Rat Insulin-like Growth Factor Binding Research Articles

Dideoxy sequencing and structural analysis of the rat insulin-like growth factor binding protein-1 gene

Insulin-like growth factor binding protein-1 (IGFBP-1) is an important modulator of IGF bioavailability. To facilitate studies of IGFBP-1 regulation and function in rodent models, we cloned the rat IGFBP-1 gene and analyzed its structure by dideoxy sequencing. The rat IGFBP-1 gene is relatively small (∼ 5 kb) and contains 4 exons and 3 introns, similar to the human IGFBP-1 gene.

Insulin and Glucocorticoids Regulate IGFBP-1 Expression via a Common Promoter Region

Hepatic expression of insulin-like growth factor binding protein-1 is regulated by insulin and glucocorticoids. To study underlying mechanisms, rat hepatocytes in primary culture were transfected with deletion mutants and heterologous promoter constructs, identifying a 41 bp region of the rat insulin-like growth factor binding protein-1 promoter which is sufficient to mediate regulation by both insulin and glucocorticoids. Half maximal suppression of promoter activity by insulin occurred at a physiologic concentration, 5 × 10-10 M, and regulation by insulin was dominant in that insulin suppressed promoter activity at all dexamethasone concentrations. Transfection of rat hepatocytes in primary culture should be a useful approach for exploring the regulation of gene expression by insulin.

Structural Characterization of the Rat Insulin-like Growth Factor Binding Protein-6 Gene

Recently a family of six distinct insulin-like growth factor binding proteins (IGFBPs) have been identified and the gene structures of the first five (IGFBP-l, -2, -3, -4 and -5) characterized. We now isolated the IGFBP-6 gene from a rat genomic library and determined its organization as well as the DNA sequence at the 5′ flanking region of the gene. The rat IGFBP-6 gene spans 5.1 kb of the genomic sequence and contains four exons interrupted by three introns of approximately 2.4, 0.2 and 1.2 kb in length, respectively. Primer extension analysis and nbonuclease protection assay using RNA from rat lung tissues demonstrated two transcriptional start sites located at 85 and 82 nucleotides upstream of the ATG translational initiation codon. The promoter region of the rat IGFBP-6 gene is devoid of a TATA or a CAAT consensus sequence motif, but putative regulatory cis elements, including a Sp1, an estrogen receptor binding site and a retinoic acid responsive element, are present in the promoter region.

Structure of the Rat Insulin-like Growth Factor Binding Protein-4 Gene

A family of six distinct insulin-like growth factor binding proteins (IGFBPs) have been isolated and their cDNA sequences characterized from rat and human species. Recently, the gene structures of the first three IGFBPs (IGFBP-l, -2 and -3) have also been determined. We now report the isolation of the rat IGFBP-4 gene and its genomic organization, as well as the DNA sequence of the promoter region. Rat IGFBP-4 gene spans at least 12 kilobases (kb) of the genomic sequence. It consists of four exons separated by three introns with approximate size of 6.4, 0.6 and 2.7 kb. A single transcriptional start site is located at 249 nucleotides upstream of the translational initiation ATG codon. The rat IGFBP-4 gene possesses a typical TATA box and a CAAT box, as well as multiple potential cis elements, including three cAMP responsive elements, three AP- 1 binding sites and one progesterone receptor binding site in the 5′ flanking region. The presence of these potential cis elements supports the tissue-specific expression of the IGFBP-4 gene.

Cloning of the Rat Insulin-like Growth Factor Binding Protein-5 Gene and DNA Sequence Analysis of Its Promoter Region

To understand the regulation of the insulin-like growth factor binding protein-5 (IGFBP-5) gene expression, we have cloned the IGFBP-5 gene from rat genomic libraries and determined its genomic organization as well as the DNA sequence at the 5′ flanking region of the gene. The rat IGFBP-5 gene spans at least 17 kilobases (kb) of the genome and contains 4 exons interrupted by 3 introns of approximately 10, 0.6 and 0.7 kb, respectively. Southern blot analysis of the rat chromosome DNA revealed a single copy gene for IGFBP-5 in the haploid genome. Primer extension experiments demonstrated a single transcriptional start site located at 772 nucleotides 5 of the ATG translational start codon. In addition to a TATA box and a CAAT box, multiple putative cis-regulatory elements, including an AP- 1, an AP-2 and a binding site for progesterone receptor are present in the promoter region. This finding suggests that the IGFBP-5 gene is one of those that are expressed in a tissue-specific manner.

Molecular cloning of the acid-labile subunit of the rat insulin-like growth factor binding protein complex

The insulin-like growth factors, IGF-I and IGF-II, circulate in both humans and rats as part of a 125–150 kDa complex comprising IGFs, the IGF binding protein IGFBP-3, and an acid-labile subunit. Clones encoding rat acid-labile subunit have been isolated from a rat liver cDNA library probed with a human acid-labile subunit cDNA. Two overlapping clones encode a leucine-rich protein of 576 amino acids preceded by a 27-residue signal sequence, with 78% homology to the human acid-labile subunit. Northern analysis of mRNA from adult rat brain, kidney, heart, lung, spleen, muscle and liver shows a major species of about 4.4 kb and minor bands of about 2 kb, 1.4 kb and 1 kb. The tissue distribution of this protein may therefore be wider than previously recognized.

Cloning of the rat insulin-like growth factor binding protein-1 gene and analysis of its 5′ promoter region

To understand specific mechanisms involved in the regulation of insulin-like growth factor binding protein-1 (IGFBP-1), an important modulator of IGF bioactivity, we cloned the rat IGFBP-1 gene and sequenced a 1.5 kb Sph1-Sph1 fragment containing 1110 bases upstream from the translation start site. computer analysis reveals the presence of ATA, CACCC, and CCAAT elements, and putative homeodomain, AP-1, insulin and glucocorticoid response elements in the 5′ promoter. Primer extension and ribonuclease protection studies reveal a single cap site in RNA from rat hepatoma cells and both control and diabetic rat liver.

Developmental Expression of Rat Insulin-Like Growth Factor Binding Protein-2 by Astrocytic Glial Cells in Culture*

Brain astrocytes were established in primary culture from postnatal and adult rats to characterize the developmental expression of secreted proteins. Astrocytes cultured from 21-day rat brain, but not 1-day rat brain, secreted a distinct group of proteins with Mr of 35,000 as determined by analysis of [35S]methionine-labeled proteins using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal amino acid sequence analysis of this protein group showed 100% identity to rat insulin-like growth factor binding protein-2 (rIGFBP-2), the BRL-3A IGFBP purified from a fetal rat liver cell line. An antiserum was generated against this astrocyte 35,000 Mr protein, and immunoblot analysis revealed a dramatic increase in rIGFBP-2 secretion in astrocytes cultured from 14-day, 21-day, and adult rat brain compared to astrocytes from 1-day and 7-day rat brain. Similar analysis of neonatal rat brain neurons in culture failed to show immunoreactive rIGFBP-2 in cell lysates or secreted protein. Ligand Western blot analysis demonstrated [125I]IGF-II binding to a single protein band which comigrated with a prominant rIGFBP-2 immunoreactive species in nonreduced conditioned medium from 21-day astrocytes. In comparison [125I]IGF-II binding proteins were detected only at low levels in medium from astrocytes cultured from 1-day rat brain and were undetectable in neuron-conditioned media. Northern blot analysis using a rIGFBP-2 complementary DNA revealed 5-fold greater messenger RNA levels in astrocytes from 21-day rat brain compared with astrocytes from 1-day brain, whereas neonate neurons showed no transcripts. Thus, rIGFBP-2 exhibits a pattern of developmental and cell-specific expression in cultured rat brain cells.

Complementary DNA structure of the high molecular weight rat insulin-like growth factor binding protein (IGF-BP3) and tissue distribution of its mRNA

Insulin-like growth factors (IGFs) found in extracellular fluids are bound to specific binding proteins. Recently a high molecular weight IGF-binding protein (IGF-BP3) has been isolated from porcine ovarian follicular fluid based on its inhibition of follicle stimulating hormone-stimulated estradiol production in rat granulosa cells. The complete primary structure of the porcine IGF-BP3 was deduced by molecular cloning. Using the porcine cDNA as a probe, we have now isolated and characterized cDNAs encoding rat IGF-BP3 from a pregnant mare serum gonadotropin-stimulated ovarian library. The predicted amino acid sequence revealed a mature polypeptide consisting of 265 amino acids with 18 cysteines and 4 potential Asn-linked glycosylation sites. Northern analysis of the IGF-BP3 mRNA in rat tissues showed a single 2.6 kb band in liver, kidney, stomach, heart, adrenal, ovary, testis, spleen, lung, small and large intestine in varying amounts, but the message is below the limit of detection in hypothalamus and brain cortex.

Heterogeneity of insulin-like growth factor binding proteins in swine

Proteins in porcine amniotic fluid and sera (both fetal and adult) were separated electrophoretically in sodium dodecyl sulfate-containing polyacrylamide gels and transferred to nitrocellulose sheets. Western blots were analysed for proteins that would bind (a) radioiodinated insulin-like growth factor-I (IGF-I) and (b) antibodies to a rat insulin-like growth factor binding protein. Multiple insulin-like growth factor binding proteins were identified in sera. The binding proteins ranged in size from M r 192000 to 26000. One immunologically cross-reactive protein ( M r 36000) was detected. No binding proteins were detected routinely in amniotic fluids. Sera from adult swine were fractionated by preparative isoelectric focusing. Two binding proteins ( M r 192000, 46000) were located in acidic fractions which also contained IGF-I and IGF-II. Two binding proteins ( M r 36000, 26000) were located in neutral to basic fractions which contained primarily IGF-II. Immunoglobulin-sized material from adult sera fractionated over Sephadex G-200 contained two binding proteins ( M r 46000, 42000) whereas albumin-sized material contained one ( M r 36000). Porcine insulin-like growth factor binding proteins are as heterogeneous as those from humans.

Insulin-Like Growth Factor (IGF) Binding Protein Complementary Deoxyribonucleic Acid from Human HEP G2 Hepatoma Cells: Predicted Protein Sequence Suggests an IGF Binding Domain Different from Those of the IGF-I and IGF-II Receptors

The primary structure of an insulin-like growth factor (IGF) binding protein produced by human HEP G2 hepatoma cells has been deduced from the cDNA sequence. The 234 amino acid protein has a predicted molecular mass of 25,274 and contains a single, distinctive cysteine-rich region. The N-terminal sequence of this protein is quite similar to the limited sequence data available for a rat IGF binding protein produced by BRL-3A cells and suggests a common ancestral origin. In contrast, the HEP G2 IGF binding protein sequence bears no similarity to the N-terminal 15 amino acids of a 53 kilodalton binding protein purified from human plasma. Comparison of full-length protein sequences for the IGF-I and IGF-II receptors with that of the HEP G2 IGF binding protein also fails to demonstrate any significant similarities among these three proteins, and suggests that each contains a unique binding domain for the IGF peptides.

Binding proteins for insulin-like growth factors in adult rat serum. Comparison with other human and rat binding proteins

Insulin-like growth factor (IGF) binding protein has been purified from adult rat serum by affinity chromatography on agarose-IGF-II and high performance reverse-phase chromatography. The final preparation contains two components, of apparent molecular mass 50 and 56 kDa nonreduced, or 44 and 48 kDa reduced, both of which specifically bind IGF-I and IGF-II. Competitive binding data indicate association constants of 5-10 X 10(10) l/mol for both IGFs, with a slightly higher affinity for IGF-II than IGF-I. Amino-terminal sequence analysis yields a unique sequence, identical in 11 of the first 15 amino acids with that of a human plasma IGF binding protein (Martin, J. L., and Baxter, R. C. (1986) J. Biol Chem. 261, 8754-8760), and with slight homology to other human and rat IGF binding proteins characterized to date. By analogy with the binding protein from human plasma, it is likely that the rat protein is part of the growth-hormone dependent complex which appears to carry most or all of the circulating IGFs.