Steen Gammeltoft

Two types of receptor for insulin-like growth factors in mammalian brain.

Two types of receptor for insulin‐like growth factors (IGFs) have been identified on adult rat and human brain plasma membranes by competitive binding assay, affinity labelling, receptor phosphorylation and interaction with antibodies to insulin receptors. The type I IGF receptor consists of two species of subunits: alpha‐subunits (mol. wt. approximately 115 000), which bind IGF I and IGF II with almost equal affinity and beta‐subunits (mol. wt. approximately 94 000), the phosphorylation of which is stimulated by IGFs. The alpha‐subunits of type I IGF receptors in brain and other tissues differ significantly (mol. wt. approximately 115 000 versus 130 000), whereas the beta‐subunits are identical (mol. wt. approximately 94 000). The type II IGF receptor in brain is a monomer (mol. wt. approximately 250 000) like that in other tissues. Two antibodies to insulin receptors, B2 and B9, interact with type I but not with type II IGF receptors. B2 is more potent than B9 in inhibiting IGF binding and in immunoprecipitating type I IGF receptors, in contrast to their almost equal effects on insulin receptors. This pattern is characteristic for IGF receptors in other cells. The presence of two types of IGF receptor in mammalian brain suggests a physiological role of IGFs in regulation of nerve cell function and growth. Since IGF II, but not IGF I, is present in human brain, we propose that IGF II interacts with both types of IGF receptor to induce its biological actions.

Insulin-like growth factor I in cultured rat astrocytes: expression of the gene, and receptor tyrosine kinase.

Gene expression, receptor binding and growth‐promoting activity of insulin‐like growth factor I (IGF I) was studied in cultured astrocytes from developing rat brain. Northern blot analysis of poly(A)+ RNAs from astrocytes revealed an IGF I mRNA of 1.9 kb. Competitive binding and receptor labelling techniques revealed two types of IGF receptor in astroglial cells. Type I IGF receptors consist of alpha‐subunits (Mr 130,000) which bind IGF I with significantly higher affinity than IGF II, and beta‐subunits (Mr 94,000) which show IGF I‐sensitive tyrosine kinase activity. Type II IGF receptors are monomers (Mr 250,000) which bind IGF II with three times higher affinity than IGF I. Both types of IGF receptor recognize insulin weakly. DNA synthesis measured by cellular thymidine incorporation was stimulated 2‐fold by IGF I and IGF II. IGF I was more potent than IGF II, and both were significantly more potent than insulin. Our findings suggest that IGF I is synthesized in fetal rat astrocytes and acts as a growth promoter for the same cells by activation of the type I IGF receptor tyrosine kinase. We propose that IGF I acts through autocrine or paracrine mechanisms to stimulate astroglial cell growth during normal brain development.

Receptor Binding, Endocytosis, and Mitogenesis of Insulin-Like Growth Factors I and II in Fetal Rat Brain Neurons

Abstract: Cell surface binding, internalization, and biological effects of insulin‐like growth factors (IGFs) I and II have been studied in primary neuronal cultures from developing rat brain (embryonic day 15). Two types of IGF binding sites are present on the cell surface. The IGF‐I receptor α‐subunit (Mr 125,000) binds IGF‐I with a KD of 1 nM and IGF‐II with 10 times lower affinity. The mannose‐6‐phosphate (Man‐6‐P)/IGF‐II receptor (Mr 250,000) binds IGF‐II with a KD of 0.5 nM and IGF‐I with 100 times lower affinity. Surface‐bound IGF‐I and IGF‐II are internalized by their respective receptors and degraded to amino acids. Man‐6‐P increases the receptor binding and internalization of IGF‐II but not those of IGF‐I. Neuronal synthesis of RNA and DNA is increased twofold by IGF‐I with 10 times higher potency than IGF‐II. Antibody 3637, which blocks receptor binding of IGF‐II, has no effect on the DNA response to IGF‐I or IGF‐II. Double immunocytochemical staining with antibodies to bromodeoxyuridine and neurofilament shows that >80% of the bromodeoxyuridine‐positive cells become neurofilament positive. It is concluded that IGF‐I and IGF‐II bind to two receptors on the surface of neuronal precursor cells that mediate endocytosis and degradation of IGF‐I and IGF‐II. Proliferation of neuronal precursor cells is stimulated by IGF‐I and IGF‐II via activation of the IGF‐I receptor.

Identification of IGF-1 receptors in primitive vertebrates

This is the first report of the existence of insulin-like growth factor (IGF-1) receptors in three representatives of lower vertebrates: the osteichtyes, chondrichtyes and cyclostomi. Competitive binding studies and affinity labelling of brain membranes from Cottus scorpius (sea scorpion), Raja clavata (ray) and Myxine glutinosa (atlantic hagfish) identified a mammalian type 1 or IGF-1 receptor by its binding specificity and the molecular size of its alpha-subunit. IGF-1 and IGF-2 are almost equally potent in displacing receptor-bound 125I-IGF-1 or 125I-IGF-2, and the proteins labeled with both tracers have a molecular size of 100,000-120,000 under reducing conditions. There was no evidence for the presence of a mammalian type 2 or IGF-2/mannose 6-phosphate receptor in brains of Cottus, Raja or Myxine. In all three species the binding of 125I-IGF-1 and 125I-IGF-2 was significantly higher in brain compared with liver and gastrointestinal tract, and the IGF-1 receptor could only be identified with certainty in Raja liver. It is concluded that the brain of three lower vertebrates express mammalian IGF-1 receptors, whereas IGF-2-mannose 6-phosphate receptors could not be detected.

Functional receptors for insulin-like growth factors I and II in rat thymocytes and mouse thymoma cells.

Functional receptors for insulin-like growth factors (IGF) I and II have been identified in rat thymocytes and mouse thymoma cell lines R1.1 and S49.1. IGF-I receptor alpha-subunit (MW 130,000) bind IGF-I and IGF-II with equal affinity (Kd approximately 4-7 nM), and insulin with approximately 100 times lower affinity. Tyrosine kinase activity and autophosphorylation of the IGF-I receptor beta-subunit (MW 95,000) are stimulated by IGF-I and IGF-II with equal potency (ED50 approximately 0.5 nM). IGF-II receptors (MW 250,000) bind IGF-II with Kd approximately 0.3 nM and IGF-I with 30 times lower affinity, but not insulin. IGF-I and IGF-II do not cross-react with the insulin receptor to which insulin binds with an apparent Kd approximately 1 nM, and stimulates its tyrosine kinase activity with ED50 approximately 3 nM. In thymocytes, alpha-aminoisobutyric acid transport is stimulated 2-fold by IGF-I and IGF-II with identical potency (ED50 approximately 2 nM), and by insulin with ED50 approximately 10 nM. Activation of thymocytes by concanavalin A increased the number of IGF-II receptors 2-fold, whereas IGF-I receptor binding and IGF-stimulated amino acid transport were unaltered. We conclude that the effect of IGF-I and IGF-II in thymocytes is mediated via binding to the IGF-I receptor and stimulation of its tyrosine kinase. The presence of functional IGF receptors on thymocytes and thymoma cells suggests that IGF-I and IGF-II play a role in the regulation of thymic functions.

Regional Distribution of Neuropeptide Y and Its Receptor in the Porcine Central Nervous System

Abstract: The regional distribution of neuropeptide Y (NPY) immunoreactivity and receptor binding was studied in the porcine CNS. The highest amounts of immunoreactive NPY were found in the hypothalamus, septum pellucidum, gyrus cinguli, cortex frontalis, parietalis, and piriformis, corpus amygdaloideum, and bulbus olfactorius (200–1,000 pmol/g wet weight). In the cortex temporalis and occipitalis, striatum, hippocampus, tractus olfactorius, corpus mamillare, thalamus, and globus pallidus, the NPY content was 50–200 pmol/g wet weight, whereas the striatum, colliculi, substantia nigra, cerebellum, pons, medulla oblongata, and medulla spinalis contained <50 pmol/g wet weight. The receptor binding of NPY was highest in the hippocampus, corpus fornicis, corpus amygdaloideum, nucleus accumbens, and neurohypophysis, with a range of 1.0–5.87 pmol/mg of protein. Intermediate binding (0.5–1.0 pmol/mg of protein) was found in the septum pellucidum, columna fornicis, corpus mamillare, cortex piriformis, gyrus cinguli, striatum, substantia grisea centralis, substantia nigra, and cerebellum. In the corpus callosum, basal ganglia, corpus pineale, colliculi, corpus geniculatum mediale, nucleus ruber, pons, medulla oblongata, and medulla spinalis, receptor binding of NPY was detectable but <0.5 pmol/mg of protein. No binding was observed in the bulbus and tractus olfactorius and adenohypophysis. In conclusion, immunoreactive NPY and its receptors are widespread in the porcine CNS, with predominant location in the limbic system, olfactory system, hypothalamoneurohypophysial tract, corpus striatum, and cerebral cortex.

Gene expression and secretion of insulin-like growth factor-II and insulin-like growth factor binding protein-2 from cultured sheep choroid plexus epithelial cells

The gene expression of insulin-like growth factor II (IGF-II) and insulin-like growth factor binding protein-2 (IGFBP-2) has previously been demonstrated in rat and human choroid plexus by in situ hybridization analysis. In the present study we have characterized IGF-II and IGFBP-2 transcripts and proteins in primary cultures of epithelial cells from lateral choroid plexus of sheep brain. Northern blot analysis of total RNA showed one major IGF-II mRNA of 4.8 kb and four minor IGF-II transcripts of 1.5, 2.0, 3.0 and 6.0 kb as well as one IGFBP-2 transcript of 1.7 kb. Radioreceptor assay of conditioned medium from the cultured choroid plexus epithelial cells showed inhibition of [125I]IGF-I and [125I]IGF-II binding to mouse NIH 3T3 fibroblasts, the displacement curves being identical to that of unlabelled IGF-II. The conditioned medium was fractionated by gel filtration on a Bio-Gel P-60 column, and analysis by IGF-II radioreceptor assay showed two peaks of IGF-II-binding inhibitory activity of M(r) 7.5-10 and 25 kDa, suggesting the presence of both IGF-II, and an IGFBP. Western immunoblot analysis of conditioned medium with antibodies toward IGF-II and IGFBP-2 demonstrated proteins with M(r) 6 kDa and 32 kDa, respectively. Protein binding assays of the conditioned medium with [125I]IGF-I or [125]IGF-II demonstrated that the IGFBP present in the conditioned medium preferentially binds IGF-II. In conclusion, cultured sheep choroid plexus epithelial cells synthesize and secrete IGF-II and IGFBP-2, suggesting that the choroid plexus epithelium is the main source of these polypeptides in the cerebrospinal fluid.

Insulin-like growth factors are mitogens for rat pheochromocytoma PC 12 cells

We have addressed the issue of a mitogenic effect of insulin-like growth factors IGF-I and IGF-II on the PC 12 line of rat pheochromocytoma cells. The proliferation of PC 12 cells cultured in serum-free medium is stimulated threefold by IGF-I and IGF-II with significantly higher potency than epidermal growth factor, whereas platelet-derived growth factor, nerve growth factor, growth hormone and bombesin are inactive. Two types of IGF receptor are present in PC 12 cells and the dose-response curves suggest that the mitogenic responses to IGFs are mediated by the IGF-I receptor. These results suggest that IGF-I and IGF-II act as mitogens on pluripotent chromaffin cells in the development of the peripheral nervous system and adrenal medulla as well as in promotion of in vivo growth of neural crest-derived tumors.

Elevated concentration of IGF II in brain tissue from an infant with macrencephaly

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Chromaffin cells express two types of insulin-like growth factor receptors

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