Functional Properties of an Isolated αβ Heterodimeric Human Placenta Insulin-like Growth Factor 1 Receptor Complex

Treatment of human placenta membranes at pH 8.5 in the presence of 2.0 mM dithiothreitol (DTT) for 5 min, followed by the simultaneous removal of the DTT and pH adjustment to pH 7.6, resulted in the formation of a functional αβ heterodimeric insulin-like growth factor 1 (IGF-1) receptor complex from the native α₂β₂heterotetrameric disulfide-linked state. The membrane-bound αβ heterodimeric complex displayed similar curvilinear¹²⁵I-IGF-1 equilibrium binding compared to the α₂β₂heterotetrameric complex. Triton X-100 solubilization of the alkaline pH and DTT-pretreated placenta membranes, followed by Bio-Gel A-1.5m gel filtration chromatography, was found to effectively separate the α₂β₂heterotetrameric and αβ heterodimeric IGF-1 receptor species.¹²⁵I-IGF-1 binding to both the isolated α₂β₂heterotetrameric and αβ heterodimeric complexes demonstrated a marked straightening of the Scatchard plots, compared to the placenta membrane-bound IGF-1 receptors, with a 2-fold increase in the high-affinity binding component. Similar to the membrane-bound IFG-1 receptor species, the¹²⁵I-IGF-1 binding properties between the α₂β₂heterotetrameric and αβ heterodimeric complexes were not significantly different. IGF-1 stimulation of IGF-1 receptor autophosphorylation indicated that the ligand-dependent activation of αβ heterodimeric protein kinase activity occurred concomitant with the reassociation into a covalent α₂β₂heterotetrameric state. These data demonstrate that (i) a combination of alkaline pH and DTT treatment of human placenta membranes results in the formation of an αβ heterodimeric IGF-1 receptor complex, (ii) unlike the insulin receptor, high-affinity homogeneous IGF-1 binding occurs in both the α₂β₂heterotetrameric and αβ heterodimeric complexes, and (iii) IGF-1-dependent autophosphorylation of the αβ heterodimeric IGF-1 receptor complex correlates with an IGF-1-dependent covalent reassociation into an α₂β₂heterotetrameric disulfide-linked state.