A Novel Binding Site for the Human Insulin-like Growth Factor-II (IGF-II)/Mannose 6-Phosphate Receptor on IGF-II

John C Wallace,Carlie Delaine,Lisbeth Gauguin,James Brown,E Yvonne Jones,Terrance D Mulhern,Kerrie A Mcneil,Briony E Forbes,Clair L Alvino,Pierre De Meyts

doi:10.1074/jbc.m700531200

John C Wallace, Carlie Delaine + Show 8 more

Open Access

https://doi.org/10.1074/jbc.m700531200

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Abstract

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 Phe(19) and Leu(53) are critical for IGF2R binding, as are residues Thr(16) and Asp(52). Furthermore, Thr(16) was identified as playing a major role in determining why IGF-II, but not IGF-I, binds with high affinity to the IGF2R.

Highlights

Via the trans-Golgi network and from the cellular membrane to lysosomes for degradation [2]
A distinct insulin-like growth factor (IGF)-II binding site is located on domain 11, and the additional presence of domain 13 is required for high affinity IGF-II binding equivalent to intact IGF2R (9 –11)
Residues corresponding to the mannose 6-phosphate binding site of the cationdependent mannose 6-phosphate receptor were mutated to Ala, and Tyr1542, Thr1570, Phe1567, and Ile1572 within this hydrophobic patch were shown to be critical for IGF-II binding

Summary

EXPERIMENTAL PROCEDURES

Materials—Oligonucleotides (Table 1) were purchased from Geneworks Pty. Ltd. (Adelaide, South Australia). The IGF2R fragment to be bound was prepared in 10 mM sodium acetate, pH 4.6, at 18 ␮g/ml and injected over the NHS/EDC activated chip surface in HBS running buffer (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.005% surfactant P20, pH 7.4). This model describes a 1:1 binding interaction with a conformational change upon binding, resulting in two association and dissociation rates This model resulted in the best fitting of the data and has been previously used for analysis of binding to IGF2R fragments [13]. The affinity constant (KA) derived from eight separate experiments on two separate chips for IGF-II on IGF2R-d10 –13 was 1.5 Ϯ 0.4 ϫ 108 MϪ1, and a similar variation between experiments was seen on all surfaces with all mutants analyzed

RESULTS

DISCUSSION

Forbes