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

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.

Fig 1

(A) Interaction of [¹²⁵I]-monoiodotyrosyl-Tyr2-IGF2 (in violet with a ¹²⁵I atom shown as a yellow sphere) with His-tagged D11 of IGF-2R (in green). The side-chains of IGF2 residues involved in D11 binding are shown as sticks. Created based on 2L29.pdb structure [26]. (B) Schematic representation of the principle of IGF2:D11 radioligand binding assay developed in this study. iBodies® [33], consisting of N-(2-hydroxypropyl) methacrylamine (HPMA) copolymer bearing covalently attached biotin molecules and Ni²⁺-chelating nitrilotriacetic acid (NTA) molecule, are immobilized on solid support (polystyrene wells) coated with neutravidin. Recombinant His-tagged D11 is immobilized in wells by binding of its His-tag to NTA-ligated Ni²⁺ atom of iBodies®. Binding affinity of non-labeled ligands is determined through competition for D11 with [¹²⁵I]-IGF2 radiotracer.

Fig 2. A typical saturation binding curve of [¹²⁵I]-monoiodotyrosyl-Tyr2-IGF2 to immobilized domain D11 of IGF2R.

Total binding is in blue, non-specific binding is in red and specific binding is shown in green.

Fig 3. Representative binding curves of IGF hormones on immobilized D11.

Inhibition of binding of human [¹²⁵I]-monoiodotyrosyl-Tyr2-IGF2 to D11 by human IGF2 (in red), by human IGF1 (in blue), and by Leu19-IGF2 analog (in green).