Hung Pham

Insulinlike growth factor binding proteins

Publisher Summary This chapter provides an overview of insulin-like growth factor-binding proteins (IGF-BP). The insulin-like growth factors (IGFs)—or somatomedins—constitute a family of growth hormone (GH)-dependent peptides with both anabolic and mitogenic activities for a wide variety of tissues and cell lines. IGF-I and -II are characterized by a striking structural homology with human proinsulin. The human gene for IGF-II is located on the short arm of chromosome 11 in close proximity to the gene for insulin and spans over 30 kb of DNA. This gene is composed of at least eight exons. The preprohormone consists of a 24-amino-acid signal peptide, a 67-amino-acid mature peptide, and a carboxy-terminal peptide of 89 amino acids. Significant diversity exists in the 5-untranslated regions, where different mRNA species arise as a result of distinct promoters and alternative RNA splicing. Both IGF-I and -II are synthesized in a wide variety of tissues. The close relationship between insulin and IGF-I receptors is not surprising, given the binding affinities and structural similarities of the two receptors. Unlike the insulin and IGF-I receptors, the type 2 IGF receptor is characterized by a long extracellular domain, containing 15 repeat sequences of approximately 150 residues, a 23-residue transmembrane domain, and a small 164-residue cytoplasmic domain. No homology exists between the type 2 IGF receptor and the insulin or type 1 receptor. The initial characterization of IGF-BPs was done by gel filtration chromatography. The chapter discusses radioreceptor assays, affinity labeling, western ligand blotting, and immune-precipitation among other methods of detection.

Insulin-like growth factor binding proteins and their regulation

Insulin and the insulin-like growth factors (IGFs) share including gel chromatography, radioreceptor assays, significant structural homology, as is evident from the affinity cross-linking, Western ligand blotting (5, 6), ability of insulin to compete for occupancy of the type 1 immunoblotting and, recently, specific radioimmunoIGF receptor. The IGFs differ from insulin, however, in assays (RIAs). It has been the study of the molecular one important respect: in contrast to insulin, the IGFs biology of the IGFBPs, however, that has provided the circulate in plasma complexed to a family of binding most information concerning their structural interproteins (1-3). Although both IGF-I and IGF-I1 bind to relationship. these binding proteins (IGFBPs) with high affinity, insulin is totally incapable of competing for occupancy. These carrier proteins significantly extend the serum half-life of the IGF peptides, transport the IGFs to target cells and modulate the interaction of the IGFs with their surface membrane receptors. Furthermore, recent evidence suggests that under certain conditions, IGFBPs may be capable of acting independently, affecting cellular metabolism and replication, even in the absence of IGF peptides. The existence of IGFBPs was first inferred 20 years ago from chromatographic studies of the size distribution of IGF peptides in serum (4). It has only been in the last few years, however, that the complexity of the interactions among the IGFs, IGFBPs and IGF receptors has been fully appreciated. This has been accompanied by the realization that a full understanding of IGF action at a cellular level must take into account the modulatory role of IGFBPs. The identification and characterization of IGFBPs in various body fluids and in conditioned media from cultured cells have been facilitated by the development of a number of biochemical and assay techniques,

Demonstration and structural comparison of receptors for insulin-like growth factor-I and -II (IGF-I and -II) in brain and blood-brain barrier

Specific receptors for insulin-like growth factors (IGF) I and II on microvessel-free rat brain cell membranes (RBCM) and in the microvessels that constitute the blood-brain barrier (BBB) were identified and characterized by means of affinity cross-linking techniques and specific anti-receptor antibodies. Two different models of BBB were examined: isolated rat brain capillaries and cultured bovine brain microvessel endothelial cells. Cross-linking with 125-I-IGF-I, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), revealed an alpha subunit of apparent Mr = 138,000 in both BBB preparations, compared to 120,000 in RBCM. Cross-linking was inhibited by unlabeled IGF and insulin, but not by antibody directed against the IGF-II receptor. When 125-I-IGF-II was cross-linked, followed by SDS-PAGE under reducing conditions, a major band of apparent Mr = 250,000 was identified in RBCM and both BBB preparations. This band, which migrated with an approximately equivalent Mr in both brain and BBB membranes, was inhibited by unlabeled IGF and by antibody specific for the IGF-II receptor. Thus, both rat and bovine brain microvessels possess classical Type I and II IGF receptors. While the alpha subunit of the Type I receptor of brain is smaller than that of the BBB, the Type II receptor of brain and BBB appear to be structurally and immunologically identical.

Characterization of insulin-like growth factor binding proteins of cultured rat astroglial and neuronal cells

Insulin-like growth factor binding proteins produced by cultured rat neurons, astrocytes, and rat cell lines BRL-3A and B104 were compared to binding proteins found in rat serum, using affinity labeling, deglycosylation, and Western ligand blotting studies. Each source elaborated an unique pattern of heterogeneous binding proteins. Some of the binding proteins from different sources behaved similarly in each experimental system suggesting that subsets of these binding proteins may be structurally related. In particular, our data suggest that cultured astrocytes and neurons make the major binding protein produced by BRL-3A cells.

Purification of the insulin-like growth factor II (IGF-II) receptor from an IGF-II-producing cell line, and generation of an antibody which both immunoprecipitates and blocks the type 2 IGF receptor.

18,54-SF cells, which secrete rat insulin-like growth factor II (rIGF-II), have abundant type 2 IGF receptors. We have purified the type 2 receptor from these cells by solubilization of crude membranes in Triton X-100, followed by chromatography on agarose-immobilized rIGF-II. A partially purified receptor preparation, obtained by chromatography of solubilized membranes over wheat germ agglutinin, was used to immunize a rabbit. The antibody generated both immunoprecipitates the type 2 receptor, and specifically inhibits IGF-II binding to a variety of rat tissues, including 18,54-SF cells, BRL-3A cells and placenta. The presence of abundant type 2 receptors on an rIGF-II-secreting cell line is consistent with an autocrine role for IGF-II in select cells.

Insulin-like growth factors (IGFs): implications for aging.

The insulin-like growth factors (IGF)-I and IGF-II are peptides with structural homology to insulin and potent mitogenic and anabolic actions in vitro and in vivo. IGF-I levels are growth hormone (GH)-dependent and vary strikingly with age. IGF-I levels are typically low in infancy and childhood, increase dramatically during puberty, and then gradually decline with advancing age. Whether age-associated changes in GH production or sex steroid secretion, or other unknown factors, cause diminished IGF production in the elderly remains to be determined. In the brain, IGF-II appears to be the most prevalent IGF, but a truncated form of IGF-I also has been recognized. IGF actions are mediated by binding to a family of receptors, which includes the insulin receptor, the structurally homologous type I IGF receptor, and the IGF-II/M-6P receptor, all of which are found in the central nervous system. Additionally, the IGFs bind with high affinity to a family of IGF-binding proteins (IGFBPs). Of the six known IGFBPs, IGFBP-2 appears to be the major one in the mammalian brain and is a major component of CSF. Immunoreactive IGFBP-2 has been identified in astrocytes, and its mRNA has been identified in fetal and adult brain and choroid plexus. The IGFBPs transport the IGFs in serum and other body fluids and appear to regulate IGF access to receptors. In vivo regulation of IGFBPs includes tissue-specific proteases, which cleave specific IGFBPs, altering their affinities for IGF peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of monoclonal antibodies to the rat insulin-like growth factor II (IGF-II) receptor

BALB/c mice were immunized with rIGF-II receptors purified from 18-54, SF cells by chromatography of solubilized receptors over agarose-immobilized rIGF-II. Two fusions of splenic lymphocytes with FO mouse myeloma cells yielded 27 stable hybrids which were positive by ELISA. Cloning of seven of these hybrids yielded 30 positive clones by ELISA. At least seven of these clones (minimum of one from each parent hybrid) were capable of specifically immunoprecipitating the rIGF-II receptor.

INSULIN-LIKE GROWTH FACTOR-II (IGF-II) RECEPTORS IN RAT NEUROBLASTOMA CELLS

The presence of high concentrations of IGF-II in cerebrospinal fluid and brain, as well as the prominence of receptors in cortical tissues, make the CNS an attractive site for the study of IGF-II binding and action. B-104 is a transformed rat neuroblastoma cell line whose neuronal qualities include neurotransmitter synthesis and the ability to project axodendritic processes. We observed steady-state binding of 125-I-IGF-II to B-104 membranes between 1.5 and 4 hrs at optimal conditions of 25*C, pH 7-8. Specific binding averaged 12.2±4.0% per 100 ug/ml membrane protein, compared with 125-I-IGF-I binding of 10.1±2.9%. 125-I-IGF-II binding was minimally inhibited (<20%) by insulin concentrations as high as 100 ug/ml. In the presence of unlabeled IGF-II (0.5-5ng/ml), 125-I-IGF-II binding was increased by as much as 50% over baseline; at higher concentrations, 125-I-IGF-II binding was inhibited, with 50% displacement at 50 ng/ml. We observed a similar increase in 125-I-IGF-II binding in the presence of unlabeled IGF-I at concentrations ranging from 1-400 ng/ml. When 125-I-IGF-II was cross-linked to membranes, solubilized, and immunoprecipitated with a specific antibody for the rat IGF-II receptor, a classical type II receptor (Mr∼240K) was visualized upon electrophoresis. These results demonstrate the presence in B-104 cells of a specific high affinity type II receptor, as well as a membrane-bound binding protein, whose existence in conditioned medium was confirmed by activated charcoal assay. B-104 should serve as an appropriate and intriguing in vitro model for future study of IGF receptors and action in the CNS.