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

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 (125)I-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 (125)I-labeled insulin and (125)I-labeled IGF-II and significantly less with (125)I-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 (125)I-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 (125)I-labeled insulin and IR, and (125)I-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.