IGF-II Receptor Research Articles

Insulin-like growth factors in human breast cancer.

Several protooncogenes and suppressor genes and a variety of growth factors and their receptors have been shown to be mutated, deleted, or activated in human breast cancer. These changes may account for the unregulated growth of breast carcinoma cells. Insulin-like growth factors I and II (IGF-I, IGF-II) belong to a family of polypeptides with growth promoting properties and structural homology to insulin. They exert their mitogenic effects by binding to the IGF-I receptor and activating its tyrosine protein kinase. Other proteins that specifically bind the IGFs include the plasma membrane IGF-II receptor, which also binds lysosomal hydrolases, and several IGF-binding proteins which may serve to modulate IGF interactions with receptors. Breast cancer cell lines express IGF-I and IGF-II receptors and different patterns of binding proteins. IGF-I and IGF-II are each mitogenic for subsets of breast cancer cell lines. This effect is inhibited by antibodies directed against the IGF-I receptor. In breast tumors, IGF-I is expressed by stromal cells, but not carcinoma cells; it is not expressed by breast cancer cell lines. IGF-I is therefore a potential paracrine regulator of breast cancer cell growth. Similarly, IGF-II is expressed in breast tumors, predominantly in stromal cells, but sometimes also in carcinoma cells and in a subset of cell lines. Thus, IGF-II is also a potential paracrine regulator of breast cancer cell growth; in addition, it can be an autocrine regulator in some breast cancer cells.

Receptors for insulin-like growth factor II in the rat uterus: characterization and variation throughout the estrus cycle

This study was undertaken to identify uterine insulin-like growth factor II receptors and examine the regulation of these receptor levels throughout the estrus cycle. We have demonstrated IGF-II receptors in crude uterine membranes by binding and cross-linking experiments. IGF-II binding to the rat uterine membranes displayed time and temperature dependence and maximum binding was achieved by 2 h at 22 degrees C. Uterine IGF-II binding sites were specific for binding IGF-II peptide and demonstrated negligible binding affinity for IGF-I and no affinity for insulin. The specific anti-IGF-II receptor antibody, R-II-PAB1, blocked the specific [125I]IGF-II binding to uterine membranes in a dose-dependent manner. The characteristics of uterine IGF-II receptor are similar to those reported for other tissues, with a single class of high-affinity binding sites with an apparent dissociation constant of 1.2 +/- 0.5 nmol/l and Beta max of 2.65 +/- 0.41 pmol/mg protein. Affinity cross-linking experiments indicated that the specific binding of [125I]IGF-II in the uterus is associated with a single band of protein with a mol wt of 250 kD. In mature cycling rats, the proestrus uterus had the lowest level of [125I]IGF-II binding per mg membrane protein, without changes in receptor affinity. However, because of greater yield of protein from proestrus uteri, the total [125I]IGF-II binding capacity of the uterus was similar to the other stages of the estrus cycle. These studies demonstrate the presence of authentic IGF-II receptors in the rat uterus and illustrate variations in the concentration of these receptors in the uterus throughout the estrus cycle.

Measurement of Insulin-Like Growth Factor-II in Physiological Fluids and Tissues. II. Extraction and Quantification in Rat Tissues*

The tissue distribution and developmental patterns of insulin-like growth factor-II (IGF-II) have not been investigated in rat tissues, primarily because of the lack of an efficient extraction method for IGF-II and a sensitive RIA. IGF-II was extracted from rat tissues by formic acid, and the extract was heated at an acidic pH and treated with acetone. The removal of binding proteins was demonstrated by fast protein liquid chromatography size exclusion column and the elimination of a dilutional bias in the RIA. Using rat IGF-II as standard, we optimized a RIA for the quantification of IGF-II in rat tissues. In adult rats, IGF-II was found in all 15 tissues examined, with the highest concentration in the pituitary, followed by kidney, seminal vesicles, intestine, and serum. This distribution is not only different from that of IGF-I, but also differs from that reported for IGF-II mRNA and IGF-II receptors, suggesting that the rates of synthesis and/or metabolism of IGF-II are tissue dependent. Developmentally, IGF-II levels fell postnatally in most tissues, a pattern similar to that of IGF-II mRNA and IGF-II receptor. This developmental pattern supports the hypothesis that IGF-II is important in early growth and development. A relatively homogeneous distribution was observed in the adult rat brain, a distribution also different from that reported for IGF-II mRNA. In the pituitary, the highest concentration was found in the posterior pituitary, followed by the intermediate and anterior pituitary. In conclusion, IGF-II is found in many tissues of adult rats. This observation supports an autocrine and/or paracrine roles for IGF-II.

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

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 alpha-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 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 greater than 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.

Mitogenic effects of insulin and insulin-like growth factors on PA-III rat prostate adenocarcinoma cells: characterization of the receptors involved.

Four transplantable cell lines (PA-I, II, III, and IV) derived from four Lobund-Wistar (L-W) rats that manifested spontaneous prostate cancer have demonstrated metastatic capacity in visceral organs. Interestingly, PA-III cells, when deposited over the scapula or calvarium of the Lobund-Wistar rat, could produce lytic and blastic reactions on rat skeleton. Since growth factors and growth factor receptors have been implicated in bone remodeling, cancer biology, and metastatic growth of cancer cells, we have examined 1) the effects of insulin and insulin-like growth factors (IGF-I and IGF-II) on the proliferation of PA-III cells; and 2) the presence of specific receptors for these peptides. IGF-I (0.5 to 100 ng/ml), IGF-II (0.5 to 100 ng/ml), and insulin (0.5 to 10 micrograms/ml) stimulated tritiated thymidine uptake and increased the number of PA-III cells in culture. Receptor studies demonstrated the presence of specific bindings sites for IGF-I and II but not for insulin. The number and affinity of the receptor sites were: IGF-I (nb = 675 fmol/100 g protein, Kd = 0.56 nmol) and IGF-II (nb = 225 fmol/100 g protein, Kd = 0.71 nmol). Molecular characterization of IGF binding sites by polyacrylamide gel electrophoresis under denaturing conditions indicated only the presence for the type I IGF receptor. The presence of the IGF-I receptor and the absence of IGF-II and insulin receptors are discussed in relation to the capacity of PA-III cells to produce bone lesions on the L-W rat.

Reciprocal modulation of binding of lysosomal enzymes and insulin-like growth factor-II (IGF-II) to the mannose 6-phosphate/IGF-II receptor.

The discovery by Morgan et al.1 that the human IGF-II receptor is 80% homologous to the bovine cation-independent mannose 6-phosphate (Man-6-P) receptor led them to propose that the receptor is bifunctional, binding both a large number of lysosomal enzymes through the Man-6-P recognition site, and the growth factor, IGF-II. Later, Komfeld’s and Czech’s laboratories reported that the avian and amphibian Man-6-P receptors did not bind IGF-II, suggesting that the bifunctional property of the receptor may be confined to mammals.2,3 Did the mammalian receptor gene simply pick up a nucleotide sequence encoding an IGF-II binding site, enabling the receptor to provide a degradative pathway for IGF-II, or are there important interactions of the two disparate classes of ligands for binding to the receptor? These interactions could result in reciprocal modulation of the targeting of lysosomal enzymes by IGF-II, on the one hand, and the modulation of IGF-II degradation and IGF-II stimulated biologic responses by lysosomal enzymes, on the other. We will briefly summarize our experimental results which provide evidence for reciprocal inhibition of binding of the two classes of ligands for the mammalian Man-6-P/ IGF-II receptor.KeywordsCellular UptakeLysosomal EnzymeReciprocal ModulationHigh Extracellular ConcentrationDisuccinimidyl SuberateThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Demonstration of Insulin-Like Growth Factors I, II and Heterogeneous Insulin-Like Growth Factor Binding Proteins in the Cyst Fluid of Patients with Craniopharyngioma

Insulin-like growth factor (IGF)-II and its binding proteins were demonstrated to be present in human craniopharyngioma cyst fluid using gel filtration and ligand blot analyses. Immunoreactive IGF-II in 3 patients was found to be 274, 232 and 310 ng/ml after gel chromatography whereas IGF-I concentrations were 13, 8 and 15 ng/ml. The IGF-II levels were severalfold higher in cyst fluid than in spinal fluid while the IGF-I levels in both fluids did not differ significantly. The binding proteins showed high affinities for [125I]IGF-II which could be displaced by unlabelled IGF-II. With the ligand blot analysis, [125I]IGF-II shows bands at 300, 175 and 46/43 kilodaltons probably representing IGF-II receptor and IGFBP-3. IGFBP-1 levels 17, 22 and 45 ng/ml, respectively, were undetectable by ligand blot.

Cathepsin D: A protease involved in breast cancer metastasis

Cathepsin D is an acidic lysosomal protease present in all cells. In estrogen receptor positive and negative breast cancer cell lines, the mRNA coding for pro-cathepsin D is overexpressed and sorting and maturation of the pro-enzyme are altered, via possibly saturation of the Man-6-P/IGF-II receptor, leading to accumulation of the active proteinase in large endosomes and to secretion of the precursor (52K protein). In MCF7 cells, the cathepsin D mRNA is induced directly and transcriptionally by estrogens and indirectly by growth factors. In patients, there is a significant correlation between high cathepsin D concentrations in the cytosol of primary breast cancer and development of metastasis. This marker is independent of other prognostic factors and appears to be particularly useful in axillary node-negative tumors. Transfection of a human cDNA cathepsin D expression vector under the control of SV40 promoter increases the metastatic potential of 3YA1-Ad12 rat tumorigenic cells when intravenously injected into nude mice. The mechanism of cathepsin D-induced metastasis is currently unknown. These results indicate that overexpression of cathepsin D might facilitate breast cancer metastasis, suggesting new possible therapeutic approaches.

Experimental Diabetes Increases Insulinlike Growth Factor I and II Receptor Concentration and Gene Expression in Kidney

Insulinlike growth factor I (IGF-I) is a mitogenic hormone with important regulatory roles in growth and development. One of the target organs for IGF-I action is the kidney, which synthesizes abundant IGF-I receptors and IGF-I itself. To study the involvement of IGF-I and the IGF-I receptor in the development of nephropathy, one of the major complications of diabetes mellitus, we measured the expression of these genes in the kidney and in other tissues of the streptozocin-induced diabetic rat. The binding of 125I-labeled IGF-I to crude membranes was measured in the same tissues. We observed a 2.5-fold increase in the steady-state level of IGF-I-receptor mRNA in the diabetic kidney, which was accompanied by a 2.3-fold increase in IGF-I binding. In addition to this increase in IGF-I binding to the IGF-I receptor, there was also binding to a lower-molecular-weight material that may represent an IGF-binding protein. No change was detected in the level of IGF-I-peptide mRNA. Similarly, IGF-II-receptor mRNA levels and IGF-II binding were significantly increased in the diabetic kidney. IGF-I- and IGF-II-receptor mRNA levels and IGF-I and IGF-II binding returned to control values after insulin treatment. Because the IGF-I receptor is able to transduce mitogenic signals on activation of its tyrosine kinase domain, we hypothesize that, among other factors, high levels of receptor in the diabetic kidney may also be involved in the development of diabetic nephropathy. Increased IGF-II-receptor expression in the diabetic kidney may be important for the intracellular transport and packaging of lysosomal enzymes, although a role for this receptor in signal transduction cannot be excluded. Finally, the possible role of IGF-binding proteins requires further study.

Increased Expression of Insulin-Like Growth Factor-II/ Mannose-6-Phosphate Receptor in Regenerating Rat Liver*

This study examined the effect of two thirds hepatectomy on rat liver insulin-like growth factor-II (IGF-II) receptors and IGF-II receptor messenger RNA (mRNA) levels. IGF-II receptor levels were determined in liver plasma membrane and Golgi/endosome fractions from sham-operated and hepatectomized rats by ligand binding and immunoblotting after electrophoresis. After hepatectomy, IGF-II receptors increased initially in the plasma membrane (within 24 h of surgery) to 4-fold control levels, remained elevated for 72 h, and declined to control levels at 120 h when liver regeneration is near complete. However, Golgi/endosome levels of IGF-II receptors did not increase until 48 h, showing a maximum increase of 3.5-fold at 72 h after surgery and returning to control values at 120 h. The 9-kilobase mRNA for IGF-II receptor, determined by slot blotting with a complementary DNA probe, increased 2.5-fold within 24 h of surgery, attaining a maximum stimulation of 4-fold at 48 h, and then decreasing to normal levels by 120 h. These changes show that, after partial hepatectomy, IGF-II receptors increase rapidly at the cell surface, possibly due to receptor translocation from intracellular pools. This is followed by an increase of IGF-II receptor mRNA and increased receptor synthesis, resulting in an increase in total cellular receptors. These results suggest a role for IGF-II receptors in regenerating liver, perhaps in cell proliferation and/or in tissue remodeling.

Insulin-Like Growth Factor-II: Possible Local Growth Factor in Pheochromocytoma*

Pheochromocytomas, neural crest tumors, express an abundance of insulin-like growth factor-II (IGF-II). To assess further the potential for IGF-II to play an autocrine role for these tumors, we measured 1) IGF-II content by RRA in 7 pheochromocytomas and peripheral blood in these patients, 2) IGF-II receptors by Western analysis, and 3) characterized the tumor binding proteins by ligand blot studies. IGF-II levels in the tumors varied from 2.8-41 micrograms/g. Chromatography revealed that 60% of the peptide eluted as a large mol wt form of IGF-II (8.7-10 kDa); the remainder coeluted with mature peptide (7.5 kDa). This was in contrast to IGF-II levels in normal adrenal tissue (0.225 +/- 0.005 micrograms/g) or another neural crest-derived tumor, medullary carcinoma of the thyroid (0.63 +/- 0.02 micrograms/g). Serum IGF-II levels in the 7 patients with pheochromocytoma (720 +/- 71 ng/mL) were similar to those in 35 normal controls (762 +/- 69 ng/mL). Radiolabeled IGF-II (9 +/- 1%) and IGF-I (20 +/- 2%) bound specifically to pheochromocytoma membranes. Western analysis of these membranes using a specific antiserum directed against the type II receptor demonstrated a band at 210 kDa. Affinity cross-linking studies with [125I]IGF-I demonstrated a specific band at 140 kDa. Ligand blot analysis was performed on the void volume pools from the Sephadex G-75 column and demonstrated bands at about 30 and 25 kDa. In conclusion, these data 1) confirm that pheochromocytomas have increased levels of IGF-II; 2) demonstrate that despite high IGF-II concentrations in the tumors, peripheral levels are not elevated, suggesting that very little tumoral IGF-II is released into the circulation, unlike catecholamines; 3) demonstrate the presence of IGF-II and IGF-I receptors; 4) describe binding protein species similar to those present in other tissues. Thus, the presence of high levels of IGF-II and both type I and type II receptors suggests that IGF II may act through both receptors to alter tumor growth.

Distinctive regulation of the functional linkage between the human cation-independent mannose 6-phosphate receptor and GTP-binding proteins by insulin-like growth factor II and mannose 6-phosphate.

The rat insulin-like growth factor II (IGF-II) receptor develops transmembrane signaling functions by directly coupling to a guanine nucleotide-binding protein (G protein) having a 40-kDa alpha subunit, Gi-2, whereas recent studies have indicated that the IGF-II receptor is a molecule identical to the cation-independent mannose 6-phosphate receptor (CI-MPR), a receptor implicated in lysosomal enzyme sorting. In this study, by using vesicles reconstituted with the clonal human CI-MPR and G proteins, we indicated that the CI-MPR could stimulate guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding and GTPase activities of Gi proteins in response to IGF-II. The stimulatory effect of IGF-II on Gi-2 depended on the reconstituted amount of the CI-MPR; it could not be found in vesicles reconstituted with Gi-2 alone; and it was also observed on Gi-1 reconstituted with the CI-MPR in phospholipid vesicles. Of interest, such stimulatory effect was not reproduced by Man-6-P in CI-MPR vesicles reconstituted with either G protein. Furthermore, the affinity for Man-6-P-mediated beta-glucuronidase binding to several kinds of native cell membranes was not reduced by 100 microM GTP gamma S. Instead, however, Man-6-P dose-dependently inhibited IGF-II-induced Gi-2 activation with an IC50 of 6 microM in vesicles reconstituted with the CI-MPR and Gi-2. The action of 100 nM IGF-II was completely abolished by 1 mM Man-6-P. Such an inhibitory effect of Man-6-P was reproduced by 4000 times lower concentrations of beta-glucuronidase or similar concentrations of fructose 1-phosphate, but not by mannose or glucose 6-phosphate. These results indicate that the human CI-MPR has two distinct signaling functions that positively or negatively regulate the activity of Gi-2 in response to the binding of IGF-II or Man-6-P.

Liver Regeneration Is Associated with Increased Expression of the Insulin-Like Growth Factor-II/Mannose-6-Phosphate Receptor

The process of liver regeneration involves the concerted action of certain growth factors, which stimulate hepatocyte proliferation, and other antiproliferative factors, which prevent uncontrolled growth of this organ. Some of the biological actions of insulin-like growth factor-II (IGF-II), a mitogenic polypeptide closely related to insulin, may be mediated by the IGF-II receptor. This receptor consists of a single chain extracellular domain and a very small cytoplasmic domain, and can bind lysosomal enzymes that contain mannose-6-phosphate (M-6-P) residues. Since these enzymes may be involved in remodelling processes in certain tissues, we measured the expression of the IGF-II/M-6-P receptor in the liver after subtotal hepatectomy. Binding of [125I]IGF-II to crude plasma membranes from regenerating liver was maximal 2 days after hepatectomy (4.9% specific binding/60 micrograms protein) and subsequently decreased. Both control livers (livers removed at the time of operation) and sham-operated control livers demonstrated specific [125I]IGF-II binding of 1.1% throughout the experimental period. This increase in binding in regenerating liver was shown to be associated with an increase in the concentration of IGF-II receptor protein by means of Western blot analysis using a polyclonal anti-IGF-II/M-6-P receptor antiserum (3637). Similarly, steady state levels of IGF-II/M-6-P receptor mRNA, measured by solution hybridization/RNase protection assays, were significantly increased in the regenerating liver (2.0-fold over the control value 2 days after hepatectomy). Five and 10 days postsurgery, the levels of IGF-II receptor mRNA were markedly reduced, and they were even lower than the levels in control livers.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of genes for insulin and insulin‐like growth factors and receptors in early postimplantation mouse embryos and embryonal carcinoma cells

The expression of genes for insulin and insulin-like growth factors (IGFs) and their receptors was examined in early postimplantation mouse embryos and differentiating F9 embryonal carcinoma cells using mRNA phenotyping. Messenger RNA phenotyping involves the reverse transcription of RNA followed by amplification of specific target cDNA sequences using the polymerase chain reaction (PCR). The identities of the resulting PCR fragments were confirmed using at least two of the following methods: 1) size determination by agarose gel electrophoresis, 2) the presence of diagnostic restriction sites, 3) hybridization with radiolabeled cDNA probes, 4) sequencing of the PCR fragment. Transcripts for insulin receptors, IGF-I receptors, and IGF-II receptors were detected in RNA samples from day 7.5 to day 9.5 mouse embryos and in F9 cells, although the level of insulin receptor mRNA in F9 cells was very low. Transcripts for both IGF-I and IGF-II ligands were also detectable in the embryo and F9 RNA samples, but transcripts for insulin ligand were undetectable in either set of material. The results suggest that insulin does not act as a paracrine or autocrine growth factor in early postimplantation embryos or F9 cells but that both embryos and F9 cells have the potential to respond to exogenous (e.g., maternal) sources of insulin. Both IGF-I and IGF-II could act as paracrine or autocrine growth factors, and IGF-II is the more abundant growth factor in differentiating F9 cells.

Modulation of the insulin growth factor II/mannose 6-phosphate receptor in microvascular endothelial cells by phorbol ester via protein kinase C.

Phosphorylation of hormone receptors by protein kinase C (PKC) may be involved in the regulation of receptor recycling. We have studied the recycling and the phosphorylation state of the insulin growth factor (IGF) II/mannose 6-phosphate (Man-6-P) receptor in microvascular endothelial cells from rat adipose tissue. Scatchard analysis showed these cells have over 2 x 10(6) receptors/cell with an affinity constant of 1 x 10(9) M-1. In the presence of phorbol myristate acetate (PMA), an activator of PKC and analog of diacylglycerol, IGF-II receptor number increased in the plasma membrane by 60% without changes in the binding affinity. This increase in cell surface receptor number was confirmed by affinity cross-linking and 125I-surface labeling studies, occurred with a half-time of 20 min, and was reversible upon withdrawal of PMA. The redistribution of IGF-II/Man-6-P receptors was not due to an inhibition of internalization which was in fact stimulated by PMA. The effect of PMA on IGF-II receptor recycling correlated with its stimulation of PKC activity. Furthermore, after down-regulation of cellular PKC levels by preincubation with PMA, PMA was unable to activate residual PKC activity in the membranous pool or increase IGF-II receptor number at the cell surface. The phosphorylation state of the IGF-II/Man-6-P receptor was determined by 32P labeling of intact cells and immunoprecipitation with anti-receptor antibodies. In the basal state, the receptor was phosphorylated only on serine residues which was increased by 75% after treatment with PMA. In contrast, IGF-II decreased receptor phosphorylation and plasma membrane binding in a parallel and dose-dependent manner. Thus, PKC-stimulated serine phosphorylation of IGF-II/Man-6-P receptor may promote the translocation of the receptor to the cell surface, whereas IGF-II-stimulated dephosphorylation of the receptor may lead to a decrease in the number of cell surface receptors. These data suggest a role for PKC-mediated serine phosphorylation in the regulation of intracellular trafficking of receptors in endothelial cells.

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

The receptor binding, internalization and tyrosine kinase activation of insulin-like growth factors, IGF-I and IGF-II have been investigated in cultured adult bovine chromaffin cells. IGF-I receptor α-subunits (Mr ∼130,000) bound IGF-I and IGF-II with identical affinity (Kd ∼1nM) and insulin with about 1000 times lower affinity. IGF-II receptors (Mr ∼250,000) bound IGF-II with aKdof0.5nM, IGF-I with about 10 times lower affinity and insulin with10,000 times lower affinity. The amounts of IGF-I and IGF-II receptors on the cell surface were8 × 104and4 × 104 sites per cell, respectively. Insulin bound to a specific receptor withKd ∼2nM and the amount of receptors was1.5 × 104 sites per cell. IGF-I and IGF-II stimulated tyrosine kinase activity and autophosphorylation of the IGF-I receptor ß-subunit (Mr ∼94,000) with equal potency (ED50 ∼1nM), whereas insulin was∼5 times less potent. Both IGF-I and IGF-II were internalized after their binding to cell surface receptors. Mannose-6-phosphate, which binds to the IGF-II receptor, did not alter the binding or internalization of IGF-II. It is concluded that IGF-I and IGF-II can exert their biological effects in chromaffin cells by activation of the IGF-I receptor tyrosine kinase or by interaction with the IGF-II receptor.

Identification and Localization of Insulin‐Like Growth Factor Receptors in Human Infant Pituitary Gland—Similar Distribution to Somatotrophs

Abstract Insulin-like growth factors(IGF) are involved in feedback regulation of growth hormone(GH) secretion from the pituitary. Though receptors for IGF-I and IGF-II have been identified on particulate preparations of rat pituitary, their localization and relationship to GH-secreting acidophils has not been determined. We used quantitative in vitro autoradiography and immunocytochemistry to simultaneously determine the distribution of IGF receptors and GH-secreting cells in human infant pituitary gland. Frozen or fixed post-mortem human infant pituitary glands were sectioned for binding studies, or immunocytochemistry. Binding for IGF-I and IGF-II showed characteristic specificity for respectively Type I and Type II receptors. Binding sites were visualized throughout the pituitary gland, with similar density and distribution for IGF-I and IGF-II receptor sites. Receptor density was two-fold higher in anterior than posterior pituitary, with highest density in the lateral horns of the anterior pituitary. The distribution of GH-containing cells (acidophils) was similar to IGF receptor distribution. Increased density of IGF receptors in regions of GH-secreting cells may point to the mechanism whereby IGF uniquely inhibits synthesis of human GH in contrast to its promotion of synthetic processes in other cells.

The insulin-like effect of growth hormone on insulin-like growth factor II receptors is opposed by cyclic AMP. Evidence for a common post-receptor pathway for growth hormone and insulin action

The counter-regulatory effects of beta-adrenergic stimulation and cyclic AMP on the insulin-like action of growth hormone (GH) on the subcellular distribution of insulin-like growth factor II (IGF-II) receptors were studied in fat cells from hypophysectomized (Hx) and sham-operated rats. For comparison, the effect of insulin on this process was also studied. Basal IGF-II binding was increased by approx. 2-fold in cells from Hx as compared with sham-operated animals. The stimulatory effect of insulin was decreased in Hx cells, mainly due to a basal redistribution but also to a reduced total number of receptors. GH exerted an acute insulin-like effect in cells from Hx rats and stimulated the translocation of IGF-II receptors from an intracellular pool to the plasma membrane. beta-Adrenergic stimulation with isoprenaline or addition of the non-metabolizable cyclic AMP-analogue N6-monobutyryl cyclic AMP induced a cellular resistance to both GH and insulin and also reduced the responsiveness to these hormones. Adenosine exerted a modulatory effect on both hormones. Binding of 125I-labelled GH to its receptors was not significantly changed by any of these factors. It is concluded that: (1) beta-adrenergic stimulation and cyclic AMP induce a cellular GH resistance at a level distal to the GH-binding site, and (2) the insulin-like effect of GH shares a common pathway with insulin which occurs at the post-binding level.

Insulin-Like Growth Factor-II/Mannose-6-Phosphate Receptors Are Increased in Hepatocytes from Regenerating Rat Liver*

Insulin-like growth factor-II (IGF-II) receptor levels were determined in hepatocytes from sham-operated and two thirds hepatectomized rats. [125I]IGF-II binding to confluent cultures increased 2-fold in cells isolated 24 and 48 h after hepatectomy compared to that in cells from sham-operated rats. Receptor levels increased from 1.74 +/- 0.14 x 10(4)/cell (sham-operated) to 3.60 +/- 0.31 x 10(4)/cell (hepatectomized; P = 0.002), with no change in affinity of IGF-II binding (Ka = 6.4 x 10(9) M-1). As previously reported, receptor levels increased in cells plated at low density, but this effect was decreased in cells from hepatectomized rats (80% increase) compared to that in cells from control rats (300% increase). Serum IGF-I levels decreased 50% 24 h after partial hepatectomy (P less than 0.001), but returned to normal levels by 48 h. However, IGF-I synthesis was not decreased in hepatocytes isolated 24 h after partial hepatectomy, suggesting that decreased serum levels are due to decreased liver mass. Circulating IGF-II levels were not altered by partial hepatectomy, and IGF-II production was not detected in hepatocytes from sham-operated or hepatectomized rats. Transforming growth factor-beta is thought to terminate hepatocyte proliferation upon complete liver regeneration. In hepatocytes from sham-operated or hepatectomized rats transforming growth factor-beta totally blocked DNA synthesis, but had no effect on elevated IGF-II receptor levels after partial hepatectomy. Concomitant with increased IGF-II receptor levels, hepatocytes from hepatectomized rats were more sensitive to IGF-II stimulation of DNA synthesis. [3H]Thymidine incorporation in the presence of epidermal growth factor (50 ng/ml) was stimulated 66% by IGF-II (300 ng/ml) in control cells compared with 220% in cells from hepatectomized animals. These results suggest that IGF-II and the IGF-II receptor may play a role in liver regeneration.

β‐Galactosidase decreases the binding affinity of the insulin‐like‐growth‐factor‐II/mannose‐6‐phosphate receptor for insulin‐like‐growth‐factor II

The insulin-like growth-factor-II/mannose-6-phosphate (IGF-II/Man6P) receptor binds two classes of ligands, insulin-like growth factors and lysosomal enzymes. We have examined the ability of the lysosomal enzyme, beta-galactosidase, to modulate the binding of 125I-IGF-II to the receptor. beta-Galactosidase purified from bovine testis was fractionated on a DEAF-Sephacel ion-exchange column. Column fractions were assayed for enzymatic activity and for ability to inhibit the binding of 125I-IGF-II to the IGF-II/Man6P receptor. Enzyme fractions eluting at higher NaCl concentrations which had previously been shown to exhibit greater uptake by cells in culture, exhibited greater potency in inhibiting the binding of 125I-IGF-II to the receptor. A pool of these fractions from the DEAE-Sephacel column inhibited 125I-IGF-II binding to pure receptor by 80% with the concentration required for half-maximal inhibition being 25 nM. The inhibition of binding by beta-galactosidase was completely blocked by simultaneous incubation with Man6P. Inhibition of the enzymatic activity of beta-galactosidase with D-galactonic acid gamma-lactone did not affect the ability of beta-galactosidase to inhibit the binding of 125I-IGF-II to the receptor. Scatchard analysis of IGF-II binding to pure receptor in the presence and absence of beta-galactosidase showed that beta-galactosidase decreased the binding affinity for IGF-II (Kd 0.26 nM versus 1.0 nM in the presence of 57 nM beta-galactosidase). We confirmed the observations of others that Man6P alone actually increases the binding of 125I-IGF-II to the IGF-II/Man6P receptor, but we found that this phenomenon was dependent upon the method of preparation of the IGF-II/Man6P receptor. Microsomal membrane preparations, solubilized membranes, and receptors purified on an IGF-II-Sepharose column all exhibited stimulation of 125I-IGF-II binding by Man6P, whereas receptors purified on lysosomal enzyme affinity columns showed little or no stimulation of 125I-IGF-II binding by Man6P. We conclude that beta-galactosidase decreases the binding affinity of the IGF-II/Man-6-P receptor for IGF-II by binding with high affinity to the Man6P-recognition site.