A radioligand binding assay for the insulin-like growth factor 2 receptor.

Pavlo Potalitsyn,Jiří Jiráček,Kateřina Nováková,Irena Selicharová,Jelena Radosavljević,Kryštof Sršeň,Aleš Marek,Lenka Žáková,Haim Werner

doi:10.1371/journal.pone.0238393

Pavlo Potalitsyn, Jiří Jiráček + Show 7 more

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https://doi.org/10.1371/journal.pone.0238393

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Abstract

Insulin-like growth factors 2 and 1 (IGF2 and IGF1) and insulin are closely related hormones that are responsible for the regulation of metabolic homeostasis, development and growth of the organism. Physiological functions of insulin and IGF1 are relatively well-studied, but information about the role of IGF2 in the body is still sparse. Recent discoveries called attention to emerging functions of IGF2 in the brain, where it could be involved in processes of learning and memory consolidation. It was also proposed that these functions could be mediated by the receptor for IGF2 (IGF2R). Nevertheless, little is known about the mechanism of signal transduction through this receptor. Here we produced His-tagged domain 11 (D11), an IGF2-binding element of IGF2R; we immobilized it on the solid support through a well-defined sandwich, consisting of neutravidin, biotin and synthetic anti-His-tag antibodies. Next, we prepared specifically radiolabeled [125I]-monoiodotyrosyl-Tyr2-IGF2 and optimized a sensitive and robust competitive radioligand binding assay for determination of the nanomolar binding affinities of hormones for D11 of IGF2. The assay will be helpful for the characterization of new IGF2 mutants to study the functions of IGF2R and the development of new compounds for the treatment of neurological disorders.

Highlights

Insulin-like growth factor 2 (IGF2) is a 7.5 kDa mitogenic peptide hormone expressed predominantly in liver, but by other tissues as well [1]
Here we developed a new and straightforward methodology based on a competition binding assay between a “cold” ligand and 125I-radiolabeled IGF2 for domain 11 (D11) protein immobilized on a solid surface through iBodies1 [33], an artificial polymer that can
This study was inspired by the growing evidence of the high importance of the IGF2/IGF2 receptor (IGF2R) axis in the brain and its newly discovered functions in neural tissues

Summary

Introduction

Insulin-like growth factor 2 (IGF2) is a 7.5 kDa mitogenic peptide hormone expressed predominantly in liver, but by other tissues as well [1]. IGF2 shares 50 % sequence homology and similar 3-D organization with IGF1 and insulin. IGF2 is a major regulator of cell growth, survival, migration and differentiation, especially during fetal development [2, 3]. In spite of recent significant improvement in the knowledge about the physiological and pathological roles of IGF2, there is still a lack of information about the full spectrum of its functions in the organism. The significance of IGF2 is supported by its high level in the serum of human adults, which is three times higher than the concentration of IGF1 and much higher than the concentration of insulin [4,5,6]. IGF2 gene expression is maternally imprinted and is tightly regulated [7].

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