Serum Factors Inhibit Melanoma Cell Surface Expression of Type I and Type II IGF Receptors

Abstract

We have identified one class of IGF-I-binding sites and two classes of IGF-II-binding sites at the surface of the melanoma cell line IGR39. By means of affinity labeling with ¹²⁵I-IGF-I, a 290–300 kDa form was characterized. Using ¹²⁵I-IGF-II, a 270 kDa polypeptide was labeled, corresponding to the type II IGF receptor. In the two serials of experiments, the order of potency in inhibiting 125I-IGF-I or ¹²⁵I-IGF-II labeling of IGF-related peptides and αIR3, an antibody directed against type I receptor α subunit, was the same as in competition experiments. When IGR39 cells were cultured in a serum-free medium, the number of both high affinity IGF-II and IGF-I binding sites was increased, by 8-and 5-fold respectively, without any significant change in K_d values. In both culture conditions, we found IGFBP-2, -3, -4 and a 30 kDa form which Mr was consistent with IGFBP-5 or -6. Except for IGFBP-2, the amount of secreted IGFBPs was modified depending on culture conditions: in conditioned medium from cells cultured with 10% FCS, the amount of IGFBP-3 or -4 was higher, and the amount of the 30 kDa IGFBP was lower when compared to conditioned medium from cells cultured in serum-free medium.     

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.     

Association of the insulin-like growth factor II (IGF2) gene with human cognitive functions

Active search for candidate genes whose polymorphisms are associated with human cognitive functions has been in progress in the past years. The study focused on the role that the insulin-like growth factor II (IGF2) gene may play in the variation of cognitive processes related to executive functions. The ApaI polymorphism of the IGF2 gene was tested for association with selective attention during visual search, working memory/mental control, and semantic verbal fluency in a group of 182 healthy individuals. The ApaI polymorphism was associated with the general cognitive index and selective attention measure. Carriers of genotype AA displayed higher values of the two parameters than carriers of genotype GG. It was assumed that the ApaI polymorphism of the IGF2 gene influences the human cognitive functions, acting possibly via modulation of the IGF-II level in the central nervous system.     

Cholecystokinin and insulin regulate insulin-like growth factor II binding to pancreatic receptors; Evidence of a role for intracellular calcium

The binding of ¹²⁵I-labeled insulin-like growth factor II (¹²⁵I-IGF II) to mouse pancreatic acini was stimulated (45%) by insulin and inhibited (30%) by cholecystokinin octapeptide (CCK₈). When CCK₈ and insulin were added together, the effect on IGF II binding was similar to that seen when CCK₈ was added alone. Two lines of evidence suggest that this effect of cholecystokinin on basal and insulin-stimulated ¹²⁵I-IGF II binding was mediated via a change in intracellular calcium: (1) the cholinergic agent carbachol inhibited IGF II binding to its receptors; (2) addition of the Ca²⁺ ionophore A23187 mimicked the effects of CCK₈ and carbachol. In contrast to its effects on IGF II binding to acini, CCK₈ had only small effects on IGF I binding and no effects on insulin binding.     

Role of protein phosphatases in insulin-like growth factor II (IGF II)-stimulated mannose 6-phosphate/IGF II receptor redistribution.

The regulated expression of mannose 6-phosphate/insulin-like growth factor II (M6P/IGF II) receptors in plasma membranes has previously been shown to be accompanied by marked changes in the phosphorylation state of the receptors (Corvera, S., Folander, K., Clairmont, K. B., and Czech, M. P. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7567-7571). In the present study we show that protein phosphatase 2A dephosphorylates the human M6P/IGF II receptor in vitro. Incubation of human fibroblasts with okadaic acid, a specific inhibitor of this phosphatase, resulted in a depletion of M6P/IGF II receptors at the cell surface without affecting their internalization kinetics. The phosphorylation state of the remaining cell surface receptors was 3-fold increased. Thus, the endocytosis rate of M6P/IGF II receptors appears to be unaltered by increased phosphorylation. While the decreased cell surface expression of receptors was reversible upon removal of okadaic acid the IGF II-induced redistribution of M6P/IGF II receptors to the plasma membrane (Braulke, T., Tippmer, S., Neher, E., and von Figura, K. (1989) EMBO J. 8, 681-686) was irreversibly inhibited by the phosphatase inhibitor. Receptor redistribution in response to protein kinase C activation was not affected by okadaic acid. These results suggest that the cell surface expression of M6P/IGF II receptor can be regulated by phosphatase-dependent and -independent pathways. In addition, the phosphorylation state and the steady-state cell surface number of transferrin receptors were not affected by okadaic acid, whereas it impaired the IGF II-stimulated receptor redistribution similarly as for M6P/IGF II receptors. The data indicate that okadaic acid-sensitive protein phosphatases may play a general role in terms of IGF II-modulated receptor recycling.     

Non-additive effects of RBP4, ESR1 and IGF2 polymorphisms on litter size at different parities in a Chinese-European porcine line

Background

The aim of this work was to study the effects on litter size of variants of the porcine genes RBP4, ESR1 and IGF2, currently used in genetic tests for different purposes. Moreover, we investigated a possible effect of the interaction between RBP4-MspI and ESR1-PvuII polymorphisms. The IGF2-intron3-G3072A polymorphism is actually used to select lean growth, but other possible effects of this polymorphism on reproductive traits need to be evaluated.

Methods

Detection of polymorphisms in the genomic and cDNA sequences of RBP4 gene was carried out. RBP4-MspI and IGF2-intron3-G3072A were genotyped in a hyperprolific Chinese-European line (Tai-Zumu) and three new RBP4 polymorphisms were genotyped in different pig breeds. A bivariate animal model was implemented in association analyses considering the number of piglets born alive at early (NBA₁₂) and later parities (NBA₃₊ ) as different traits. A joint analysis of RBP4-MspI and ESR1-PvuII was performed to test their possible interaction. In the IGF2 analysis, paternal or maternal imprinting effects were also considered.

Results

Four different RBP4 haplotypes were detected (TGAC, GGAG, GAAG and GATG) in different pig breeds and wild boars. A significant interaction effect between RBP4-MspI and ESR1-PvuII polymorphisms of 0.61 ± 0.29 piglets was detected on NBA₃₊. The IGF2 analysis revealed a significant increase on NBA₃₊ of 0.74 ± 0.37 piglets for the paternally inherited allele A.

Conclusions

All the analyzed pig and wild boar populations shared one of the four detected RBP4 haplotypes. This suggests an ancestral origin of the quoted haplotype. The joint use of RBP4-MspI and ESR1-PvuII polymorphisms could be implemented to select for higher prolificacy in the Tai-Zumu line. In this population, the paternal allele IGF2-intron3-3072A increased litter size from the third parity. The non-additive effects on litter size reported here should be tested before implementation in other pig breeding schemes.     

Malony-CoA inhibits the S113L variant of carnitine-palmitoyltransferase II

Carnitine palmitoyltransferases (CPT), located both in the outer (CPT I) and inner membrane (CPT II) of mitochondria, are the key players for an efficient transport of long chain fatty acids into this cell compartment. The metabolite malonyl-CoA is known to inhibit CPT I, but not CPT II. His₆-N-hCPT2 (wild type) and His₆-N-hCPT2/S113L (variant) were produced recombinantly in prokaryotic host, purified and characterized according to their functional and regulatory properties. The wild type and the variant showed the same enzymatic activity and were both inhibited by malonyl-CoA and malonate in a time-dependent manner. The inhibition was, however, significantly more pronounced in the mutated enzyme. The residual activities were 40% and 5% at temperatures of 4 °C and 30 °C, respectively. The inhibitory effect proceeded irreversibly with no recovery after post-incubation of palmitoyl-CoA (Pal-CoA) as native substrate. A model of malonyl-CoA and malonate binding to human CPT II was suggested by docking studies to explain the action of the inhibitors regarding to the effect of the mutation on the protein conformation. Results indicated that not only CPT I, but also CPT II can be inhibited by malonyl-CoA. Thus, the complete inhibition of total CPT (i.e. CPT I and CPT II) in muscle homogenates by an established assay is not due to a lack of enzymatically active CPT II, but rather due to an abnormal regulation of the enzyme.     

Peptidic models for the binding of Pb(II), Bi(III) and Cd(II) to mononuclear thiolate binding sites

Herein, we evaluate the binding of Pb(II) and Bi(III) to cysteine-substituted versions of the TRI peptides [AcG-(LKALEEK)₄G-NH₂] which have previously been shown to bind Hg(II) and Cd(II) in unusual geometries as compared with small-molecule thiol ligands in aqueous solutions. Studies of Pb(II) and Bi(III) with the peptides give rise to complexes consistent with the metal ions bound to three sulfur atoms with M–S distances of 2.63 and 2.54 Å, respectively. Competition experiments between the metal ions Pb(II), Cd(II), Hg(II) and Bi(III) for the peptides show that Hg(II) has the highest affinity, owing to the initial formation of the extremely strong HgS₂ bond. Cd(II) and Pb(II) have comparable binding affinities at pH > 8, while Bi(III) displays the weakest affinity, following the model, M(II) + (TRI LXC)₃ ^3? → M(II)(TRI LXC)₃ ^?. While the relevant equilibria for Hg(II) binding to the TRI peptides corresponds to a strong first step forming Hg(TRI LXC)₂(HTRI LXC), followed by a single deprotonation to give Hg(TRI LXC)₃ ^?, the binding of Cd(II) and Pb(II) is consistent with initial formation of M(II)(TRI LXC)(HTRI LXC)₂ ⁺ at pH < 5 followed by a two-proton dissociation step (pK _a2) yielding M(II)(TRI LXC)₃ ^?. Pb(II)(TRI LXC)(HTRI LXC)₂ ⁺ converts to Pb(II)(TRI LXC)₃ ^? at slightly lower pH values than the corresponding Cd(II)–peptide complexes. In addition, Pb(II) displays a lower pK _a of binding to the “d”-substituted peptide, (TRI L12C, pK _a2 = 12.0) compared with the “a”-substituted peptide, (TRI L16C, pK _a2 = 12.6), the reverse of the order seen for Hg(II) and Cd(II). Pb(II) also showed a stronger binding affinity for TRI L12C (K _bind = 3.2 × 10⁷ M^?1) compared with that with TRI L16C (K _bind = 1.2 × 10⁷ M^?1) at pH > 8.     

Epitope-Specific Mechanisms of IGF1R Inhibition by Ganitumab

Background

Therapeutic antibodies targeting the IGF1R have shown diverse efficacy and safety signals in oncology clinical trials. The success of these agents as future human therapeutics depends on understanding the specific mechanisms by which these antibodies target IGF1R signaling.

Methodology/Principal Findings

A panel of well-characterized assays was used to investigate the mechanisms by which ganitumab, a fully human anti-IGF1R antibody undergoing clinical testing, inhibits IGF1R activity. Epitope mapping using IGF1R subdomains localized the ganitumab binding site to the L2 domain. Binding of ganitumab inhibited the high-affinity interaction of IGF-1 and IGF-2 required to activate IGF1R in cells engineered for IGF1R hypersensitivity and in human cancer cell lines, resulting in complete blockade of ligand-induced cellular proliferation. Inhibition of IGF1R activity by ganitumab did not depend on endosomal sequestration, since efficient ligand blockade was obtained without evidence of receptor internalization and degradation. Clinically relevant concentrations of ganitumab also inhibited the activation of hybrid receptors by IGF-1 and IGF-2. Ganitumab was not an agonist of homodimeric IGF1R or hybrid receptors in MCF-7 and COLO 205 cells, but low-level IGF1R activation was detected in cells engineered for IGF1R hypersensitivity. This activation seems biologically irrelevant since ganitumab completely inhibited ligand-driven proliferation. The in vivo efficacy profile of ganitumab was equivalent or better than CR and FnIII-1 domain-specific antibodies, alone or in combination with irinotecan. CR domain-specific antibodies only blocked IGF-1 binding to IGF1R but were more potent than ganitumab at inducing homodimer and hybrid receptor downregulation in vitro, however this difference was less obvious in vivo. No inhibition of hybrid receptors was observed with the FnIII-1 domain antibodies, which were relatively strong homodimer and hybrid agonists.

Conclusions/Significance

The safety and efficacy profile of ganitumab and other anti-IGF1R antibodies may be explained by the distinct molecular mechanisms by which they inhibit receptor signaling.     

Radiosynthesis and evaluation of IGF1R PET ligand [11C]GSK1838705A

Radiosynthesis and evaluation of [¹¹C]GSK1838705A in mice using microPET and determination of specificity in human GBM UG87MR cells are described herein. The radioligand was synthesized by reacting desmethyl-GSK1838705A with [¹¹C]CH₃I using GE FX2MeI module in ～5% yield (EOS), >95% radiochemical purity and a specific activity of 2.5 ± 0.5 Ci/μmol. MicroPET imaging in mice indicated that [¹¹C]GSK1838705A penetrated blood brain barrier (BBB) and showed retention of radiotracer in brain. The radioligand exhibited high uptake in U87MG cells with >70% specific binding to IGF1R. Our experiments suggest that [¹¹C]GSK-1838705A can be a potential PET radiotracer for the in vivo quantification of IGF1R expression in GBM and other brain tumors.     

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).     

Functional Class I and II Amino Acid-activating Enzymes Can Be Coded by Opposite Strands of the Same Gene

Aminoacyl-tRNA synthetases (aaRS) catalyze both chemical steps that translate the universal genetic code. Rodin and Ohno offered an explanation for the existence of two aaRS classes, observing that codons for the most highly conserved Class I active-site residues are anticodons for corresponding Class II active-site residues. They proposed that the two classes arose simultaneously, by translation of opposite strands from the same gene. We have characterized wild-type 46-residue peptides containing ATP-binding sites of Class I and II synthetases and those coded by a gene designed by Rosetta to encode the corresponding peptides on opposite strands. Catalysis by WT and designed peptides is saturable, and the designed peptides are sensitive to active-site residue mutation. All have comparable apparent second-order rate constants 2.9–7.0E-3 m⁻¹ s⁻¹ or ∼750,000–1,300,000 times the uncatalyzed rate. The activities of the two complementary peptides demonstrate that the unique information in a gene can have two functional interpretations, one from each complementary strand. The peptides contain phylogenetic signatures of longer, more sophisticated catalysts we call Urzymes and are short enough to bridge the gap between them and simpler uncoded peptides. Thus, they directly substantiate the sense/antisense coding ancestry of Class I and II aaRS. Furthermore, designed 46-mers achieve similar catalytic proficiency to wild-type 46-mers by significant increases in both k_cat and K_m values, supporting suggestions that the earliest peptide catalysts activated ATP for biosynthetic purposes.     

Energy transfer between Photosystem II and Photosystem I in chloroplasts

A model for the photochemical apparatus of photosynthesis is presented which accounts for the fluorescence properties of Photosystem II and Photosystem I as well as energy transfer between the two photosystems. The model was tested by measuring at ?196 °C fluorescence induction curves at 690 and 730 nm in the absence and presence of 5 mM MgCl₂ which presumably changes the distribution of excitation energy between the two photosystems. The equations describing the fluorescence properties involve terms for the distribution of absorbed quanta, α, being the fraction distributed to Photosystem I, and β, the fraction to Photosystem II, and a term for the rate constant for energy transfer from Photosystem II to Photosystem I,k_T(II→I). The data, analyzed within the context of the model, permit a direct comparison of α andk_T(II→I) in the absence (?) and presence (+) of Mg²⁺:α/^?α⁺= 1.2andk/^?_T(II→I)k⁺_T(II→I)= 1.9. If the criterion thatα + β = 1 is applied absolute values can be calculated: in the presence of Mg²⁺,a⁺ = 0.27 and the yield of energy transfer,φ⁺_T(II→I) varied from 0.065 when the Photosystem II reaction centers were all open to 0.23 when they were closed. In the absence of Mg²⁺,α^? = 0.32 andφ_T(II→I) varied from 0.12 to 0.28.The data were also analyzed assuming that two types of energy transfer could be distinguished; a transfer from the light-harvseting chlorophyll of Photosystem II to Photosystem I,k_T(II→I), and a transfer from the reaction centers of Photosystem II to Photosystem I,k_t(II→I). In that caseα/^?α⁺= 1.3,k/^?_T(II→I)k⁺_T(II→I)= 1.3 andk/^?_t(II→I)k⁺_(tII→I)= 3.0. It was concluded, however, that both of these types of energy transfer are different manifestations of a single energy transfer process.     

Purification and refolding of recombinant human IGF II from silkworms infected with recombinant Bombyx mori nuclear polyhedrosis virus.

We have reported that insulin-like growth factor II (IGF II) was produced as a fusion protein in Bombyx mori (silkworm) larval bodies infected with recombinant B. mori nuclear polyhedrosis virus [J. Gen. Virol., 68, 2599-2606 (1987)]. In this study, the purification of IGF II from the infected silkworms is reported. The fusion protein was extracted with 6.0 M guanidine-HCl from the infected larval bodies homogenized in water. The use of organic solvents to remove the impurities, such as lipid derived from the larval bodies, was a very effective method of purification. IGF II was released from the partially purified fusion protein by treatment with CNBr, purified by HPLC, and refolded by air-oxidization. Refolded IGF II had an identical primary structure including disulfide bonds and showed identical thymidine uptake stimulation activity with human IGF II. Furthermore, protein disulfide-isomerase was shown to be able to refold scrambled IGF II rapidly.     

Contrasting effects of IGF binding protein-3 expression in mammary tumor cells and the tumor microenvironment

IGFBP-3 has both stimulatory and inhibitory effects on cancer progression. The growth of EO771 mammary carcinoma cells as syngeneic tumors in C57BL/6 mice is reduced in Igfbp3-null (BP3KO) mice, suggesting that systemic IGFBP-3 enhances tumor progression. In this study we assessed the growth of EO771 cells expressing human IGFBP-3 in BP3KO mice. Cells expressing hIGFBP-3 showed decreased proliferation in vitro and increased levels of IGF-1 receptor (IGF1R) protein but not mRNA, consistent with sequestration of endogenous IGF by IGFBP-3. The growth rate of these cells was restored by exposure to IGF-1 or analogues with reduced affinity for IGFBP-3 (long Arg³-IGF-1) or IGF1R (Leu²⁴-IGF-1). In EO771 cells implanted orthotopically into mice, hIGFBP-3 expression by the cells inhibited tumor establishment in BP3KO but not wild-type mice. For tumors that successfully established, final weight was not affected significantly by hIGFBP-3 expression. However, final tumor weight was inversely related to intratumoral T cell counts, and sera from BP3KO mice with tumors showed low-titer immunoreactivity against IGFBP-3. The contrasting effects on tumor establishment and progression of IGFBP-3 expressed by mammary carcinoma cells, compared to systemic stromal and circulating IGFBP-3, highlights the complexity of growth regulation by IGFBP-3 in mammary tumors.     

An antibody that blocks insulin-like growth factor (IGF) binding to the type II IGF receptor is neither an agonist nor an inhibitor of IGF-stimulated biologic responses in L6 myoblasts

To better define the biologic function of the type II insulin-like growth factor (IGF) receptor, we raised a blocking antiserum in a rabbit by immunizing with highly purified rat type II IGF receptor. On immunoblots of crude type II receptor preparations, only bands corresponding to the type II IGF receptor were seen with IgG 3637, indicating that the antiserum was specific for the type II receptor. Competitive binding and chemical cross-linking experiments showed that IgG 3637 blocked binding of 125I-IGF-II to the rat type II IGF receptor, but did not block binding of 125I-IGF-I to the type I IGF receptor, nor did IgG 3637 block binding of 125I-insulin to the insulin receptor. In addition, IgG 3637 did not inhibit the binding of 125I-IGF-II to partially purified 150- and 40-kDa IGF carrier proteins from adult and fetal rat serum. L6 myoblasts have both type I and type II IGF receptors. IGF-I was more potent than IGF-II in stimulating N-methyl-alpha-[14C]aminoisobutyric acid uptake, 2-[3H]deoxyglucose uptake, and [3H]leucine incorporation into cellular proteins. IgG 3637 did not stimulate either 2-[3H]deoxyglucose uptake, N-methyl-alpha-[14C]aminoisobutyric acid uptake, or [3H]leucine incorporation into protein when tested alone. Furthermore, IgG 3637 at concentrations sufficient to block type II receptors under conditions of the uptake and incorporation experiments did not cause a shift to the right of the dose-response curve for stimulation of these biologic functions by IGF-II. We conclude that the type II IGF receptor does not mediate IGF stimulation of N-methyl-alpha-[14C]aminoisobutyric acid and 2-[3H]deoxyglucose uptake and protein synthesis in L6 myoblasts; presumably, the type I receptor mediates these biologic responses. The anti-type II receptor antibody inhibited IGF-II degradation in the media by greater than 90%, suggesting that the major degradative pathway for IGF-II in L6 myoblasts utilizes the type II IGF receptor.     

Variant Cell Lines of Haplopappus gracilis with Disturbed Activities of Nitrate Reductase and Nitrite Reductase

Selected variant cell lines of Haplopappus gracilis (Nutt) Gray that showed disturbed growth after transfer from an alanine medium to NO₃⁻ medium were characterized. The in vivo NO₃⁻ reductase activity (NRA) was lower in these lines than in the wild type. In vitro NRA assays suggest that decreased in vivo NRA was not caused by a lower amount of active enzyme. Cells of the variant lines revealed up to 75% lower extractable activity of NO₂⁻ reductase as compared with the wild type. This coincided with higher accumulation of NO₂⁻ by the variant than by the wild type cells after transfer from alanine medium to NO₃⁻ medium. NO₂⁻ accumulation was transient or continuous, depending on cell line, metabolic state of the cells, and light conditions.