[Back to Issue 1 ToC] [Back to Journal Contents] [Back to Biochemistry (Moscow) Home page]

Isolated Compared to Membrane-Bound Receptors Exhibit Altered Insulin/IGF Interaction

O. Nedić* and R. Masnikosa

Institute for the Application of Nuclear Energy (INEP), University of Belgrade, 11080 Belgrade, Serbia; fax: (+) 381-11-2618-724; E-mail: olgica@inep.co.yu; romana@inep.co.yu

* To whom correspondence should be addressed.

Received February 12, 2008; Revision received April 14, 2008
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

DOI: 10.1134/S0006297909010040