Kerrie A. McNeil

Structural basis for the lower affinity of the insulin-like growth factors for the insulin receptor.

Insulin and the insulin-like growth factors (IGFs) bind with high affinity to their cognate receptor and with lower affinity to the noncognate receptor. The major structural difference between insulin and the IGFs is that the IGFs are single chain polypeptides containing A-, B-, C-, and D-domains, whereas the insulin molecule contains separate A- and B-chains. The C-domain of IGF-I is critical for high affinity binding to the insulin-like growth factor I receptor, and lack of a C-domain largely explains the low affinity of insulin for the insulin-like growth factor I receptor. It is less clear why the IGFs have lower affinity for the insulin receptor. In this study, 24 insulin analogues and four IGF analogues were expressed and analyzed to explore the role of amino acid differences in the A- and B-domains between insulin and the IGFs in binding affinity for the insulin receptor. Using the information obtained from single substituted analogues, four multiple substituted analogues were produced. A “quadruple insulin” analogue ([PheA8, SerA10, ThrB5, GlnB16]Ins) showed affinity as IGF-I for the insulin receptor, and a “sextuple insulin” analogue ([PheA8, SerA10, ThrA18, ThrB5, ThrB14, GlnB16]Ins) showed an affinity close to that of IGF-II for the insulin receptor, whereas a “quadruple IGF-I” analogue ([His4, Tyr15, Thr49, Ile51]IGF-I) and a “sextuple IGF-II” analogue ([His7, Ala16, Tyr18, Thr48, Ile50, Asn58]IGF-II) showed affinities similar to that of insulin for the insulin receptor. The mitogenic potency of these analogues correlated well with the binding properties. Thus, a small number of A- and B-domain substitutions that map to the IGF surface equivalent to the classical binding surface of insulin weaken two hotspots that bind to the insulin receptor site 1.

Role Of N- And C-Terminal Residues Of Insulin-Like Growth Factor (IGF)-Binding Protein-3 In Regulating IGF Complex Formation And Receptor Activation

Insulin-like growth factor-binding protein-3 (IGFBP-3), the major IGFBP in the circulation, sequesters IGF in a stable ternary complex with the acid-labile subunit. The high affinity IGF-binding site is proposed to reside within an N-terminal hydrophobic domain in IGFBP-3, but C-terminal residues have also been implicated in the homologous protein IGFBP-5. We have mutated in various combinations Leu77, Leu80, and Leu81 in the N terminus and Gly217 and Gln223 in the C terminus of IGF-BP-3. All mutants retained immunoreactivity toward a polyclonal IGFBP-3 antibody, whereas IGF ligand blotting showed that all of the mutants had reduced binding to IGFs. Both solution IGF binding assays and BIAcore analysis indicated that mutations to the N-terminal region caused greater reduction in IGF binding activity than C-terminal mutations. The combined N- and C-terminal mutants showed undetectable binding to IGF-I but retained <10% IGF-II binding activity. Reduced ternary complex formation was seen only in mutants that had considerably reduced IGF-I binding, consistent with previous studies indicating that the binary IGF·IGFBP-3 complex is required for acid-labile subunit binding. Decreased IGF binding was also reflected in the inability of the mutants to inhibit IGF-I signaling in IGF receptor overexpressing cells. However, when present in excess, IGFBP-3 analogs defined as non-IGF-binding by biochemical assays could still inhibit IGF signaling. This suggests that residual binding activity of IGFBP-3 mutants may still be sufficient to inhibit IGF biological activity and questions the use of such analogs to study IGF-independent effects of IGFBP-3.

Alanine scanning of a putative receptor binding surface of insulin-like growth factor-I.

Current evidence supports a binding model in which the insulin molecule contains two binding surfaces, site 1 and site 2, which contact the two halves of the insulin receptor. The interaction of these two surfaces with the insulin receptor results in a high affinity cross-linking of the two receptor α subunits and leads to receptor activation. Evidence suggests that insulin-like growth factor-I (IGF-I) may activate the IGF-I receptor in a similar mode. So far IGF-I residues structurally corresponding to the residues of the insulin site 1 together with residues in the C-domain of IGF-I have been found to be important for binding of IGF-I to the IGF-I receptor (e.g. Phe23, Tyr24, Tyr31, Arg36, Arg37, Val44, Tyr60, and Ala62). However, an IGF-I second binding surface similar to site 2 of insulin has not been identified yet. In this study, we have analyzed whether IGF-I residues corresponding to the six residues of the insulin site 2 have a role in high affinity binding of IGF-I to the IGF-I receptor. Six single-substituted IGF-I analogues were produced, each containing an alanine substitution in one of the following positions (corresponding insulin residues in parentheses): Glu9 (HisB10), Asp12 (GluB13), Phe16 (LeuB17), Asp53 (SerA12), Leu54 (LeuA13), and Glu58 (GluA17). In addition, two analogues with 2 and 3 combined alanine substitutions were also produced (E9A,D12A IGF-I and E9A,D12A,E58A IGF-I). The results show that introducing alanine in positions Glu9, Asp12, Phe16, Leu54, and Glu58 results in a significant reduction in IGF-I receptor binding affinity, whereas alanine substitution at position 53 had no effect on IGF-I receptor binding. The multiple substitutions resulted in a 33–100-fold reduction in IGF-I receptor binding affinity. These data suggest that IGF-I, in addition to the C-domain, uses surfaces similar to those of insulin in contacting its cognate receptor, although the relative contribution of the side chains of homologous residues varies.

Understanding the Mechanism of Insulin and Insulin-Like Growth Factor (IGF) Receptor Activation by IGF-II

Background Insulin-like growth factor-II (IGF-II) promotes cell proliferation and survival and plays an important role in normal fetal development and placental function. IGF-II binds both the insulin-like growth factor receptor (IGF-1R) and insulin receptor isoform A (IR-A) with high affinity. Interestingly both IGF-II and the IR-A are often upregulated in cancer and IGF-II acts via both receptors to promote cancer proliferation. There is relatively little known about the mechanism of ligand induced activation of the insulin (IR) and IGF-1R. The recently solved IR structure reveals a folded over dimer with two potential ligand binding pockets arising from residues on each receptor half. Site-directed mutagenesis has mapped receptor residues important for ligand binding to two separate sites within the ligand binding pocket and we have recently shown that the IGFs have two separate binding surfaces which interact with the receptor sites 1 and 2. Methodology/Principal Findings In this study we describe a series of partial IGF-1R and IR agonists generated by mutating Glu12 of IGF-II. By comparing receptor binding affinities, abilities to induce negative cooperativity and potencies in receptor activation, we provide evidence that residue Glu12 bridges the two receptor halves leading to receptor activation. Conclusions/Significance This study provides novel insight into the mechanism of receptor binding and activation by IGF-II, which may be important for the future development of inhibitors of its action for the treatment of cancer.

A novel binding site for the human insulin-like growth factor-II (IGF-II)/mannose 6-phosphate receptor on IGF-II.

The mammalian insulin-like growth factor (IGF)-II/cation-independent mannose 6-phosphate receptor (IGF2R) binds IGF-II with high affinity. By targeting IGF-II to lysosomal degradation, it plays a role in the maintenance of correct IGF-II levels in the circulation and in target tissues. Loss of IGF2R function is associated with tumor progression; therefore, the IGF2R is often referred to as a tumor suppressor. The interaction between IGF2R and IGF-II involves domains 11 and 13 of the 15 extracellular domains of the receptor. Recently, a hydrophobic binding region was identified on domain 11 of the IGF2R. In contrast, relatively little is known about the residues of IGF-II that are involved in IGF2R binding and the determinants of IGF2R specificity for IGF-II over the structurally related IGF-I. Using a series of novel IGF-II analogues and surface plasmon resonance assays, this study revealed a novel binding surface on IGF-II critical for IGF2R binding. The hydrophobic residues Phe19 and Leu53 are critical for IGF2R binding, as are residues Thr16 and Asp52. Furthermore, Thr16 was identified as playing a major role in determining why IGF-II, but not IGF-I, binds with high affinity to the IGF2R.

A Novel Approach to Identify Two Distinct Receptor Binding Surfaces of Insulin-like Growth Factor II

Very little is known about the residues important for the interaction of insulin-like growth factor II (IGF-II) with the type 1 IGF receptor (IGF-1R) and the insulin receptor (IR). Insulin, to which IGF-II is homologous, is proposed to cross-link opposite halves of the IR dimer through two receptor binding surfaces, site 1 and site 2. In the present study we have analyzed the contribution of IGF-II residues equivalent to insulins two binding surfaces toward the interaction of IGF-II with the IGF-1R and IR. Four “site 1” and six “site 2” analogues were produced and analyzed in terms of IGF-1R and IR binding and activation. The results show that Val43, Phe28, and Val14 (equivalent to site 1) are critical to IGF-1R and IR binding, whereas mutation to alanine of Gln18 affects only IGF-1R and not IR binding. Alanine substitutions at Glu12, Asp15, Phe19, Leu53, and Glu57 analogues resulted in significant (>2-fold) decreases in affinity for both the IGF-1R and IR. Furthermore, taking a novel approach using a monomeric, single-chain minimized IGF-1R we have defined a distinct second binding surface formed by Glu12, Phe19, Leu53, and Glu57 that potentially engages the IGF-1R at one or more of the FnIII domains.

Contribution of residues A54 and L55 of the human insulin-like growth factor-II (IGF-II) A domain to Type 2 IGF receptor binding specificity.

The underlying specificity of the interaction between insulin-like growth factor-II (IGF-II) and mammalian Type 2 insulin-like growth factor/cation-independent mannose 6 phosphate receptor (IGF2R) is not understood. We have mutated residues A54 and L55 of IGF-II in the second A domain helix to arginine (found in the corresponding positions of IGF-I) and measured IGF2R binding. There is a 4- and 3.3-fold difference in dissociation constants for A54R IGF-II and L55R IGF-II, respectively, and a 6.6-fold difference for A54R L55R IGF-II compared with IGF-II as measured by BIAcore analysis using purified rat IGF2R. This is also confirmed using cross-linking and soluble rat placental membrane receptor binding assays. Binding to the type 1 IGF receptor (IGF1R) and IGF binding protein-2 (IGFBP-2) is not altered. We can, therefore, conclude that residues at positions 54 and 55 in IGF-II are important for and equally contribute to IGF2R binding.

Insulin-like growth factor binding protein-2: NMR analysis and structural characterization of the N-terminal domain

The insulin-like growth factor binding proteins are a family of six proteins (IGFBP-1 to -6) that bind insulin-like growth factors-I and -II (IGF-I/II) with high affinity. In addition to regulating IGF actions, IGFBPs have IGF-independent functions. IGFBP-2, the largest member of this family, is over-expressed in many cancers and has been proposed as a possible target for the development of novel anti-cancer therapeutics. The IGFBPs have a common architecture consisting of conserved N- and C-terminal domains joined by a variable linker domain. The solution structure and dynamics of the C-terminal domain of human IGFBP-2 have been reported (Kuang Z. et al. J. Mol. Biol. 364, 690-704, 2006) but neither the N-domain (N-BP-2) nor the linker domain have been characterised. Here we present NMR resonance assignments for human N-BP-2, achieved by recording spectra at low protein concentration using non-uniform sampling and maximum entropy reconstruction. Analysis of secondary chemical shifts shows that N-BP-2 possesses a secondary structure similar to that of other IGFBPs. Although aggregation hampered determination of the solution structure for N-BP-2, a homology model was generated based on the high degree of sequence and structure homology exhibited by the IGFBPs. This model was consistent with experimental NMR and SAXS data and displayed some unique features such as a Pro/Ala-rich non-polar insert, which formed a flexible solvent-exposed loop on the surface of the protein opposite to the IGF-binding interface. NMR data indicated that this loop could adopt either of two alternate conformations in solution - an entirely flexible conformation and one containing nascent helical structure. This loop and an adjacent poly-proline sequence may comprise a potential SH3 domain interaction site for binding to other proteins.

Insulin-like growth factor-I (IGF-I): Solution properties and NMR chemical shift assignments near physiological pH

OBJECTIVE Insulin-like growth factor-I (IGF-I) plays important roles in normal growth and development, as well as in disease states, and its structure and function have been studied extensively using nuclear magnetic resonance (NMR) spectroscopy. However, IGF-I typically gives poor quality NMR spectra containing many broad peaks, because of aggregation at the protein concentrations generally required for NMR experiments as well as the internal dynamics of the molecule. The present study was undertaken to determine a reliable set of assignments under more physiological conditions. DESIGN Several reports of chemical shift assignments have been published previously for IGF-I either bound to a ligand or at relatively low pH (approximately 3-4), but there are many contradictions among them, reflecting the poor behaviour of IGF-I. Low pH conditions are also suboptimal for the analysis of interactions between IGF-I and IGF binding proteins (IGFBP) or IGFBP fragments. Spectra were recorded at low concentrations in order to identify conditions of temperature and pH where all peaks could be observed. RESULTS We show that good quality 2D (1)H-(15)N HSQC spectra of (15)N-labelled IGF-I can be obtained at pH 6 and 37 degrees C, much closer to physiological conditions, by using lower IGF-I concentrations (0.05 mM). Surprisingly, at this concentration and temperature, spectra were of better quality at pH 6 than at pH 4, in contrast to previous observations made at millimolar concentrations of IGF-I. We were then also able to assign the chemical shifts of IGF-I at pH 6 and 37 degrees C using 3D heteronuclear spectra recorded on a 0.7 mM (15)N/(13)C-labelled IGF-I sample. CONCLUSION These results provide a valuable resource for future studies of the structure, dynamics, folding, and binding interactions of IGF-I, as well as analogues thereof, by means of NMR spectroscopy.

P46 Studies on the bivalent interaction of IGF-II with the insulin and type 1 IGF receptors

demonstrated the role of the IGF system in the regulation of trophoblast proliferation and survival and alterations in the IGF ligands or the receptors could have implications in the pathogenesis of trophoblastic diseases. The aim of this study was to investigate the role of the IGF system in trophoblast cell differentiation by evaluating the expression of genes of the IGF system during the process. We utilized the HTR8/Svneo extra villous trophoblast cell line and induced differentiation by stimulation with forskolin (FSK), an agonist of adenylyl cyclase, We found that cells treated with FSK 25mM for 72 hours secreted high levels of chorionic gonadotropin (hCG), a common differentiation marker of syncitiotrophoblast. Interestingly we found a hCG related increase in IGF-II and IGF-IIR mRNAs expression, suggesting an important role of this growth factor and its receptor during differentiation. To investigate the potential role of an autocrine IGF-II/IGF-IIR loop we obtained a stable IGF-IIR silenced HTR8/Svneo cell line by shRNA tranfection and induced differentiation aspreviously. After 72 hours of treatment with FSK we found a marked reduction in IGF-II mRNA levels, in comparison with the non-silenced cell line, together with a decrease in the expression in the IGF-I and Insulin receptors. In addition we observed a direct relation between IGF-IIR silencing with a delayed kinetic pattern of differentiation. In conclusion this study presents a first approximation to the autocrine role of IGFII/IGF-IIR system in trophoblastic cell differentiation.