF-I and IGF-II), which are members of the insulin family of proteins. We report here the chromosomal mapping of the human genes for IGF-I and IGF-II. IGF-II maps to the short arm of chromosome 11, which also contains the gene for insulin and the proto-oncogene c-Ha-ras1 (ref. 9). IGF-I maps to chromosome 12, which is evolutionarily related to chromosome 11 and carries the gene for the proto-oncogene c-Ki-ras2 (refs 10,44). We have also localized the human gene for an unrelated polypeptide hormone, epidermal growth factor, to chromosome 4q, in the same region as another specialized growth factor, T-cell growth factor. We speculate that these map assignments reflect the existence of gene families involved in growth control.     

Epidermal growth factor-dependent phosphorylation of lipocortin

Lipocortin-like proteins are a family of steroid-induced inhibitors of phospholipase activity with potential anti-inflammatory activity. Related proteins have been detected in a variety of tissues and species. The best characterized form is a protein of relative molecular mass (Mr) approximately 40,000 (40K), which is phosphorylated in vivo by protein tyrosine kinases and by protein serine-threonine kinases. It has been proposed that the phospholipase inhibitory activity of lipocortin can be regulated by its phosphorylation. In the A431 cell line, a protein of approximately 35K is phosphorylated by the protein tyrosine kinase activity of the epidermal growth factor (EGF) receptor. Here we report that human lipocortin is phosphorylated near its amino terminus by the EGF receptor/kinase. By peptide mapping and immunological analyses, we show that lipocortin and the endogenous 35K substrate for the EGF receptor/kinase from A431 cells are the same protein.     

Deduced amino acid sequence from the bovine oxytocin-neurophysin I precursor cDNA

The nonapeptide hormone oxytocin-like arginine-vasopressin (AVP) is synthesized as part of a larger precursor polypeptide. The precursor also includes the neurophysin molecule with which the hormone is associated in the neurosecretory granules of the hypothalamo-pituitary tract. A protein of molecular weight (Mr) approximately 20,000 has been isolated from supraoptic nuclei of rat hypothalami which, after tryptic cleavage, released a neurophysin-like molecule of Mr approximately 10,000 and an oligopeptide related to oxytocin. This result was complemented by in vitro translation of bovine hypothalamic mRNA. Among the primary translation products a single polypeptide of Mr approximately 16,500 was shown to contain antigenic determinants recognized by specific antisera against bovine neurophysin I and oxytocin. Here we report the amino acid sequence of the bovine oxytocin-neurophysin I (OT-NpI) precursor which was derived from sequence analysis of the cloned cDNA. As is the case for the bovine arginine-vasopressin-neurophysin II (AVP-NpII) precursor, the signal sequence of the OT-NpI precursor is immediately followed by the nonapeptide hormone which is connected to neurophysin I by a Gly-Lys-Arg sequence. A striking feature of the nucleic acid sequence is the 197-nucleotide long perfect homology with the AVP-NpII precursor mRNA sequence encoding the conserved middle part of neurophysins I and II.     

Human preprovasoactive intestinal polypeptide contains a novel PHI-27-like peptide, PHM-27

Vasoactive intestinal polypeptide (VIP), a 28-amino acid peptide originally isolated from porcine duodenum, is present not only in gastrointestinal tissues but also in neural tissues, possibly as a neurotransmitter, and exhibits a wide range of biological actions (for example, relaxation of smooth muscle, stimulation of intestinal water and electrolyte secretion and release of insulin, glucagon and several anterior pituitary hormones). As the structure of porcine and bovine VIP shows several similarities to those of mammalian glucagon, secretin and gastric inhibitory peptide (GIP), VIP is considered to be a member of the glucagon-secretin family. Recently, we have found that VIP is synthesized from a precursor, pro-VIP (molecular weight (Mr) 17,500), in human neuroblastoma cells and that the primary translation product of the mRNA encoding VIP is prepro-VIP (Mr 20,000). In an attempt to elucidate the primary structure of the precursor, we have now cloned the DNA sequence complementary to the mRNA coding for human VIP and analysed the nucleotide sequence. The entire amino acid sequence of the precursor, deduced from the nucleotide sequence, indicates that the precursor protein contains not only VIP but also a novel peptide of 27 amino acids. The peptide, designated PHM-27, differs by only 2 amino acids from PHI-27, a peptide recently isolated from porcine intestine, and is also closely related in sequence to VIP.     

Sequence, structure, receptor-binding domains and internal repeats of human apolipoprotein B-100

Apolipoprotein (apo) B-100, the major protein component in low density lipoprotein (LDL), is the ligand that binds to the LDL receptor. It is important in the metabolism of LDL and elevated plasma levels of LDL-apo B are strongly associated with increased risk of coronary artery disease. Although apo B-100 is of great clinical and biological importance its primary structure has defied chemical elucidation, mainly because of its enormous size, insolubility, and tendency to aggregate. Less than 5% of the apo B-100 sequence has been reported, despite the efforts of many laboratories over the past twenty years. Here we report the complete amino acid sequence of human apo B-100 as deducted by sequence analysis of complementary DNA clones; 2,366 of the 4,536 residues were also confirmed by direct sequencing of apo B-100 tryptic peptides. The distribution of trypsin-accessible and -inaccessible peptides of the protein on LDL is non-random and they can be grouped into 5 hypothetical domains. Of 20 potential N-glycosylation sites identified in the sequence, 13 were found by direct peptide sequencing to be glycosylated, and 4 unglycosylated. Examination of the primary structure of apo B-100 reveals that it contains a large number of long (greater than 70 residues) internal repeats and an even larger number of shorter ones, suggesting that the apo B-100 sequence was derived largely from internal duplications. Finally, using synthetic peptides of a specific region of apo B-100, we have identified a potential LDL receptor-binding domain (residues 3,345-3,381) which can bind to the LDL receptor and suppress 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase activities in cultured human fibroblasts.     

Molecular cloning and expression of human hepatocyte growth factor

Hepatocyte growth factor (HGF) is the most potent mitogen for mature parenchymal hepatocytes in primary culture, and seems to be a hepatotrophic factor that acts as a trigger for liver regeneration after partial hepatectomy and liver injury. The partial purification and characterization of HGF have been reported. We have demonstrated that pure HGF from rat platelets is a new growth factor effective at concentrations as low as 1 ng ml-1. The effects of HGF and epidermal growth factor (EGF) are additive. The activity of HGF is not species-specific, although it does not stimulate growth in Swiss 3T3 fibroblasts. HGF has a relative molecular mass (Mr) of 82,000 and is a heterodimer composed of a large alpha-subunit of Mr 69,000 and a small beta-subunit of Mr 34,000. Here we report the amino-acid sequence of human HGF determined by complementary DNA cloning and the expression of biologically active human HGF from COS-1 cells transfected with cloned cDNA. The nucleotide sequence of the human HGF cDNA reveals that both alpha- and beta-chains are contained in a single open reading frame coding for a pre-pro precursor protein of 728 amino acids.     

不同物种间胰岛素及其编码mRNA,DNA序列比较与分析

通过PSI-BLAST搜索与人类胰岛素原(含有86个氨基酸)相似的蛋白质序列，并进行比对，计算比对矩阵的相似得分和期望值，同时运用ClustalW算法对不同物种编码前胰岛素原mRNA及其翻译的蛋白质和DNA序列进行多重比对．结果发现，脊椎动物的胰岛素蛋白质一级结构(A链和B链)和mRNA非常相似，但部分动物C肽的部分序列有差异；系统进化分析表明，人和猴、小鼠和大鼠编码胰岛素的mRNA在进化上关系相近．各物种间编码相同氨基酸的核苷酸序列（CDS)相同，但编码胰岛素的DNA序列不同．各物种胰岛素原蛋白质序列中，A链和B链序列保守，C肽有一定的差异；DNA序列差异较大．     

Most of the coding region of rat ACTH beta--LPH precursor gene lacks intervening sequences

The peptide hormones ACTH, beta-endorphin, alpha- and beta-melanotropin(MSH) and possibly gamma-MSH are synthesized in the pituitary gland by the processing of a 32,000-molecular weight (MW) polypeptide called proopiomelanocortin (POMC). The existence of a further precursor (pre form) to POMC containing an additional N-terminal 'leader' peptide has been suggested by analysis of the in vitro translation products of poly(A)-containing RNA from AtT-20 cells, a mouse ACTH-producing cell line of pituitary origin. Nakanishi et al. cloned and sequenced a cDNA copy of the bovine prePOMC mRNA. This sequence confirmed the known structure of the carboxyl half of POMC and revealed the presence of a new MSH-like moiety, gamma-MSH, within the 16,000-MW amino half of the precursor (16K fragment). Recent experiments have suggested that this peptide may act in synergy with ACTH to increase corticosterone and aldosterone production in vivo and in vitro. We have now isolated from a rat genomic DNA library a segment of a DNA encoding most of POMC, using as probe a mouse 144-base pair cloned cDNA fragment encoding beta-MSH and beta-endorphin. The cloned rat gene is one of two (or more) closely related POMC genes. The DNA sequence obtained shows that the cloned POMC gene is not interrupted by any intervening sequence (IVS) between the codon for amino acid 19 and the presumptive poly(A) addition site. This region of POMC encodes all the biologically active peptides mentioned above. The DNA sequence encoding the putative gamma-MSH and the coding sequence that precedes it are highly conserved between rat and cow. This may indicate an as yet unrecognized biological function(s) for the NH2-terminal portion of the 16K fragment.     

Ghrelin is a growth-hormone-releasing acylated peptide from stomach

Small synthetic molecules called growth-hormone secretagogues (GHSs) stimulate the release of growth hormone (GH) from the pituitary. They act through GHS-R, a G-protein-coupled receptor for which the ligand is unknown. Recent cloning of GHS-R strongly suggests that an endogenous ligand for the receptor does exist and that there is a mechanism for regulating GH release that is distinct from its regulation by hypothalamic growth-hormone-releasing hormone (GHRH). We now report the purification and identification in rat stomach of an endogenous ligand specific for GHS-R. The purified ligand is a peptide of 28 amino acids, in which the serine 3 residue is n-octanoylated. The acylated peptide specifically releases GH both in vivo and in vitro, and O-n-octanoylation at serine 3 is essential for the activity. We designate the GH-releasing peptide 'ghrelin' (ghre is the Proto-Indo-European root of the word 'grow'). Human ghrelin is homologous to rat ghrelin apart from two amino acids. The occurrence of ghrelin in both rat and human indicates that GH release from the pituitary may be regulated not only by hypothalamic GHRH, but also by ghrelin.     

Solution structure of the major binding protein for the immunosuppressant FK506

The major FK506 binding protein (FKBP, relative molecular mass approximately 11,800; Mr 11.8K) and cyclophilin (Mr approximately 17K) belong to a class of proteins termed immunophilins. Although unrelated at the amino-acid sequence level, they both possess peptidyl-prolyl cis-trans isomerase activities which are inhibited by immunosuppressants that block signal transduction pathways leading to T-lymphocyte activation. FK506 and rapamycin strongly inhibit the peptidyl-prolyl cis-trans isomerase activity of FKBP, whereas cyclosporin A inhibits that of cyclophilin. The significance of this enzyme activity and the role of the immunophilins in immunoregulation is unknown. To understand better the function of the immunophilins and their interaction with inhibitors, we are investigating the solution structures of FKBP and FKBP-inhibitor complexes by multidimensional NMR methods. Here we report the solution conformation of FKBP, as generated by NMR, distance geometry and molecular dynamics methods. The regular secondary structure of FKBP is composed mainly of beta sheet (approximately 35%) with little helical structure (less than 10%). The hydrophobic core of the molecule, containing the buried side chains of six of the protein's nine aromatic amino acids, is enclosed by a five-stranded antiparallel beta sheet on one side, a loop and a short helix at residues 51-56 and 57-65, and an aperiodic loop at residues 81-95. Examination of the structure suggests a possible site of interaction with FK506.     

Regulation of human insulin gene expression in transgenic mice

Insulin is a polypeptide hormone of major physiological importance in the regulation of fuel homeostasis in animals (reviewed in refs 1,2). It is synthesized by the beta-cells of pancreatic islets, and circulating insulin levels are regulated by several small molecules, notably glucose, amino acids, fatty acids and certain pharmacological agents. Insulin consists of two polypeptide chains (A and B, linked by disulphide bonds) that are derived from the proteolytic cleavage of proinsulin, generating equimolar amounts of the mature insulin and a connecting peptide (C-peptide). Humans, like most vertebrates, contain one proinsulin gene, although several species, including mice and rats, have two highly homologous insulin genes. We have studied the regulation of serum insulin levels and of insulin gene expression by generating a series of transgenic mice containing the human insulin gene. We report here that the human insulin gene is expressed in a tissue-specific manner in the islets of these transgenic mice, and that serum human insulin levels are properly regulated by glucose, amino acids and tolbutamide, an oral hypoglycaemic agent.     

Insulin-like growth factor II receptor as a multifunctional binding protein

The primary structure of human insulin-like growth factor II receptor, predicted from the complementary DNA sequence, reveals a transmembrane receptor molecule with a large extracellular domain made up of fifteen repeat sequences and a small region homologous to the collagen-binding domain of fibronectin. The structural and biochemical features of the IGF-II receptor appear identical to those of the cation-independent mannose-6-phosphate receptor.     

血管紧张素Ⅰ转换酶抑制剂的合成及生理活性研究

用多肽固相合成法合成了血管紧张素Ⅰ转换酶抑制剂(ACEI),经HPLC检查其纯度达99%以上,用质谱测得分子量与理论值相同,氨基酸组分分析与预期结果相一致。动物实验表明,在体外ACEI对大鼠的ACE有明显的抑制作用;在体内能有效地降低血压。     

The cell-cycle regulated proliferating cell nuclear antigen is required for SV40 DNA replication in vitro

Cell-free extracts prepared from human 293 cells, supplemented with purified SV40 large-T antigen, support replication of plasmids containing the SV40 origin of DNA replication. A cellular protein (Mr approximately 36,000) that is required for efficient SV40 DNA synthesis in vitro has been purified from these extracts. This protein is recognized by human autoantibodies and is identified as the cell-cycle regulated protein known as proliferating cell nuclear antigen (PCNA) or cyclin.     

Expression of human growth hormone-releasing factor in transgenic mice results in increased somatic growth

The neurohumoral regulation of growth hormone secretion is mediated in part by two hypothalamic peptides that reach the anterior pituitary via the hypothalamo-hypophysial portal blood system. Somatostatin inhibits the release of growth hormone, whereas growth hormone-releasing factor (GRF) positively regulates both growth hormone synthesis and secretion. Two forms of human GRF, 40 and 44 amino acids long, have been characterized from extra-hypothalamic tumours as well as from the hypothalamus. Analysis of human GRF complementary DNA and genomic clones indicates that the GRF peptides are first synthesized as a 107- or 108-amino-acid precursor protein. To examine the physiological consequences of GRF expression, we have established strains of transgenic mice containing a fusion gene including the promoter/regulatory region of the mouse metallothionein-I (MT-I) gene and the coding region of the human GRF gene. We report that expression of the human GRF precursor protein in these animals results in measurable levels of human GRF and increased levels of mouse growth hormone in plasma and accelerated growth rates relative to control littermates. These results demonstrate a direct role for GRF in the positive regulation of somatic growth. Unexpectedly, female transgenic mice carrying the MT-GRF fusion gene are fertile, in contrast to female transgenic mice expressing human or rat growth hormone, which are generally infertile. These transgenic mouse strains should provide useful animal models for the study of several types of human growth disorders.