Delineation of the IGF-II C domain elements involved in binding and activation of the IR-A, IR-B and IGF-IR

Growth Hormone & IGF Research 25 (2015) 20–27

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Delineation of the IGF-II C domain elements involved in binding and activation of the IR-A, IR-B and IGF-IR S.T. Henderson a,b, G.V. Brierley a, K.H. Surinya a, I.K. Priebe a, D.E.A. Catcheside b, J.C. Wallace c, B.E. Forbes c, L.J. Cosgrove a,⁎ a b c

Commonwealth Scientific Industrial Research Organisation, Preventative Health National Research Flagship, Adelaide, South Australia 5000, Australia School of Biological Sciences, Flinders University, Adelaide, South Australia 5001, Australia School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia 5005, Australia

a r t i c l e

i n f o

Article history: Received 18 February 2013 Received in revised form 5 September 2014 Accepted 26 September 2014 Available online 30 October 2014 Keywords: IGF-I IGF-II Chimera Structure Binding Insulin receptor

a b s t r a c t Objective: Human insulin-like growth factor-I and -II (IGF-I and -II) ligands share a high degree of sequence and structural homology. Despite their similarities, IGF-I and IGF-II exhibit differential receptor binding and activation characteristics. The C domains of IGF-I and IGF-II are the primary determinants of binding specificity to the insulin-like growth factor I receptor (IGF-IR), insulin receptor exon 11− (IR-A) and exon 11+ (IR-B) isoforms. Design: Three IGF-II analogues were generated in order to delineate the C domain residues that confer the differential receptor binding affinity and activation properties of the IGFs. Chimeric IGF-II analogues IGF-IICIN and IGFIICIC contained partial IGF-I C domain substitutions (IGF-I residues underlined) GYGSSSRRSR and SRVSRRAPQT, respectively. Results: The IGF-IICIN analogue bound the IR-A and IGF-IR with high affinity but bound the IR-B with a relatively lower affinity than IGF-II, suggesting a negative interaction between the exon-11 encoded peptide in the IR-B and the C-domain. The ability of IGF-IICIN to activate receptors and elicit cell viability responses was generally proportional to its relative receptor binding affinity but appeared to act as a partial agonist equivalent to IGF-I when binding and activating the IGF-IR. In contrast, IGF-IICIC bound IGF-IR with high affinity but elicited lower receptor activation and cell viability responses. Analogue IGF-IICIS contained a truncated IGF-I C domain (GSSSRRAT) and generally displayed a relatively poor ability to bind, activate and elicit viability responses via each receptor. Conclusions: Together, the IGF analogues demonstrate that both flanks of the IGF-II C domain play important roles in the greater ability of IGF-II to bind and activate IR receptors than IGF-I. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction IGF-I and IGF-II share a high degree of sequence and structural homology [1–3]. The single-chain polypeptides are folded into four domains: B, C, A and D (in order of N to C terminus) to form mature IGF-I and IGF-II. The A and B domains of these two growth factors are structurally analogous to mature insulin. The flexible C domains are similar to the C domain in pro-insulin but are not proteolytically cleaved, whilst the D domains are unique to the IGFs [4]. Despite their structural similarities, IGF-I and IGF-II exhibit differential receptor binding and activation characteristics. The IGFs elicit a wide range of actions via Abbreviations: IGF-I, insulin-like growth factor I; IGF-II, insulin-like growth factor II; IR, insulin receptor; IR-A, insulin receptor isoform A; IR-B, insulin receptor isoform B; IGF-IR, type 1 IGF receptor; R−IR-A, R− cells expressing the human IR-A; R−IR-B, R− cells expressing the human IR-B; R−IGFIR, R− cells expressing the human IGF-IR; aa, amino acid; mqH2O, Milli-Q ultra pure water. ⁎ Corresponding author at: CSIRO Preventative Health National Research Flagship, PO Box 10041, Adelaide 5000, South Australia, Australia. Tel.: + 61 8 83038833; fax: +61 8 83038899. E-mail address: [email protected] (L.J. Cosgrove).

http://dx.doi.org/10.1016/j.ghir.2014.09.004 1096-6374/© 2014 Elsevier Ltd. All rights reserved.

interactions with the type 1 IGF receptor (IGF-IR) or insulin receptor (IR) that are vital to normal growth and development including: somatic growth, cellular proliferation, differentiation, migration, and protection from apoptosis [5–7]. Elucidating the mechanisms by which the IGFs interact with their receptors is of particular interest as these biological functions are commonly perturbed in cancer. The IR and IGF-IR are highly homologous heterotetrameric transmembrane receptor tyrosine kinases consisting of two α and two β subunits linked together by disulphide bonds [8]. The IGFs and insulin bind with high affinity to their cognate receptors (IGF-IR and IR, respectively) and with lower affinity to their non-cognate receptors. However, the IR is expressed as two different isoforms that differ at the carboxyterminus of the α-subunits by 12 amino acids and differentially bind ligands [9–11]. The IR-A (exon 11−) displays high affinity for insulin, intermediate affinity for IGF-II, and low affinity for IGF-I [9,11]. The IR-B (exon 11+) displays high affinity for insulin and only low affinities for IGF-I and IGF-II [11]. Thus, IGF-II can bind with high affinity to both the IR-A and IGF-IR [9,11], with both receptors mediating IGF-II bioactivity [12]. The mechanism for the differential binding of IGFs to the two insulin receptor isoforms has yet to be fully elucidated.

S.T. Henderson et al. / Growth Hormone & IGF Research 25 (2015) 20–27

The two binding surfaces and mechanism of ligand-receptor interaction between IGF-I and the IGF-IR are comparable to that of the insulin and IR interaction [8]. However, the molecular interactions between IGF-II and the IR and IGF-IR are considerably less well understood relative to our understanding of how insulin and IGF-I ligands interact with their cognate receptors. Site-directed mutagenesis studies have identified two receptor binding sites in IGF-II, composed of amino acids in the B and A-domains (Site 1: Val14, possibly Asp15, Gln18, Phe26, Tyr27, Phe28, and Val43; Site 2: Glu12, Phe19, Leu53, and Glu57), that are analogous to the binding sites in IGF-I and insulin although there are subtle differences in the contribution of particular residues [13,14]. Furthermore, alanine-scanning mutagenesis studies have shown that IGF-II binds to elements in the L1 domain and the CT peptide of the IGF-IR, but unlike IGF-I, IGF-II does not interact with elements in the cysteine-rich (CR) domain, suggesting that IGF-I and IGF-II utilise different mechanisms to bind to the same receptor binding site [15]. Support for this is also evident from the lack of inhibition of IGF-II binding to the IGF-IR by monoclonal antibodies with binding epitopes in the IGF-IR CR domain [16]. It has been demonstrated that the majority of free energy of IGF-I and IGF-II receptor binding is due to two sites located primarily in the B and A domains [14,17]. However, IGF-II and IGF-I chimeras with reciprocally swapped C domains and/or D domains exhibit receptor binding affinities similar to those of the donor molecules, thereby demonstrating that the IR and IGF-IR binding specificity of the IGFs is primarily determined by their C domains and, to a lesser extent, the D-domains of the IGFs [11]. In addition, an IGF-I chimera containing the IGF-II C domain induced autophosphorylation of the IR-A and IR-B and activated downstream IRS-1 and Akt/PKB signalling pathways to a similar extent as IGF-II [11] further signifying the importance of the IGF C domain in modulating IGF-receptor interactions. The mechanism by which the IGF-II C domain determines receptor binding specificity and activation is unknown. The IGF-II C domain consists of 8 amino acids, whilst the IGF-I C domain has 12 amino acids. Thus it is possible that the smaller IGF-II C domain permits greater steric accessibility to the ligand binding pocket of the IR. However, it has been shown that an IGF-I analogue with four insulin substitutions in the B and A-domains had insulin-like binding affinity to the IR-A, suggesting that the IR-A ligand binding pocket is capable of accepting the bulkier IGF-I C domain [18]. This assertion is supported by the finding that a single-chain insulin analogue with the IGF-I C domain was able to bind to the IR with a similar affinity to insulin [19].

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Identifying the IGF C domain residues involved in differential receptor binding and activation could provide valuable insights into how IGF-II and IGF-I exert their biological effects. Elucidating the mechanism of high affinity IGF-II binding is of particular interest as the biological effects of this ligand are mediated through both the IR-A and IGF-IR and both receptors are implicated in cancer [5]. As such we aimed to delineate the residues within the C domain of IGF-II which conferred high affinity binding and potent activation of the IR-A. Three IGF-II analogues were produced to identify the C domain regions which confer differential receptor binding affinity and activation. All three IGF-II analogues included the C domain residues that are conserved in both IGF-I and IGF-II; Ser36/35, Arg37/36 and Arg38/37 (numbering refers to IGF-II/IGF-I amino acid positions, respectively). The IGF-II analogues IGF-IICIN and IGF-IICIC contain IGF-I C domain residues from either the N-terminal flank or C-terminal flank of the conserved SRR motif (Fig. 1). The IGF-IICIS analogue contained a shortened IGF-I C domain so as to be similar in size to the IGF-II C domain whilst retaining the charge characteristics of the IGF-I C domain to investigate if the larger size of the IGF-I C domain sterically hindered IR binding.

2. Materials and methods 2.1. Materials General chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Cell culture reagents were purchased from Life Technologies (Carlsbad, CA). Oligonucleotides were purchased from Geneworks Pty. Ltd (Adelaide, South Australia). Restriction enzymes were purchased from New England Biolabs (Hitchin, UK). pGEM-T-Easy Vector system and CellTiter-Blue Cell Viability Assay were purchased from Promega Corp. (Madison, WI). Human IGF-I and IGF-II were from GroPep Bioreagents (Adelaide, South Australia). Human insulin was purchased from Novo Nordisk (Bagsvaerd, Denmark). The generation of IGF-IICI (IGF-II containing the C domain of IGF-I) is described by Denley et al. [11]. DELFIA europium-labelling kit, DELFIA enhancement solution and europium-conjugated antiphosphotyrosine antibody PY20 were purchased from PerkinElmer Life Sciences (Waltham, MA). Europium-labelled IGF-I and insulin were produced as described previously [11]. Greiner Lumitrac 600 96-well plates were from Omega Scientific (Tarzana, CA). Antibodies 83-7 and 24-31 were kind gifts from Professor K. Siddle (Cambridge, UK).

Fig. 1. (A) Amino acid sequence alignment of mature human insulin, IGF-I and IGF-II proteins. Numbering of amino acids is above the sequence for both insulin and IGF-I, and below the sequence for IGF-II. The C domains of both IGF-I and IGF-II are highlighted in the exploded view and bold type face indicates the conserved residues. (B) Amino acid sequences of the C domain chimeras. IGF-I residues are indicated by a grey shaded box, while IGF-II residues are indicated by an open box. Conserved residues between the IGF-I and IGF-II C domains are indicated by bold type face. Numbering of amino acids is above the sequence of each C domain chimera.

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2.2. Construction of expression plasmids encoding human IGF-II C domain chimeras The Escherichia coli codon optimized IGF-IICI coding sequence was PCR amplified (forward primer 5′–GTTAACCCGGCACCGATG-3′ and reverse primer 5′-AAGCTTTTATTCGCTTTTTGC-3′) from plasmid pGH(1-11) VNPAPM-BCADII(I) [11] and cloned into the pGEM-T-easy vector as a template for site-directed mutagenesis [20]. The IGF-II C domain chimeras were generated by utilising oligonucleotide pairs (IGF-IICIN: 5′-CTCGTC CGGCGGGCTATGGCAGCAGCAGCCGGCGTTCTCGCGG-3′ and 5′-CCGCGA GAACGCCGGCTGCTGCTGCCATAGCCCGCCGGACGAG-3′; IGF-IICIC: 5′CTCGCGTTAGCCGGCGTGCGCCACAGACCGGAATCGTGG-3′ and 5′-CCAC GATTCCGGTCTGTGGCGCACGCCGGCTAACGCGAG-3′; IGF-IICIS: 5′-CTCG TCCGGCGGGCTCTAGCAGCCGGCGTGCGACCGGAATCGTGG-3′ and 5′CCACGATTCCGGTCGCACGCCGGCTGCTAGAGCCCGCCGGACGAG-3′) to introduce the desired C domain sequence changes into the IGF-IICI coding sequence which were PCR amplified and subcloned into pGH(1-11). The pGH(1-11) expression vector facilitates efficient heterologous expression of wildtype and mutant IGF-I and IGF-II proteins in E. coli [21]. The IGF-IICIN, IGF-IICIC and IGF-IICIS coding sequences (Fig. 1B) were fused in-frame to the coding sequence for the first eleven amino acids of porcine growth hormone and a PAPM linker sequence [22]. The IGF-IICIN, IGF-IICIC and IGF-IICIS fusion proteins were expressed in E. coli JM101 after isopropyl β-D-thiogalactoside induction and inclusion bodies were isolated as described by King et al. [21]. 2.3. Purification of IGF-II C domain chimeras The procedures for protein purification from inclusion bodies containing the IGF-IICIN, IGF-IICIC and IGF-IICIS fusion peptides were similar to those described previously [23]. Purified IGF-IICIN, IGF-IICIC and IGFIICIS proteins were analysed by mass spectrometry and N-terminal sequencing and were determined to have the correct molecular weight and to be greater than 95% pure. The C domain chimeras were quantitated by comparing analytical rpHPLC profiles with profiles of a reference standard of receptor grade human IGF-I [24]. 2.4. Cell lines and cell culture conditions P6 cells (BALB/c3T3 cells overexpressing the human IGF-IR) [25] and R− cells (mouse 3T3-like cells with a targeted ablation of the IGF-IR gene) [26] were a kind gift from Professor R. Baserga (Philadelphia, PA). The construction of R− cells expressing human IR-A (R−IR-A), IR-B (R−IR-B) and IGF-IR (R−IGF-IR) cDNAs has been previously reported [11]. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (v/v) foetal calf serum, 50 units/ml penicillin, 50 μg/ml streptomycin and 0.4 mg/ml geneticin at 37 °C/5%CO2 unless otherwise stated.

addition of 100 μl/well of DELPHIA enhancement solution and incubation at room temperature for 30 min. Time-resolved fluorescence was measured using a Wallac Victor3V 1420 Multilabel Counter (Perkin Elmer) with 340 nm excitation and 615 nm emission filters. The data were calculated as a percentage of either bound Eu-insulin or Eu-IGF-I in the absence of competing ligand (% specific binding = bound/total × 100) and plotted using Prism 5 (Graphpad) with IC50's calculated by curve-fitting with a one-site competition model. 2.6. Receptor phosphorylation assays Receptor tyrosine phosphorylation was measured essentially as described by Chen et al. [27]. R−IR-A, R−IR-B and R−IGF1R cells were plated in Falcon 96 well flat bottom plates at 8 × 103 cells/well and grown overnight at 37 °C/5%CO2 prior to 4 h serum starvation in serum-free DMEM. Cells were then treated with various concentrations (0.1–100 nM) of either insulin, IGF-II, IGF-I or C domain chimeras for 10 min at 37 °C/5%CO2. Treatment medium was aspirated and cells lysed in lysis buffer (20 mM HEPES, 150 mM NaCl, 1.2 mM MgCl2, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 10 mM NaF, 10% (v/v) glycerol, 1% (v/v) Triton X-100, pH 7.5) prior to capturing receptors overnight at 4 °C on either anti-IR antibody 83-7 or anti-IGF-IR 24-31 antibody coated white Greiner Lumitrac 600 96-well plates. Plates were washed thrice with 1 × PBST and phosphorylated receptors detected with europium-labelled anti-phosphotyrosine antibody PY20. DELFIA enhancement solution was added and time-resolved fluorescence measured with a Wallac Victor3V 1420 Multilabel Counter (Perkin Elmer) with 340 nm excitation and 615 nm emission filters. 2.7. Cell viability assays R−IR-A, R−IR-B and R−IGFIR cells were serum starved for 5 h at 37 °C/5%CO2 prior to treatment with various concentrations (0.1– 100 nM) of IGF-I, IGF-II, Insulin and C domain chimeras in serum-free DMEM medium containing 0.1%BSA/5 mM sodium butyrate. After 48 h, cell viability was determined by measuring the conversion of the redox dye resazurin to the fluorescent end product resorufin by the CellTiterBlue Cell Viability Assay according to the manufacturer's instructions. 2.8. Statistics Data were analysed using GraphPad Prism 5, version 5.02 (GraphPad, California, USA). IC50 values for competition ligand binding were calculated by curve-fitting with a one-site competition model. Statistical significance of differences in each ligand's ability to activate receptors and elicit viability responses was determined by ANOVA with Tukey's multiple comparison test. Statistical significance was defined as p b 0.05. 3. Results

2.5. Binding analysis of C domain chimeras to IR isoforms and IGF-IR 3.1. IR-A interaction with the IGF-II analogues The binding affinities of the purified IGF-II C domain chimeras to the IR-A, IR-B and IGF-IR were determined by a competitive binding assay with europium labelled insulin (Eu-insulin) or IGF-I (Eu-IGF-I) as described by Denley et al. [11]. Briefly, serum-starved R−IR-A, R−IR-B or P6 cells were lysed with lysis buffer (20 mM HEPES, 150 mM NaCl, 1.2 mM MgCl2, 1 mM EDTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 1/1000 protease inhibitor cocktail, pH 7.5) for 1 h at 4 °C to provide sources of human IR-A, IR-B and IGF-IR receptors. Lysates were cleared of debris by centrifugation at 2800 g for 5 min at 4 °C prior to capturing receptors overnight at 4 °C on either anti-IR antibody 83-7 or anti-IGFIR 24-31 antibody coated white Greiner Lumitrac 600 96 well plates. Approximately 1 × 106 counts of either Eu-insulin or Eu-IGF-I were added to wells containing captured receptors along with various amounts of unlabelled competitor ligands and incubated overnight at 4 °C. Plates were washed thrice with 1× PBST and twice with mqH2O before the

Three analogues were constructed to delineate residues within the IGF-II C domain that confer high affinity receptor binding and potent activation of the IR-A. The IGF-IICIN and IGF-IICIC analogues bound the IR-A with affinities similar to IGF-II (IC50s of 7.5 ± 0.8 nM, 6.8 ± 1.3 nM and 5.9 ± 0.7 nM, respectively) and with greater affinity than IGF-IICI (IC50: 12.0 ± 1.8 nM) indicating that the C domain configurations of both IGF-IICIN and IGF-IICIC facilitated high affinity binding to the IR-A (Fig. 2A and Table 1). In contrast, IGF-IICIS (IC50: 13.8 ± 3. 0nM) bound the IR-A with a 13% lower affinity than IGF-IICI. Despite the equipotent IR-A binding affinities of IGF-IICIN and IGFIICIC, the IR-A activation response to IGF-IICIN was comparable to IGFII whereas IGF-IICIC induced a phosphorylation response similar to IGF-IICIS, IGF-IICI and IGF-I (Fig. 3A and see Table 2). Cell viability assays were conducted to assess the ability of the IGF-II analogues to induce

S.T. Henderson et al. / Growth Hormone & IGF Research 25 (2015) 20–27

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Fig. 2. Competitive ligand binding assays of IR-A, IR-B and IGF-IR by insulin, IGF-I, IGF-II and IGF-II chimeras. Immunocaptured IR-A (A), IR-B (B) or IGF-IR (C) were incubated with Eulabelled ligand (Eu-insulin for IR-A and IR-B, Eu-IGF-I for IGF-IR) in the presence or absence of increasing concentrations of insulin, IGF-I, IGF-II, IGF-IICI or IGF C domain chimeras. Results are expressed as a percentage of Eu-labelled ligand bound in the absence of competing ligand, and the data points are the mean ± SEM of triplicate samples from three independent experiments. Error bars are shown when greater than the size of the symbols. Relative binding is expressed as a percent of IGF-II and whether it is significant p b 0.05 (*) or not significant (ns) p N 0.05. Ligands are denoted as follows: insulin [■], IGF-I [▼], IGF-II [●], IGF-IICI [◆], IGF-IICIN [△], IGF-IICIC [□], and IGF-IICIs [▽]. All binding curves are represented by a solid line, except for IGF-II where a dashed line is used.

protection from butyrate-stimulated apoptosis. The addition of 5 mM butyrate resulted in a 2-fold reduction in R−IR-A cell viability, and all ligands were capable of providing some protection from butyrateinduced apoptosis (Fig. 4A and D). The capacity of each ligand to increase R−IRA cell viability, with the exception of insulin, was generally proportional to their relative ability to induce IR-A phosphorylation. IGF-IICIN, IGF-IICIC, IGF-IICIS, IGF-II and insulin stimulated similar cell viability responses (EC50: 1.88 ± 0.47 nM, 3.31 ± 0.58 nM, 3.80 ± 0.79 nM, 4.9 ± 1.90 nM, 3.15 ± 0.74 nM), whereas the IGF-IICI (EC50: 10.2 ± 2.23 nM viability responses were similar to IGF-I 10.5 ± 6.36 nM) (Fig. 4A & D and Table 3). A summary of the ligand binding, receptor phosphorylation and survival data for the IR-A is shown in Tables 1 2 and 3, respectively.

Insulin induced the highest phosphorylation levels corresponding with the high binding affinity of this ligand to the IR-B. Butyrate treatment reduced R−IR-B cell viability 2-fold and all ligands were able to provide protection from the effects of butyrate (Fig. 4B and E, and Table 3). Both IGF-IICIN (EC50: 2.1 ± 0.45 nM) and IGF-II (EC50: 1.79 ± 0.29 nM) were similar in their ability to elicit increases in R−IR-B cell viability. The IGF-IICIC and IGF-IICIS ligands (EC50: 3.75 ± 0.76 nM and 2.92 ± 0.71 nM, respectively) elicited similar responses to those of IGF-IICI (EC50: 8.06 ± 3.85 nM) and IGF-I (EC50: 6.14 ± 2.59 nM), but were less effective than IGF-IICIN and IGF-II ligands at increasing R−IR-B cell viability. With the exception of insulin (EC50: 7.93 ± 2.14 nM), the cell viability responses generally reflected the ability of each ligand to induce IR-B phosphorylation. Insulin bound the IR-B with the highest affinity and was the most potent activator of this receptor but induced relatively low increases in R−IR-B cell viability.

3.2. IR-B interaction with the IGF-II analogues 3.3. IGF-IR interaction with the IGF-II analogues Insulin bound the IR-B with a 2-fold higher affinity than the IR-A, whereas IGF-II (2-fold), IGF-I (1.5-fold) and IGF analogues (1.5–3-fold) all bound the IR-A with higher affinity than the IR-B (Table 1), which is consistent with our previous work [11]. The IGF-IICIC and IGF-II ligands had similar IR-B binding affinities (IC50s of 15.8 ± 2.3 c and 11.5 ± 0.9 nM, respectively) (Fig. 2B) whereas IGF-IICIN (IC50: 23.0 ± 3.6 nM) bound the IR-B with a lower affinity similar to that of IGF-IICI (IC50: 24.5 ± 2.2 nM) and IGF-IICIS (IC50: 20.0 ± 2.7 nM) (Table 1). Despite demonstrating a binding affinity similar to that of IGF-II, the IGF-IICIs ligand was relatively poor at inducing IR-B phosphorylation with a phosphorylation response similar to that of IGF-IICI (Fig. 3B and E, and Table 2). Interestingly, the IR-B binding affinity of IGF-IICIN was comparable to the binding affinity of IGF-IICI but induced an IR-B phosphorylation response similar to that of IGF-II. The IGF-IICIS ligand both bound and activated the IR-B with a similar affinity to that of IGF-IICI.

The competition binding curves for IGF-I, IGF-II, insulin and IGF-II analogues binding the IGF-IR are shown in Fig. 2C and IC50 values are presented in Table 1. Surprisingly, the IGF-IICIN (IC50: 0.12 ± 0.02 nM) and IGF-IICIC (IC50: 0.10 ± 0.03 nM) analogues bound the IGF-IR with similar affinity to IGF-I (IC50: 0.09 ± 0.01 nM) and slightly higher affinity than IGF-IICI (IC50: 0.14 ± 0.02 nM). IGF-II (IC50: 0.19 ± 0.02 nM) bound the IGF-IR with moderate affinity, whereas IGF-IICIS (IC50: 0.91 ± 0.48 nM) and insulin (IC50: 18.0 ± 7.0 nM) had relatively poor IGF-IR binding affinities. Despite having similar binding affinities for the IGF-IR, the IGF-IICIN and IGF-IICIC IGF-II analogues stimulated different levels of IGF-IR phosphorylation. At sub-nanomolar concentrations, IGF-I was the most potent activator of IGF-IR phosphorylation overall (Fig. 3C) though, IGF-IICIN at 3 nM induced the greatest IGF-IR phosphorylation equivalent to IGF-I (Fig. 3F,and Table 2). IGF-IICIC induced

Table 1 Inhibition of europium-labelled ligand from binding to the IR-A, IR-B and IGF-IR by Insulin, IGF-I, IGF-II, IGF-IICI and IGF C domain chimeras. IR-A

IR-B

IGF-IR

Ligand

IC50 (nM)

IC50 relative to IGF-II (%)

IC50 (nM)

IC50 relative to IGF-II (%)

IC50 (nM)

IC50 relative to IGF-II (%)

IGF-II IGF-IICIN IGF-IICIC IGF-IICIS IGF-IICI IGF-I Insulin

5.9 7.5 6.8 13.8 12.0 26.2 1.2

100 79a 87a 43a 49b 23b 492b

11.5 23.0 15.8 20.0 24.5 40.6 0.6

100 50a 73a 58b 47b 28b 1917b

0.19 0.12 0.10 0.91 0.14 0.09 18.0

100 158a 190a 21b 136a 211b 1b

± ± ± ± ± ± ±

0.7 0.8 1.3 3.0 1.8 4.3 0.2

± ± ± ± ± ± ±

0.9 3.6 2.3 2.7 2.2 4.5 0.03

± ± ± ± ± ± ±

0.02 0.02 0.03 0.48 0.02 0.01 7.0

The IC50 values are the mean ± SEM from three independent experiments. The relative IC50 refers to the relative binding affinity of each ligand for the stated receptor compared to IGF-II. a Not significant when compared to IGF-II. b p value b 0.05.

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S.T. Henderson et al. / Growth Hormone & IGF Research 25 (2015) 20–27

Fig. 3. Phosphorylation of IR-A, IR-B and IGF-IR by insulin, IGF-I, IGF-II and IGF-II chimeras. R− cells overexpressing either the human IR-A (A, D), IR-B (B, E), or IGF-IR (C, F) were serum starved prior to stimulation with increasing concentrations of either insulin, IGF-I, IGF-II, IGF-IICI or IGF C domain chimeras for 10 min. Cells were lysed and activated receptors were immunocaptured on 96-well plates and then incubated with Eu-PY20 antibody to detect phosphorylated tyrosine residues using time-resolved fluorescence. The data points are mean ± SEM of triplicate samples from four independent experiments. Error bars are shown when greater than the size of the symbols. Ligands are denoted as follows: insulin [■], IGF-I [▼], IGF-II [●], IGF-IICI [◆], IGF-IICIN [△], IGF-IICIC [□], and IGF-IICIs [▽]. All activation curves are represented by a solid line, except for IGF-II where a dashed line is used (A, B, C). Receptor activation at 10 nM ligand (D and E) and 3 nM ligand (F) is represented as grey bars and data are mean ± SEM. The statistical significance of differences between the ability of each ligand to activate each receptor was determined by ANOVA with Tukey's multiple comparison test; in all cases ns = not significant p N 0.05. For (D), a, b = p b 0.01; c, d = p b 0.001. For (E), a, c = p b 0.01; d, e, f, h = p b 0.05; b, g = p b 0.001. For (F); a, b, c, d, e, f, g, h, i, j, k, l, m = p b 0.001.

lower levels of IGF-IR phosphorylation relative to IGF-IICIN but was more potent than IGF-II (Fig. 3C and F, and Table 2). Of the IGF-II analogues, IGF-IICIS (EC50: 14.7 ± 3.93 nM) was the poorest at stimulating IGF-IR phosphorylation correlating with its low IGF-IR binding affinity. The relative order of potency in inducing IGF-IR phosphorylation was IGF-I (1.71 ± 0.74 nM) followed by IGF-IICIN, IGF-IICI, IGFIICIC, IGF-II, and IGF-IICIS with insulin being the poorest activator of IGF-IR. Treatment with butyrate reduced R−IGF-IR cell viability to levels seen in both R−IR-A and R−IR-B cells. All ligands were able to provide some protection from the effects of butyrate and with the exceptions of IGF-IICIS and insulin generally reflected the ability of each ligand to induce IGF-IR phosphorylation (Fig. 4C and F, and Table 3). As such, IGF-I stimulated the greatest increase in R−IGF-IR cell viability followed

by IGF-IICIN, IGF-IICIC, IGF-II, IGF-IICI, IGF-IICIS, and lastly insulin,(EC50: 0.25 ± 0.13 nM, 0.41 ± 0.13 nM, 0.63 ± 0.21 nM, 0.87 ± 0.21 nM, 1.01 ± 0.39 nM, 2.44 ± 0.47 nM, 18.50 ± 8.79 nM respectively). 4. Discussion IGF-II and IGF-I have two receptor binding surfaces composed of residues in the B and A domains which provide the majority of receptor binding free energy [14,17]. Characterization of IGF-II and IGF-I chimeras with swapped C and/or D domains indicated the C domain is largely responsible for IR and IGF-IR binding specificity and activation [11]. The IGF-IICI chimera characterized in this previous study consisted of the IGF-I C domain incorporated into IGF-II and this conferred more IGF-I like receptor binding affinities and activation responses in the IR-

Table 2 Activation of IR-A, IR-B, and IGF-IRs by Insulin, IGF-I, IGF-II, IGF-IICI and IGF C domain chimeras. IR-A

IR-B

IGF-IR

Ligand

EC50 (nM)

EC50 relative to IGF-II (%)

EC50 (nM)

EC50 relative to IGF-II (%)

EC50 (nM)

EC50 relative to IGF-II (%)

IGF-II IGF-IICIN IGF-IICIC IGF-IICIS IGF-IICI IGF-I Insulin

41.0 63.2 127 102 125 216 30.2

100 64.87a 32.28b 40.19b 32.8b 18.98b 135.7a

24.1 38.5 27.0 65.4 81.9 44.2 5.49

100 62.6a 89.25a 36.85b 29.42b 54.52a 438.97a

10.3 1.46 5.90 14.7 2.80 1.71 170

100 705b 174.57b 70a 367.86b 602.33b 6.05c

± ± ± ± ± ± ±

7.53 7.06 57 17.2 30.2 84.2 18.2

± ± ± ± ± ± ±

4.74 26.1 7.27 16.6 35.6 17.1 2.65

± ± ± ± ± ± ±

1.13 0.26 1.16 3.93 5.46 0.74 72.7

The EC50 values are the mean ± SEM from three independent experiments. The relative EC50 refers to the relative binding affinity of each ligand for the stated receptor compared to IGF-II. a Not significant when compared to IGF-II. b p value b 0.05. c An estimate, unable to be accurately determined.

S.T. Henderson et al. / Growth Hormone & IGF Research 25 (2015) 20–27

A

B

25

C 3 3

F o ld c h a n g e in c e ll v ia b ility

1

2

1

0

0 - 1 0 .5

- 1 0 .0

- 9 .5

- 9 .0

- 8 .5

- 8 .0

- 7 .5

D

- 1 0 .0

- 9 .5

- 9 .0

E ns

acd

- 8 .5

- 7 .5

- 7 .0

- 1 0 .5

- 1 0 .0

- 9 .5

- 9 .0

70 60 50 40 30 20

e

c c

- 7 .5

- 7 .0

ns ns

ad

e

f

90 % Maximal survival response relative to 100nM IGF-II

% Maximal survival response relative to 100nM IGF-II

ae

- 8 .0

d

100

a

90 cf

F ns

c de

- 8 .5

L ig a n d [ M ]

ns

90 dg

- 8 .0

ns 100

efg

80

1

L ig a n d [ M ]

ns

100

2

0

- 1 0 .5

- 7 .0

L ig a n d [ M ]

% Maximal survival response relative to 100nM IGF-II

o v e r c e lls t r e a t e d w it h 5 m M B u A

F o ld c h a n g e in c e ll v ia b ility

2

o v e r c e lls t r e a t e d w it h 5 m M B u A

F o ld c h a n g e in c e ll v ia b ility

o v e r c e lls t r e a t e d w it h 5 m M B u A

3

80 70 60 50 40 30 20

cdef 80 70 60 50 40 30 20

10

10

10

0

0

0

Fig. 4. R− cells overexpressing either the human IR-A, IR-B or IGF-IR were incubated with increasing concentrations of ligand in the presence of 5 mM butyrate. Results are expressed as fold change increase in cell viability relative to cells treated with 5 mM butyrate alone. The data points are mean ± SD of triplicate samples from four independent experiments. Error bars are shown when greater than the size of the symbols. Ligands are denoted as follows: insulin [■], IGF-I [▼], IGF-II [●], IGF-IICI [◆], IGF-IICIN [△], IGF-IICIC [□], and IGF-IICIs [▽]. All viability curves are represented by a solid line, except for IGF-II where a dashed line is used (A, B, C). Note the difference in the scale of the Y-axis for Fig. 4B, IR-B graph. Cell viability at 10 nM ligand (D and E) and 3 nM ligand (F) is represented as grey bars and data are mean ± SEM. The statistical significance of differences between each ligand to increase cell viability was determined by ANOVA with Tukey's multiple comparison test. For (D), a = p b 0.05; c, e = p b 0.01; b, d, f, g = p b 0.001. For (E), a, c = p b 0.05; c = p b 0.05; b, d, e = p b 0.001. For (F), a, c, e, h, I, j, k, l, m = p b 0.001; b, d, f, g = p b 0.05.

A, IR-B and the IGF-IR [11]. Using IGF-IICI as a reference point, three IGFII analogues IGF-IICIN, IGF-IICIC, and IGF-IICIS were generated with partial IGF-I/IGF-II chimeric C domains to delineate C domain elements which conferred differential receptor binding and activation of the IR isoforms and the IGF-IR. The IGF-IICIN and IGF-IICIC chimeras contained partial IGF-I C domain substitutions flanking the conserved Ser36, Arg37 & Arg38 residues (Fig. 1). The IGF-IICIS analogue contained a shortened IGF-I C domain so as to be similar in size to the IGF-II C domain, whilst retaining the charge characteristics of the IGF-I C domain, to specifically address the possibility of steric hindrance being responsible for the decreased ability of the IGF-IICI chimera to bind and activate the IR isoforms [11,28]. It should be noted though that insulin analogues with only partial agonist activity are almost unknown, and the biological activity of insulin analogues (at least in terms of receptor tyrosine

phosphorylation and acute biological responses) largely parallels their relative binding affinities [29]. There are substantial variations in competitive receptor binding assay formats and absolute receptor binding affinities of the IGFs reported in the literature. This could be due largely to the different assay formats typically used in receptor binding studies including the use of intact cells or immunopurified receptors and use of iodine-125 (125I) or europium labelled homologous or heterologous tracers. A study which directly compared the binding affinities of IGF-II and IGF-II analogues in IR-A and IGF-IR binding assays with whole cells and immunocaptured receptors indicated there was up to a ~10-fold variation in binding affinities of ligands between the two types of assays [14]. Whereas direct comparison of 125I-labelled and europium-labelled IGF-I and IGF-II tracers in immunopurified IGF-IR homologous competitive binding assays indicated

Table 3 Cell viability of IR-A, IR-B, and IGF-IRs by Insulin, IGF-I, IGF-II, IGF-IICI and IGF C Domain Chimeras. IR-A

IR-B

IGF-IR

Ligand

EC50 (nM)

EC50 relative to IGF-II (%)

EC50 (nM)

EC50 relative to IGF-II (%)

EC50 (nM)

EC50 relative to IGF-II (%)

IGF-II IGF-IICIN IGF-IICIC IGF-IICIS IGF-IICI IGF-I Insulin

4.9 1.88 3.31 3.80 10.2 10.5 3.15

100 260a 148a 128.94a 48a 46.66a 155a

1.79 2.10 3.75 2.92 8.06 6.14 7.93

100 85.23a 47.73b 61.3a 22.2b 29.15b 22.57b

0.87 0.41 0.63 2.44 1.01 0.25 18.50

100 212.53a 136.65a 35.45 85.64a 337.89b 4.67c

± ± ± ± ± ± ±

1.90 0.47 0.58 0.79 2.23 6.36 0.74

± ± ± ± ± ± ±

0.29 0.45 0.76 0.71 3.85 2.59 2.14

± ± ± ± ± ± ±

0.21 0.13 0.21 0.47 0.39 0.13 8.79

The EC50 values are the mean ± SEM from three independent experiments. The relative EC50 refers to the relative binding affinity of each ligand for the stated receptor compared to IGF-II. a Not significant when compared to IGF-II. b p value b 0.05. c An estimate, unable to be accurately determined.

26

S.T. Henderson et al. / Growth Hormone & IGF Research 25 (2015) 20–27

that there was little difference between the binding curves for each label [30]. On this basis, the competitive receptor binding assay results obtained in this study were compared with published IGF binding studies using immunopurified receptors and europium or 125I-labelled ligand tracers. The IGF-IICIN and IGF-IICIC analogues bound the IR-A with similar affinity to that of IGF-II (Fig. 2A). This indicates that either flank of the IGF-II C domain is sufficient to permit high affinity binding to the IR-A, whilst the presence of partial IGF-I C domain residues in IGF-IICIN and IGF-IICIC is tolerated with no significantly adverse affect on IR-A binding. In contrast, the IGF-IICI chimera containing the entire IGF-I C domain had an IR-A binding affinity intermediate between IGF-II and IGF-I which was consistent with previous studies [11]. The reduced IR-A binding affinity of IGF-IICI relative to IGF-II, IGF-IICIN and IGF-IICIC was unlikely to be due solely to steric hindrance of the larger IGF-I C domain (12 amino acids) as the IGF-IICIS analogue had a shortened IGF-I C domain (8 amino acids) and bound the IR-A with a lower affinity than IGF-IICI (Table 1). In addition, two previous studies with ligands containing the entire IGF-I C domain consisting of an IGF-I mutant containing B and A-domain substitutions [31] and a single chain hybrid combining insulin with the IGF-I C domain [19], have been shown to bind the IRA with high affinity. An alternative possibility for the differential IR-A binding of IGF-II, IGF-IICIN and IGF-IICIC relative to IGF-IICI could be due to charge differences between the C domains of IGF-II and IGF-I. Previous mutagenesis studies with IGF-I [18,32] and IGF-II [33] have shown substantial differences in IR and IGF-IR binding affinities when the conserved Arg36/ Arg37 (IGF-I) and Arg37/Arg38 (IGF-II) C domain residues were substituted with neutral amino acids. These studies have demonstrated that the presence or absence of these charged residues in the IGF C domain can play important roles in receptor binding specificity. The three IGFII analogues generated in this study contained two arginine residues within the conserved Ser36-Arg37-Arg38 segment of the C domain (Fig. 1). In addition, IGF-IICIN and IGF-IICIC also contained Arg40 and Arg34, respectively, which may have facilitated the higher IR-A binding affinities of these analogues. In contrast, the C domains of IGF-IICIS and IGF-IICI only contained the conserved Arg37 and Arg38 residues and were observed to bind the IR-A with lower affinity. Thus, the Arg34 and Arg40 residues may contribute to the IR-A binding specificity of IGF-II. IGF-I, IGF-II and the IGF-II analogues bound the IR-B with lower affinity relative to the IR-A which is consistent with other studies [34,11] (Table 1). Interestingly, IGF-II and IGF-IICIC had comparable IR-B binding affinities whereas IGF-IICIN bound the IR-B with a proportionally lower affinity similar to IGF-IICI. This indicated that IGF-IICIN lacked one or more IGF-II residues beneficial to IR-B binding and/or contained IGF-I residues which were detrimental to IR-B but not IR-A binding. Despite demonstrating equivalent binding affinities to IGF-IICI, the IGF-IICIN analogue elicited greater IR-B phosphorylation and cell viability responses. This indicated that the chimeric C domain of IGF-IICIN was more amenable to receptor conformational changes facilitating greater kinase domain autophosphorylation than that permitted by the full IGF-I C domain in IGF-IICI. In contrast, the IR and IGF-IR phosphorylation and cell viability responses induced by IGF-IICIC were similar to those obtained with IGF-IICI but were discordant with the high affinity binding of this analogue to all three receptors indicating that IGF-IICIC was a partial agonist of the IR isoforms and the IGF-IR. Surprisingly, the IGF-IR binding affinities of both IGF-IICIN and IGFIICIC were similar to IGF-I and were greater than IGF-IICI and IGF-II indicating that the individual flanks of the IGF-I C domain are better tolerated than the presence of the full IGF-I C domain. It cannot be determined from this study if the increased IGF-IR binding affinities of IGF-IICIN and IGF-IICIC relative to IGF-IICI and IGF-II were facilitated by the presence of favourable IGF-I/IGF-II C domain residues, the absence of unfavourable IGF-II amino acids, or a combination of both. The higher IGF-IR binding affinity of IGF-IICIC is unlikely to be due solely to the presence of IGF-I C domain residues as this chimera lacks the Tyr31 residue which has

been shown to have a significant role in IGF-I binding to the IGF-IR [35]. The IGF-I Arg36/37 C domain residues are known to interact with the cysteine rich (CR) region of the IGF-IR [18]. Despite the conservation of these arginine residues in IGF-II, alanine scanning mutagenesis of the IGF-IR has shown that IGF-I interacts with the CR domain IGF-IR [36] whereas IGF-II does not [15]. Additionally, monoclonal antibodies with binding epitopes within the IGF-IR CR domain impaired IGF-I and IGFIICI binding to the IGF-IR whilst IGF-II was unaffected [16]. Of the three IGF-II analogues generated in this study, IGF-IICIS is most similar to IGF-IICI as the C domain residues were derived solely from IGF-I with the omission of residues Gly30, Tyr31, Pro39 and Glu40. Interestingly, IGF-IICIS bound the IGF-IR with the lowest affinity of the IGF analogues. The 6.5-fold lower IGF-IR binding affinity of IGFIICIS relative to IGF-IICI is most likely attributable to the absence of the Tyr31 residue which has been shown by alanine substitution to reduce IGF-I binding to the IGF-IR by 6-fold [35]. The relatively low IGF-IR binding affinity of IGF-IICIS suggested that the absence of unfavoured IGF-II C domain residues (viz. G30, Y31, P39, Q40) was not solely responsible for the higher IGF-IR binding affinity of IGF-IICI relative to IGF-II. In summary, our characterization of the IGF-IICIN and IGF-IICIC chimeras has indicated that both flanks of the IGF-II C domain play important roles in receptor binding and activation. The affinity fold changes seen in this study are close to the 2-fold threshold generally considered being significant for disruption of receptor interaction [36]. This however indicates that the different interactions between the Cdomains of the IGF analogues and the IR/IGF-IR are subtle and need to be interpreted in this context. The IGF-IICIC analogue had similar binding to IGF-II to both IR isoforms and the IGF-IR, whereas the IGF-IICIN C domain made different contributions to IR-A and IR-B binding, presumably due to a negative interaction with the exon-11 encoded peptide in the IR-B. Characterization of the IGF-IICIS analogue indicated that the larger size of the IGF-I C domain was unlikely to be sterically hindering ligand binding with the IR suggesting that side chain interactions between receptor and the IGF-II and IGF-I C domains are likely to influence differential receptor binding affinities of the IGFs. Conflict of interest The authors declare that there is no conflict of interest. Acknowledgements We thank Ms Kerrie McNeil for her assistance in protein purification, Dr Adam Denley for his assistance with protein purification and the europium-based competition assay, Dr Chris Bagley for mass spectrometry analysis and Mr Chris Cursaro for N-terminal sequencing analysis. We thank Dr Colin Ward for his support of this project, Professor Ken Siddle for providing monoclonal antibodies to the IR and IGF-IR, and Professor Renato Baserga for providing P6 and R− cells. This project was supported by an Australian Postgraduate Award and CSIRO Postgraduate Top-up scholarship awarded to S.T.H., and by funding to L.J.C's laboratory from CSIRO Preventative Health National Research Flagship. The authors declare no conflict of interest. References [1] A. Sato, S. Nishimura, T. Ohkubo, et al., Three-dimensional structure of human insulin-like growth factor-I (IGF-I) determined by 1H-NMR and distance geometry, Int. J. Pept. Protein Res. 41 (5) (1993) 433–440. [2] A.M. Torres, B.E. Forbes, S.E. Aplin, et al., Solution structure of human insulin-like growth factor II. Relationship to receptor and binding protein interactions, J. Mol. Biol. 248 (2) (1995) 385–401. [3] R.M. Cooke, T.S. Harvey, I.D. Campbell, Solution structure of human insulin-like growth factor 1: a nuclear magnetic resonance and restrained molecular dynamics study, Biochemistry 30 (22) (1991) 5484–5491. [4] E. Rinderknecht, R.E. Humbel, The amino acid sequence of human insulin-like growth factor I and its structural homology with proinsulin, J. Biol. Chem. 253 (8) (1978) 2769–2776.

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