A mutant of human insulin-like growth factor II (IGF II) with the processing sites of proinsulin. Expression and binding studies of processed IGF II.

A mutant of human insulin-like growth factor II (IGF II) was constructed by site-directed mutagenesis: the nucleotides coding for Ser33 and Ser39 were changed to yield Arg and Lys, respectively, thus creating two pairs of basic residues, Arg-Arg and Lys-Arg, as flanking sequences of the remaining C domain. [Arg33, Lys39]IGF II was expressed in NIH-3T3 cells as a processed two-chain peptide with a deletion of amino acid residues 37-40 and crosslinked by three disulfide bonds. This des(37-40)[Arg33]IGF II showed 3.6-fold and 7.4-fold reduced affinities to the type 1 and type 2 IGF receptor overexpressing cells, respectively, whereas the thymidine incorporation potency was the same as that of wild-type IGF II. We speculate that the discrepancy between the reduced binding to the type 1 IGF receptor and the full thymidine incorporation potency is due to the 6.1-fold reduced affinity of the expressed mutant to the co-expressed IGF binding protein 3 (IGFBP-3). The results suggest that des(37-40)[Arg33]IGF II assumes a conformation very similar to IGF II, and that the entire length of the C domain is not essential for biological activity.

The N-terminal hexapeptide fragment of IGF II stimulates thymidine incorporation into fibroblasts.

We synthesized the N-terminal hexapeptide fragment of IGF II to study potential binding to NMDA receptors in analogy to the N-terminal tripeptide of IGF I. The amino acid sequence of the hexapeptide is furthermore identical with the C-terminal sequence of the casiragua insulin B chain. The hexapeptide did not bind to the NMDA receptors, but was found to promote [3H]-thymidine incorporation into fibroblasts at concentrations of 10(-8) - 10(-5) M in a dose-dependent manner. Since [125I]-hexapeptide did not bind to IGF receptors, indirect competition studies using either labelled IGFs or insulin had to be used. The competition of hexapeptide at a concentration of 10(-5) M with labelled IGF I or II was about equal to that of 10(-9) M IGF I or II. IGF receptors were apparently up-regulated by the hexapeptide, as has also been described for insulin. When using casiragua insulin as labelled ligand, IGF II and casiragua insulin competed with equal potency, whereas the hexapeptide at 10(-7) M caused an apparent up-regulation of the casiragua insulin binding sites. Our results that the hexapeptide stimulates [3H]-thymidine incorporation and up-regulates IGF II and casiragua insulin binding sites may be connected to one or several of the following findings: the hystricomorph insulins--of which the casiragua insulin is a member--stimulate DNA synthesis to a greater extent than other insulins; the insulin and type 1 IGF receptor binding regions are localized predominantly in the C-terminal region of the insulin B chain; and the "cooperative" site regulating the affinity of the insulin receptor is also located in the C-terminal region of the insulin B chain. Further experiments will be needed to clarify the exact mechanism.

Mutants of human insulin-like growth factor II (IGF II). Expression and characterization of truncated IGF II and of two naturally occurring variants.

Insulin-like growth factor II (IGF II) and four structural analogs, constructed by site-directed mutagenesis, were expressed as protein A fusion proteins in Escherichia coli BL21pLysS cells, cleaved with cyanogen bromide and purified by affinity chromatography and HPLC. Two mutants (Ser29 substituted by Arg-Leu-Pro-Gly, and Ser33 substituted by Cys-Gly-Asp) represent two naturally occurring variants of IGF II. The other two mutants, (7-67)IGF II and (9-67)IGF II, are truncated at the amino-terminus in analogy to the naturally occurring des(1-3)IGF I ('truncated IGF I'). These mutants were tested for their binding affinities to type-1 and type-2 IGF receptors, to IGF binding protein-3 (IGFBP-3) and for their stimulation of thymidine incorporation into DNA. The affinities of the Ser29 and Ser33 mutants to the type-1 IGF receptor were 85% and 39%, respectively, compared to wild-type IGF II, those of (7-67)IGF II and (9-67)IGF II 96% and 15%, respectively. The potencies of the Ser33 and the (9-67) mutant to stimulate thymidine incorporation into DNA correlated closely with the affinities to the type-1 IGF receptor, whereas the bioavailability of the Ser29 mutant was lower and that of the (7-67) mutant higher than the type-1 receptor binding, possibly due to interferences with endogenously secreted IGFBPs. The affinities of the Ser29 and Ser33 mutants to the type-2 IGF receptor were 110% and 71%, respectively, those of the two truncated mutants 25% and 23%, respectively. The affinity of the Ser29 mutant to IGFBP-3 was increased to 171%, whereas those of the Ser33 mutant and the two truncated mutants were reduced (34%, 10% and 19%, respectively).

Mutants of human insulin-like growth factor II: expression and characterization of analogs with a substitution of TYR27 and/or a deletion of residues 62-67.

Five structural analogs of human insulin-like growth factor II (IGF II), [Leu27]IGF II, [Glu27]IGF II, des(62-67)IGF II, des(62-67)[Leu27]IGF II and des(62-67)[Glu27]IGF II were constructed by site-directed mutagenesis and expressed as protein A fusion proteins in E. coli BL21 pLysS cells, cleaved with CNBr and purified by affinity chromatography and HPLC. These mutants were tested for their binding affinities to type 1 and type 2 IGF receptors, to IGF binding protein-3 (IGFBP-3) and for their stimulation of thymidine incorporation into DNA. [Leu27]IGF II exhibits an affinity to the type 2 IGF receptor close to that of wild-type IGF II, but has lost completely the affinity to the type 1 IGF receptor. The results further suggest that the D domain, which is close to Tyr27, forms part of the binding region for the type 1 IGF receptor.

Mutants of human insulin-like growth factor II with altered affinities for the type 1 and type 2 insulin-like growth factor receptor.

With the aim to produce insulin-like growth factors (IGF) with enhanced specificity for the type 1 or type 2 IGF receptors, three mutants of IGF II have been prepared and expressed in NIH-3T3 cells. IGF II mutated at Tyr27 to Leu and Glu showed a 25- and 54-fold decrease in affinity for the type 1 IGF receptor and a 3.4- and 9.2-fold decrease in affinity for the type 2 IGF receptor. IGF II mutated at Phe48 to Glu showed a 18-fold decrease in affinity for the type 2 IGF receptor and a 2.8-fold decrease in affinity for the type 1 IGF receptor. These affinities were measured in radioreceptor assays using type 1 or 2 IGF receptor overexpressing cells. Data obtained on receptor cross-linking and thymidine incorporation assays confirmed the results of the radioreceptor assays. It is concluded that mutations of Tyr27 preferentially decrease binding to the type 1 IGF receptor and of Phe48 to the type 2 IGF receptor, either by the loss of a residue involved in receptor binding or by preferentially destabilizing the region involved in receptor binding.

Expression of a human insulin-like growth factor II cDNA in NIH-3T3 cells.

Recombinant human insulin-like growth factor II (IGF-II) was produced in NIH-3T3 cells transfected with a plasmid containing a construct encoding the signal peptide and the sequence of mature human IGF II. Successfully transfected clones secreted correctly processed recombinant human IGF II at rates of about 100ng per 24 hours per 10(6) cells. The biological activity of the purified recombinant human IGF II exhibited similar potencies as standard human IGF II isolated from serum in radio-receptorassays as well as in thymidine incorporation assays.

Structural alteration of the insulin-like growth factor II-gene in Wilms tumour.

In this study messenger ribonucleic acid (mRNA) and DNA of five Wilms tumours were investigated. As expected, the level of insulin-like growth factor (IGF) II-mRNA was elevated up to 50 times in tumour tissue as compared to normal adjacent kidney tissue. In addition, genomic DNA was isolated and digested with appropriate restriction enzymes. Southern blots were prepared and hybridized to IGF II-cDNA probes. Additional bands were present in one of the five Wilms tumours compared to normal tissue. The results indicate a rearrangement of the IGF II-gene on one of the two chromosomes. It is speculated, that this change is responsible for the elevated IGF II expression which may be a factor contributing to tumour growth.

Mannose 6-phosphate/insulin-like growth factor II-binding proteins in human serum and urine. Their relation to the mannose 6-phosphate/insulin-like growth factor II receptor.

Human serum and urine contain polypeptides which bind mannose 6-phosphate (M6P) and insulin-like growth factor II (IGF II) and crossreact with antibodies against the M6P/IGF II receptor. These polypeptides are considered to be fragments of the M6P/IGF II receptor. The major Mr approx. 205,000 fragment in serum and urine is about 10 kDa smaller in size than the membrane-associated receptor and is accompanied by minor forms with Mr values ranging from 104,000 to 180,000. The presence of receptor fragments in biological fluids indicates that shedding is one of the mechanisms contributing to the turnover of the M6P/IGF II receptor and that receptor fragments are part of the heterogenous group of serum proteins whic bind IGF II.

Mannose 6-phosphate/insulin-like growth factor II receptor: distinct binding sites for mannose 6-phosphate and insulin-like growth factor II.

Pentamannosyl phosphate substituted bovine serum albumin (PMP-BSA) and insulin like growth factor II (IGF II) bind specifically to immobilized mannose 6-phosphate/insulin like growth factor II receptor. An excess of IGF II inhibited binding of PMP-BSA by less than or equal to 20%, and an excess of PMP-BSA inhibited binding of IGF II by less than or equal to 10%. Polyclonal antibodies against the receptor purified from human liver inhibited preferentially the binding of PMP-BSA, and a monocloncal antibody 2C2 inhibited only the binding of IGF II to the receptor. Similar results were obtained for binding of PMP-BSA and IGF II to human skin fibroblasts. These results suggest that the binding sites for mannose 6-phosphate and IGF II reside in different portions of the receptor.

Tissue-specific expression of insulin-like growth factor II mRNAs with distinct 5' untranslated regions.

We have used RNA from human hypothalamus as template for the production of cDNAs encoding insulin-like growth factor II (IGF-II). The prohormone coding sequence of brain IGF-II RNA is identical to that found in liver; however, the 5' untranslated sequence of the brain cDNA has no homology to the 5' untranslated sequence of the previously reported liver cDNAs. By using hybridization to specific probes as well as a method based on the properties of RNase H, we found that the human IGF-II gene has at least three exons that encode alternative 5' untranslated regions and that are expressed in a tissue-specific manner. A probe specific to the brain cDNA 5' untranslated region hybridizes to a 6.0-kilobase transcript present in placenta, hypothalamus, adrenal gland, kidney, Wilms tumor, and a pheochromocytoma. The 5' untranslated sequence of the brain cDNA does not hybridize to a 5.3-kilobase transcript found in liver or to a 5.0-kb transcript found in pheochromocytoma. By using RNase H to specifically fragment the IGF-II transcripts into 3' and 5' fragments, we found that the RNAs vary in size due to differences in the 5' end but not the 3' end.

Insulin-like growth factor II in human adrenal pheochromocytomas and Wilms tumors: expression at the mRNA and protein level.

Two forms of insulin-like growth factor (IGF) II with molecular masses of 10 and 7.5 kDa, respectively, were found in tumor tissue from human adrenal pheochromocytomas. The tumors contained 5.3-7.1 micrograms of immunoreactive IGF-II per g of tissue, which is about 20 times more than in adrenal medulla. The total bioactive IGF in the pheochromocytomas exceeded that in normal liver or kidney, which contained only the 7.5-kDa IGF-II species, by a factor of approximately equal to 100. By contrast, the amount of IGF-I was just measurable and did not vary significantly between tumor and normal tissue. The high amounts of IGF-II in the pheochromocytomas were not reflected, however, by a corresponding increase of mRNA. The opposite situation was found in Wilms tumors, where IGF-II content was in the same range as in nontumor tissues despite increased expression of IGF-II mRNA.

Insulin-like growth factors I and II in fetal and adult bovine serum. Purification, primary structures, and immunological cross-reactivities.

Insulin-like growth factors I and II (IGF-I and IGF-II) were prepared from fetal calf serum and adult bovine serum by gel filtration, immunoaffinity chromatography, chromatofocusing, and reverse-phase high pressure liquid chromatography, and their complete amino acid sequences determined. IGF-I and -II are found in both adult and fetal serum. The sequence of bovine IGF-I is found to be identical to that of human IGF-I, whereas 3 out of 67 amino acid residues are found to be different between bovine and human IGF-II. The differences are located in the C-peptide region of the molecule. Bovine IGF-II shows less than 10% immunological cross-reactivity with antisera against human and rat IGF-II, but is equipotent to human IGF-II in displacing human 125I-labeled IGF-II from human placental receptor. Bovine IGF-I was equipotent to human IGF-I in both radioimmunoassays and radioreceptor assays within the limits of the assay.

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.

Amino acid sequence of a variant pro-form of insulin-like growth factor II.

Human serum contains, in addition to the "classical" 7.5-kDa insulin-like growth factors (IGFs) I and II, small amounts of larger IGF-II. A 10-kDa IGF-II was isolated by gel filtration, immunoaffinity chromatography, and reversed-phase HPLC. Upon amino acid sequence determination, a substitution of Cys-Gly-Asp for Ser-33 was found as well as a COOH-terminal extension of 21 residues (E peptide). These sequence differences suggest that 10-kDa IGF-II is a precursor of a variant IGF-II. Since the substitution is not located at a known intron/exon hinge region, the finding of this variant IGF-II is evidence for the presence of more than one gene for IGF-II.

Insulin-like growth factor II (IGF II) in human brain: regional distribution of IGF II and of higher molecular mass forms.

Twenty-four distinct areas of human brain were analyzed for the presence of insulin-like growth factor (IGF). As reported for cerebrospinal fluid, only IGF II-like immunoreactivity, but no significant amounts of IGF I-like immunoreactivity, could be found. Upon gel permeation chromatography, two to five distinct size classes were separated on the basis of their immunoreactivity. The smallest component had an apparent molecular mass of 7.5 kDa, identical to the one of purified IGF II from human serum. Radioimmunoassays and a bioassay also gave results indistinguishable from those of serum IGF II. The highest amounts of IGF II-like immunoreactivity occur in the anterior pituitary--namely, 20-25 pmol equivalents/g of wet weight. This is up to 100 times more than in most other brain regions analyzed. The higher molecular mass immunoreactive species were partially characterized. After immunoaffinity purification, the 38- and 26-kDa species are active in a bioassay. Specific IGF-binding protein activity could be shown after purification of the 38- and 26-kDa species on an IGF-affinity column. The 13-kDa species released significant amounts of 7.5-kDa material. The results are interpreted as evidence for the presence of IGF II synthesized locally in human brain. The structure of the larger forms of IGF II-like immunoreactive material as well as the function of IGF II in brain are not yet known.

Tertiary structures, receptor binding, and antigenicity of insulinlike growth factors.

Three-dimensional models for human insulinlike growth factors (IGF-I and IGF-II) have been constructed by using interactive molecular graphics. It is suggested that the two growth factors have structures in which the A and B chains and the hydrophobic cores are identical to those of insulin. The conformations of the connecting peptides and COOH-terminal extensions are predicted by statistical methods but the structures are limited by the constraints implied by the insulinlike part. The models explain the nonsuppressibility by anti-insulin antibodies of the IGFs and show that part of the insulin receptor-binding region is conserved, which explains the growth factors' ability to bind insulin receptors.

Interactions of a monoclonal antibody to the insulin receptor with receptors for insulin-like growth factors.

A monoclonal antibody to the human insulin receptor was tested for its ability to inhibit the binding of 125I-insulin-like growth factor I (IGF-I) and 125I-insulin-like growth factor II (IGF-II) to their receptors in human placenta membranes and cultured human IM-9 lymphocytes. In both placenta membranes and IM-9 cells, the antibody progressively inhibited the binding of 125I-IGF-I to its receptor with a potency that was 300-fold less than its ability to inhibit the binding of 125I-insulin to its own receptor. In contrast, in human placenta membranes, this antibody inhibited the binding of 125I-IGF-II to its receptor only slightly. These studies indicate, therefore, that this monoclonal antibody binds preferentially to the insulin receptor but also crossreacts to a lesser extent with the IGF-I receptor.

Monoclonal antibodies directed to human insulin-like growth factor I (IGF I). Use for radioimmunoassay and immunopurification of IGF.

Mouse hybridomas secreting antibodies to human insulin-like growth factor I (IGF I) were produced by fusion of spleen cells of hyperimmunised mice with FO mouse-myeloma cells. Eight clones producing antibodies against human IGF I have been isolated, two of which have been characterised. One was used in a radioimmunoassay, the other for immunopurification of IGF.