title: High affinity insulin binding in the human placenta insulin receptor requires alpha beta heterodimeric subunit interactions.
abstract: Insulin binding to human placenta membranes treated at pH 7.6 or 8.5 in the presence or absence of 2.0 mM DTT for 5 min, followed by the simultaneous removal of the DTT and pH adjustment to pH 7.6, displayed curvilinear (heterogeneous) insulin binding plots when analyzed by the method of Scatchard. However, Triton X-100 solubilization followed by Bio-Gel A-1.5m gel filtration chromatography of the placenta membranes previously treated with DTT at pH 8.5 generated a nearly straight line (homogeneous) Scatchard plot. 125I-insulin affinity crosslinking studies coupled with Bio-Gel A-1.5m gel filtration chromatography demonstrated that the alkaline pH and DTT treatment of placenta membranes followed by detergent solubilization generated an alpha beta heterodimeric insulin receptor complex from the alpha 2 beta 2 heterotetrameric disulfide-linked state. The ability of alkaline pH and DTT to produce a functional alpha beta heterodimeric insulin receptor complex was found to be time dependent with maximal formation and preservation of tracer insulin binding occurring at 5 min. These data demonstrate that (i) a combination of alkaline pH and DTT treatment of placenta membranes can result in the formation of a functional alpha beta heterodimeric insulin receptor complex. (ii) the alpha beta heterodimeric complex displays homogeneous insulin binding. (iii) the insulin receptor membrane environment maintains the alpha 2 beta 2 association state, which displays heterogeneous insulin binding, despite reduction of the critical domains that are responsible for the covalent interaction between the alpha beta heterodimers.
authors.affiliation: Department of Physiology & Biophysics, University of Iowa, Iowa City 52242.
authors.forename: M L
authors.initials: ML
authors.lastname: Swanson
authors.forename: J E
authors.initials: JE
authors.lastname: Pessin
mesh_terms: D002462:Cell Membrane / Q000032:analysis / Q000187:drug effects / Q000648:ultrastructure; D002846:Chromatography, Affinity; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000032:analysis / Q000378:metabolism*; D010920:Placenta / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure*; D011247:Pregnancy; D011972:Receptor, Insulin / Q000032:analysis / Q000378:metabolism*
chemical_list: D007328:Insulin; D011972:Receptor, Insulin; D004229:Dithiothreitol