Previous measurements of somatomedins (Sms) and insulin-like growth factors (IGFs) in maternal and fetal serum have yielded contradictory results. We have, therefore, measured maternal, fetal, and neonatal rat serum with two highly specific assays: 1) IGF-I/Sm-C RiA and 2) a highly specific IGF-II/rat placental membrane radioreceptor assay (RRA). In addition, we have made measurements with a less specific multiplication-stimulating activity (MSA)-rat placental membrane RRA. To avoid possible serious artifacts created by Sm-binding proteins, preliminary acid-ethanol extraction of serum was performed. Results are expressed in terms of a reference human serum with an assigned potency of 1 U/ml. Maternal RIA IGF-I fluctuated between 1.1-1.4 U/ml from the 17th day of pregnancy to the 25th day after delivery (nonpregnant rat serum pool, 1.25 +/- 0.22 U/ml). On day 21 of gestation, fetal serum radioimmunoassayable IGF-I was 1.03 +/- 0.03 U/ml. After birth, radioimmunoassayable IGF-I fell and reached .19 +/- 0.03 U/ml at 18 days of age, but rose to 0.71 +/- 0.04 U/ml at 25 days of age. At term, maternal radioreceptor assayable IGF-II was 2.18 +/- 0.27 U/ml (nonpregnant female pool, 1.4 +/- 0.12). By the 25th postpartum day, radioreceptor assayable IGF-II was 1.39 +/- 0.12 U/ml. Radioreceptor assayable IGF-II in fetal serum on day 19 was 3.26 +/- 0.48 U/ml and rose to 5.37 +/- 0.66 U/ml on the day of delivery. A further rise to 8.92 +/- 1.03 occurred on day 5. A subsequent fall to 2.41 +/- 0.05 U/ml was observed on day 25. The patterns of results of the MSA RRA in fetal and neonatal rat serum were similar to that obtained with the IGF-II RRA. We now conclude that radioimmunoassayable IGF-I is present in higher concentrations than previously reported in term fetal rat serum and that radioreceptor assayable IGF-II is selectively elevated in rat fetal and neonatal life and may have unique metabolic and growth-promoting significance.U

It has recently been recognized that human serum contains a protein that specifically binds human growth hormone (hGH). This protein has the same restricted specificity for hGH as the membrane-bound GH receptor. To determine whether the GH-binding protein is a derivative of, or otherwise related to, the GH receptor, we have examined the serum of three patients with Laron-type dwarfism, a condition in which GH refractoriness has been attributed to a defect in the GH receptor. The binding of 1251-labeled hGH incubated with serum has been measured after gel filtration of the serum through an Ultrogel AcA 44 minicolumn. Nonspecific binding was determined when '25I-hGH was incubated with serum in tho presence of an excess of GH. Results are expressed as percent of specifically bound 251-hGH and as specific binding relative to that of a reference serum after correction is made for endogenous GH. The mean ± SEM of specific binding of sera from eight normal adults (26-46 years of age) was 21.6 ± 0.45%, and the relative specific binding was 101.1 ± 8.6%. Sera from 11 normal children had lower specific binding of 12.5 ± 1.95% and relative specific binding of 56.6 ± 9.1%. Sera from three children with Laron-type dwarfism lacked any demonstrable GH binding, whereas sera from 10 other children with other types of nonpituitary short stature had normal relative specific binding. We suggest that the serum GHbinding protein is a soluble derivative of the GH receptor. Measurement of the serum GH-binding protein may permit recognition of other abnormalities of the GH receptor.When normal human serum is subjected to gel filtration in neutral buffers, higher-molecular-weight forms of growth hormone (GH) have been frequently reported (1, 2). While these big forms of GH have been attributed to incompletely processed precursor forms of the hormone or aggregated GH molecules, there is now evidence that complexes of GH with a specific serum binding protein exist in human serum and the serum of a number of mammals.The presence of a specific GH-binding protein (GH-BP) was first convincingly established in rabbit serum by Ymer and Herington (3). They added "25I-labeled human GH (hGH) or 125mlabeled bovine GH to normal rabbit serum, and after 2 hr of incubation, the reaction mixture was filtered through Ultrogel. As much as 45% of the added 125I-labeled hGH (125I-hGH) was bound to a component with a Mr of >100,000. Rabbit OH-BP had little affinity for either prolactin or human placental lactogen. The serum GH-BP, like a cytosolic GH-BP found in liver, exhibited 100% cross-reactivity with a monoclonal antireceptor antibody raised by Simpson et al. (4,5) against GH receptors isolated from rabbit liver membranes. These observations led Ymer and Herington to suggest that the serum GH-BP arises from the liver and might be structurally related to the membrane-bound GH receptor. Herington et al. (6) then demonstrated a similar GH-BP in human serum by gel filtration. The techniques of charcoal adsorption and PEG precipitation, which are oft...

The insulin-like growth factor II (IGF-II) gene is overexpressed in many mesenchymal tumors and can lead to non-islet-cell tumor hypoglycemia (NICTH). ProIGF-II consists of the 67 aa of IGF-II with a carboxyl 89-aa extension, the E domain. A derivative of proIGF-II containing only the first 21 aa of the E domain [proIGF-II-(E1-21)] has been isolated by others from normal serum and has 0-linked glycosylation. We found that the "big IGF-II" of normal serum, as detected by an RIA directed against residues 1-21 of the E domain of proIGF-H, was reduced in size by treatment with neuraminidase and 0-glycosidase. The big IGF-II, which is greatly increased in NICTH sera, was unaffected by neuraminidase and 0-glycosidase treatment. We have also shown that big IGF-H from normal serum is retained byjacalin lectin columns and that big IGF-H from NICTH serum was not retained, indicating that it lacked 0-glycosylation. Normal O-linked glycosylation may be required for proper peptidase processing of proIGF-H. The lack of normal 0-linked glycosylation by tumors may explain the predominance of big IGF-ll in NICTH sera. In normal serum, most of the IGF-ll is present in a 150-kDa ternary complex with IGF-U binding protein (IGFBP) 3 and a subunit. In NICTH serum, however, the complexes carrying big IGF-II are <50 kDa. We investigated whether big IGF-ll of NICTH was responsible for this abnormality. Tumor big IGF-H and IGF-HI were equally effective in forming the 150-kDa complex with purified IGFBP-3 and 12SI-labeled a subunit. Both 12SI-labeled IGF-ll and 12SI-labeled proIGF-II-(E1-21), when incubated with normal serum, formed the 150-kDa complex as detected by Superose 12 exclusion chromatography. We conclude that the nonglycosylated big IGF-II of NICTH serum can form normal complexes with serum IGFBPs. The defective binding in NICTH is attributable to defective IGFBP-3 binding.Insulin-like growth factor (IGF) II is synthesized as proIGF-II, which consists of the 67 aa of IGF-II and an 89-aa carboxyl-terminal extension, the E domain (1). Processing of proIGF-II normally occurs in a stepwise fashion, and cleavage after the single lysine at position 21 ofthe E domain yields a 10.5-kDa peptide [proIGF-II-(E1-21)] that has been isolated from serum (2). A larger variant of proIGF-II-(El-21), with 0-linked glycosylation on threonine at position 8 of the E domain, has also been isolated from serum (3).Many mesenchymal and some renal, adrenal, and hepatic cell tumors have increased expression of the IGF-II gene and synthesize and secrete IGF-II peptides (4). In some patients with large tumors, the secreted IGF-II peptides can produce hypoglycemia. However, measurement of these peptides with the IGF-II RIA has given inconsistent results (5, 6). Two recent findings provided an explanation for these inconsisThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.tencies. The predomina...

No abstract

The hydrophilic GH-binding protein of serum is a derivative of the GH receptor. Little is known how this GH binding protein is released from the receptor which is firmly anchored in the plasma membrane. The IM-9 lymphocytes provide a useful laboratory model for studying this process because they are richly endowed with GH receptors and, under special conditions, are able to shed these receptors during incubation. Incubation of IM-9 cells for 90 min at 30 C did not result in the appearance of significant [125I]hGH binding in conditioned medium as determined with an ultrogel AcA 44 minicolumn. When iodoacetamide, 20 mM, or N-ethylmaleimide, 5 mM, was added during incubation, the conditioned medium bound 20-35% of [125I]human(h)GH. p-Chloromercuriphenyl sulfonic acid was less effective in promoting shedding of GH-binding protein. In contrast, aprotinin, phenylmethylsulfonylfluoride (PMSF), bacitracin, leupeptin, pepstatin, phosphoramidon, or chloroquine did not promote release of GH binding protein and did not affect iodoacetamide-induced release. Release was not inhibited by the addition of serum lacking GH binding protein. GH binding protein release was markedly temperature sensitive and practically ceased at 4 C. GH binding protein incubated with [125I] hGH was cross-linked with disuccinimidyl suberate. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of dithiothreitol the complex migrated with an estimated molecular weight of 100,000 whereas [125I]hGH cross-linked to the membrane-bound GH receptor of the IM-9 cells migrated with an estimated molecular weight of 135,000. The smaller size of the binding protein is consistent with its derivation from the extracellular domain of the GH receptor. Because the release of this GH binding is greatly augmented by iodoacetamide and N-ethylmaleimide, two known sulfhydryl reactive reagents, we suggest that a free sulfhydryl group, either on the GH receptor or on a neighboring protein normally maintains the integrity of the receptor. The loss of this sulfhydryl group destabilizes the receptor and permits a membrane endopeptidase to release the GH binding protein. Cleavage is not dependent on lysosomal action and is not inhibited by protease inhibitors.