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The cation-independent mannose-6-phosphate/insulin-like growth factor-II receptor (IGF2R) is a membrane-bound glycoprotein consisting of 15 homologous extracellular repeat domains. The major function of this receptor is trafficking of lysosomal enzymes from the trans-Golgi network to the endosomes and their subsequent transfer to lysosomes. The IGF2R also plays a major role in binding and regulating the circulating and tissue levels of IGF-II. As this ligand is important for cell growth, survival, and migration, the maintenance of correct IGF-II levels influences its actions in normal growth and development. Deregulation of IGF2R expression has therefore been associated with growth related disease and cancer. This review highlights recent advances in understanding the IGF2R structure and mechanism of interaction with its ligands, in particular IGF-II. Recent mutagenesis studies combined with the crystal structure of domains 11-14 in complex with IGF-II have mapped the sites of interaction and explain how the IGF2R specificity for IGF-II is achieved. The role of domain 13 in high-affinity IGF-II binding is also revealed. Characterization of ligand:IGF2R interactions is vital for the understanding of the mechanism of IGF2R actions and will allow the development of specific cancer therapies in the future. [ABSTRACT FROM AUTHOR]

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-IICI(N) and IGF-IICI(C) contained partial IGF-I C domain substitutions (IGF-I residues underlined) GYGSSSRRSR and SRVSRRAPQT, respectively., Results: The IGF-IICI(N) 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-IICI(N) 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-IICI(C) bound IGF-IR with high affinity but elicited lower receptor activation and cell viability responses. Analogue IGF-IICI(S) 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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In the absence of a complete tertiary structure to define the molecular basis of the high affinity binding interaction between insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs), we have investigated binding of IGFs by discrete amino-terminal domains (amino acid residues 1-93, 1-104, 1-132, and 1-185) and carboxyl-terminal domains (amino acid residues 96-279, 136-279, and 182-284) of bovine IGFBP-2 (bIGFBP-2). Both halves of bIGFBP-2 bound IGF-I and IGF-II in BIAcore studies, albeit with different affinities ((1-132)IGFBP-2, K(D) = 36.3 and 51.8 nm; (136-279)IGFBP-2HIS, K(D) = 23.8 and 16.3 nm, respectively). The amino-terminal half appears to contain components responsible for fast association. In contrast, IGF binding by the carboxyl-terminal fragment results in a more stable complex as reflected by its K(D). Furthermore, des(1-3)IGF-I and des(1-6)IGF-II exhibited reduced binding affinity to (1-279)IGFBP-2HIS, (1-132)IGFBP-2, and (136-279)IGFBP-2HIS biosensor surfaces compared with wild-type IGF. A charge reversal at positions 3 and 6 of IGF-I and IGF-II, respectively, affects binding interactions with the amino-terminal fragment and full-length bIGFBP-2 but not the carboxyl-terminal fragment.

Insulin-like growth factors (IGFs) promote cell growth and differentiation. Their actions are regulated by six different, but related, binding proteins (IGFBPs). To investigate the molecular interactions between IGFs and IGFBPs, an Escherichia coli based production method and a phage display system has been developed. The cDNA for bovine IGFBP-2 was inserted between regions coding for the pelB signal sequence and geneIII product, g3p, of bacteriophage fd in a phagemid vector to generate pGF14. The coding sequences of IGFBP-2 and g3p were separated by an amber stop codon and a flexible linker containing the cleavage recognition site for H64A subtilisin. Using this system in BL21, a non-supE strain lacking ompT, most product, approximately 4 mg 1(-1) of IGFBP-2, was obtained in the growth medium. The bacterially derived IGFBP-2 had a correct N-terminal sequence, molecular mass on SDS-PAGE and the same affinity for IGF-1 and IGF-II as IGFBP-2 from mammalian cells. In a supE strain of E. coli, IGFBP-2 was produced as an IGF-binding fusion to g3p. Procedures for display and approximately 10000 fold enrichment of IGFBP-2 bearing phage using adsorption to IGF-II coated microtitre plates were developed. Thus IGFBP-2 can be secreted in E. coli and displayed on filamentous phage. These can be selectively enriched by binding to immobilised IGF-II.

The three-dimensional structure of human insulin-like growth factor (IGF) II in aqueous solution at pH 3.1 and 300 K has been determined from nuclear magnetic resonance data and restrained molecular dynamics calculations. Structural constraints consisting of 502 NOE-derived distance constraints, 11 dihedral angle restraints, and three disulfide bridges were used as input for distance geometry calculations in DIANA and X-PLOR, followed by simulated annealing refinement and energy minimization in X-PLOR. The resulting family of 20 structures was well defined in the regions of residues 5 to 28 and 41 to 62, with an average pairwise root-mean-square deviation of 1.24 A for the backbone heavy-atoms (N, C2, C) and 1.90 A for all heavy atoms. The poorly defined regions consist of the N and C termini, part of the B-domain, and the C-domain loop. Resonances from these regions of the protein gave stronger cross peaks in two dimensional NMR spectra, consistent with significant motional averaging. The main secondary structure elements in IGF-II are alpha-helices encompassing residues 11 to 21, 42 to 49 and 53 to 59. A small anti-parallel beta-sheet is formed by residues 59 to 61 and 25 to 27, while residues 26 to 28 appear to participate in intermolecular beta-sheet formation. The structure of IGF-II in the well-defined regions is very similar to those of the corresponding regions of insulin and IGF-I. Significant differences between IGF-II and IGF-I occur near the start of the third helix, in a region known to modulate affinity for the type 2 IGF receptor, and at the C terminus. The IGF II structure is discussed in relation to its binding sites for the insulin and IGF receptors and the IGF binding proteins.