Evolution of insulin-like growth factor I (IGF-I): structure and expression of an IGF-I precursor from Xenopus laevis

By means of a cloning strategy employing the polymerase chain reaction, we have isolated and characterized cDNAs for Xenopus laevis insulin-like growth factor I (IGF-I). These cDNAs encode a primary IGF-I translation product of 153 residues that demonstrates considerable amino acid sequence similarity with IGF-IA peptides from other species. Fifty-seven of 70 residues of the mature protein are identical among human, rat, chicken, and Xenopus IGF-I, while less amino acid conservation is found at the COOH-terminus (25/35 identities) or at the NH2-terminus (24/48 identities) of the precursor protein. Despite the lower degree of structural similarity at the NH2-terminus, in vitro studies of IGF-I biosynthesis and proteolytic processing support a conserved function for the atypically long 48 residue NH2-terminal signal sequence in directing the nascent IGF-I peptide through the secretory pathway. The 5'-untranslated region of Xenopus IGF-I mRNA matches the human, rat, and chicken sequences in greater than 90% of 279 nucleotides. IGF-I mRNAs from all four species encode a conserved upstream open reading frame of 14 amino acids starting 240-250 nucleotides 5' to the translation start site, suggesting a possible role for this region in modulating IGF-I gene expression. The X. laevis IGF-I gene is transcribed and processed into three mRNAs of 1.6, 2.1, and 3.0 kilobases in liver, and IGF-I mRNAs can be detected in liver, lung, heart, kidney, and peritoneal fat of adult animals. These studies demonstrate that both the IGF-I protein precursor and potential regulatory regions of IGF-I mRNA have been conserved during vertebrate evolution, and indicate that like several other polypeptide growth factors, IGF-I may be of fundamental importance in regulating specific aspects of growth and development in all vertebrates.     

Sequences of liver cDNAs encoding two different mouse insulin-like growth factor I precursors.

Complementary DNAs encoding mouse liver insulin-like growth factor I (IGF-I) have been isolated and sequenced. Alternative RNA splicing results in the synthesis of two types of mouse IGF-I precursor that differ in the size and sequence of the COOH-terminal peptide. The sequences of the signal peptides, IGF-I moieties and the first 16 amino acids of the COOH-terminal peptides or E-domains of the two precursors are identical. The sequence difference results from the presence in preproIGF-IB mRNA of a 52 base insertion which introduces a 17 amino acid segment into the COOH-terminal peptide of preproIGF-IB and also causes a shift in the reading frame of the mRNA. As a consequence of this insertion, the COOH-terminal 19 and 25 amino acids of mouse preproIGF-IA and -IB, respectively, are different. The sequences of mouse and human preproIGF-IA are highly conserved and possess 94% identity. In contrast, the sequences of mouse and human preproIGF-IB are quite different in the region of the COOH-terminal peptide. A comparison of the sequences of mouse and human preproIGF-IB mRNA indicates that they are generated by different molecular mechanisms.     

Structural characterization of exon 6 of the rat IGF-I gene.

In rat liver, insulin-like growth factor I (IGF-I) mRNAs exist as two major size classes of 7.5-7.0 kb and 1.2-0.9 kb. The 7.5- to 7.0-kb IGF-I mRNAs predominate in some nonhepatic tissues of the rat. Because the previously reported sequences of rat IGF-I cDNAs and genomic clones account for only 2.1 kb of sequence, the majority of the sequence of 7.5- to 7.0-kb rat IGF-I mRNAs was unknown. Using a combination of nucleotide sequencing of genomic DNA and cDNA clones and Northern hybridization and RNase protection, we have characterized a 6,354-base-long 3' exon (exon 6) of the rat IGF-I gene. The sequence of exon 6 establishes the previously unknown sequence of the 3' end of the 7.5- to 7.0-kb rat IGF-I mRNAs, comprised predominantly of an unusually long 3' untranslated sequence (3'UT). The long 3'UT contains multiple ATTTA, A(T)nA, and (T)nA sequences, as well as inverted repeats. These sequences may contribute to the shorter half-life of the 7.5- to 7.0-kb rat IGF-I mRNAs relative to the 1.2- to 0.9-kb forms that have been demonstrated previously in vitro and in vivo. We also demonstrate that the 7.5- to 7.0-kb rat IGF-I mRNAs are localized to the cytoplasm of rat liver, providing indirect evidence that they are mature and functional mRNAs.     

Structure and evolutionary origin of the gene encoding a human serum mannose-binding protein.

The N-terminal sequence of the major human serum mannose-binding protein (MBP1) was shown to be identical at all positions determined with the amino acid sequence predicted from a cDNA clone of a human liver MBP mRNA. An oligonucleotide corresponding to part of the sequence of this cDNA clone was used to isolate a cosmid genomic clone containing a homologous gene. The intron/exon structure of this gene was found to closely resemble that of the gene encoding a rat liver MBP (MBP A). The nucleotide sequence of the exons differed in several places from that of the human cDNA clone published by Ezekowitz, Day & Herman [(1988) J. Exp. Med. 167, 1034-1046]. The MBP molecule comprises a signal peptide, a cysteine-rich domain, a collagen-like domain, a 'neck' region and a carbohydrate-binding domain. Each domain is encoded by a separate exon. This genomic organization lends support to the hypothesis that the gene arose during evolution by a process of exon shuffling. Several consensus sequences that may be involved in controlling the expression of human serum MBP have been identified in the promoter region of the gene. The consensus sequences are consistent with the suggestion that this mammalian serum lectin is regulated as an acute-phase protein synthesized by the liver.     

Identification and cDNA sequence of delta-preprotachykinin, a fourth splicing variant of the rat substance P precursor.

The neuropeptides substance P and neurokinin A are synthesised from a family of precursor polypeptides encoded by the preprotachykinin A (PPT) gene. In addition to a mRNA (beta-PPT) containing all 7 exons of the gene, alternatively spliced mRNAs lacking either exon 4 (gamma-PPT) or exon 6 (alpha-PPT) have been identified. We have determined the sequences of cDNA clones encoding four variants of PPT mRNA from rat dorsal root ganglion (DRG), including a novel mRNA species (delta-PPT) in which both exons 4 and 6 are absent. The sequence of delta-PPT predicts the existence of a novel tachykinin precursor polypeptide.     

Alternative splicing of glucokinase mRNA in rat liver.

The sequences of two near full-length cDNAs encoding rat liver glucokinase are reported. One of the cDNAs is essentially identical to the cDNA cloned by Andreone, Printz, Pilkis, Magnuson & Granner. [(1989) J. Biol. Chem. 264, 363-369]. The other cDNA contains a 151 bp insertion and a downstream 52 bp deletion. The inserted block of bases has been shown to originate from an optional cassette exon, termed 2A, between the previously described exons 1 and 2. The conceptual translation product from the variant mRNA is identical to the original glucokinase protein for the first 15 amino acids. Next there is a novel polypeptide sequence of 87 residues, comprising 50 residues encoded by the cassette exon and 37 residues specified by an altered reading frame in exon 2. Due to the 52 bp deletion, 17 amino acids of the reference sequence are then missing, after which the sequence reverts to the original. Northern blot analysis with oligonucleotide probes has shown that alternatively spliced mRNA represents about 5% of total glucokinase mRNA. Alternative splicing of glucokinase mRNA in liver may explain earlier findings of minor isoforms of hepatic glucokinase.     

Structure and analysis of the bovine atrial natriuretic peptide precursor gene

The isolation and sequence analysis of the gene encoding the bovine atrial natriuretic peptide (ANP) precursor is described. The bovine-ANP coding sequences are located on three exons which are interrupted by two intervening sequences. Comparison of the bovine, human, rat and mouse ANP gene sequences reveals a common organization of introns and exons and a high degree of sequence homology in the 5'-flanking and coding regions. Examination of the pre-proANP amino acid sequence derived from the bovine gene with those from rat, mouse and human, indicates a high degree of sequence homology in both the amino-terminal and biologically-active carboxy-terminal ANP region. The latter region in the bovine sequence resembles its human counterpart except for a carboxy-terminal Arg-Arg dipeptide.     

The bovine chromogranin A gene: structural basis for hormone regulation and generation of biologically active peptides.

The structure of the gene encoding bovine chromogranin-A has been determined by characterization of two isolated genomic clones. Chromogranin-A is encoded by eight exons, which organize the coding region into several distinct structural and functional domains. Exons 1-5 represent the highly conserved signal peptide and N-terminal domain, which are separated into regions corresponding to the signal peptide, N-terminal sequence, disulfide-bonded loop, and remainder of the conserved N-terminal domain. Exon 6 represents the variable domain and encodes a region that is identical to the novel chromogranin-A-derived peptide chromostatin. Exon 7 encodes the biologically active peptide pancreastatin as well as most of the conserved C-terminal domain, with the remainder found on exon 8. The mRNA sequence obtained from the gene contains five nucleotide differences from the consensus sequence of four reported bovine chromogranin-A cDNA clones. Two of the differences in the gene result in two amino acid changes in the region encoded by exon 6. The structural organization of the chromogranin-A gene resembles that of the chromogranin-B gene in the exons corresponding to the signal peptide, N-terminal sequence, disulfide loop, and C-terminal sequence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular characterization of the murine argininosuccinate synthetase locus.

The cDNA and gene encoding murine argininosuccinate synthetase were cloned and characterized. The cDNA sequence predicts a peptide of 412 amino acids (aa) including the initiator methionine. There is 98% identity with the aa sequence of the human enzyme. The 3'-untranslated region of the cDNA includes two regions of sequence which are conserved between mouse, rat, human and cow. The murine gene contains 16 exons with the start codon occurring in exon 3. Although alternative splicing occurs in primates to include or exclude exon 2, exon 2 sequences were included in the murine mRNA in all tissues and developmental stages examined. The inclusion of exon 2 in murine mRNA, compared to the usual exclusion in human mRNA, may be explained by differences in the donor splice sequences for exon 2.     

Changing the insulin receptor to possess insulin-like growth factor I ligand specificity

To examine the role of the N-terminal part of the insulin-like growth factor I (IGF-I) receptor and insulin receptor in determining ligand specificity, we prepared an expression vector encoding a hybrid receptor where exon 1 (encoding the signal peptide and seven amino acids of the alpha-subunit), exon 2, and exon 3 of the insulin receptor were replaced with the corresponding IGF-I receptor cDNA (938 nucleotides). To allow direct quantitative comparison of the binding capabilities of this hybrid receptor with those of the human IGF-I receptor and the insulin receptor, all three receptors were expressed in baby hamster kidney (BHK) cells as soluble molecules and partially purified before characterization. The hybrid IGF-I/insulin receptor bound IGF-I with an affinity comparable to that of the wild-type IGF-I receptor. In contrast, the hybrid receptor no longer displayed high-affinity binding of insulin. These results directly demonstrate that it is possible to change the specificity of the insulin receptor to that of the IGF-I receptor and, furthermore, that the binding specificity for IGF-I is encoded within the nucleotide sequence from 135 to 938 of the IGF-I receptor cDNA. Since the hybrid receptor only bound insulin with low affinity, the insulin binding region is likely to be located within exons 2 and 3 of the insulin receptor.