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Biochemistry (Moscow)

, Volume 74, Issue 1, pp 29–35 | Cite as

Isolated compared to membrane-bound receptors exhibit altered insulin/IGF interaction

  • O. NedićEmail author
  • R. Masnikosa
Article

Abstract

Insulin and insulin-like growth factors (IGFs) bind to their cognate receptors with high affinities, but due to their homology they may cross-react with each other’s receptors. We performed a series of binding studies to reanalyze the cross-reactivity of insulin, IGF-I, and IGF-II to affinity-purified insulin (IR) and type 2 IGF receptors (IGF-2R) from human placental membranes. IR and IGF-2R were purified using insulin- and mannose-6-phosphate affinity chromatography (I-AC and M6P-AC). Binding studies were performed with 125I-labeled and unlabeled ligands. According to immunoblotting, the only receptor species isolated by I-AC was IR, whereas the only receptor isolated by M6P-AC was IGF-2R. Isolated IR reacted to similar extent with 125I-labeled insulin and 125I-labeled IGF-II and significantly less with 125I-labeled IGF-I, implicating predominance of IR-A. The affinity of IR towards heterologous ligands increased after its separation from other membrane proteins. Affinity-purified IGF-2R was almost unable to bind ligands under experimental conditions used in this work, but when incubated with 125I-labeled ligands prior to affinity chromatography, IGF-2R interacted not only with IGF-II, but to a certain extent with the other two ligands. In the competitive M6P-AC, the binding of labeled ligands was inhibited with either homologous or heterologous ligands, in a dose dependent manner. In competitive ligand-blotting, specific interactions between 125I-labeled insulin and IR, and 125I-labeled IGF-II and IGF-2R were also inhibited with all unlabeled ligands, although to a different extent. The results presented in this work imply that isolation of IR an IGF-2R from their membrane milieu increases their reactivity towards all members of the insulin/IGF ligand family.

Key words

insulin insulin-like growth factors receptors affinity chromatography ligand-binding studies 

Abbreviations

CIM6P-R

cation-independent M6P receptor

DMSO

dimethyl sulfoxide

DSS

disuccinimidyl suberate

ECL

enhanced chemiluminescence

HRP

horseradish peroxidase

HyR

hybrid receptor

I-AC

insulin-affinity chromatography

IGF

insulin-like growth factor

IGF-1R and -2R

type 1 and 2 IGF receptors, respectively

IGFBP

IGF-binding protein

IR

insulin receptor

LBA

ligand-binding assay

M6P

mannose-6-phosphate

M6P-AC

M6P-affinity chromatography

PEG

polyethylene glycol

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Copyright information

© Pleiades Publishing, Ltd. 2009

Authors and Affiliations

  1. 1.Institute for the Application of Nuclear Energy (INEP)University of BelgradeBelgradeSerbia

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