High affinity insulin binding in the human placenta insulin receptor requires alpha beta heterodimeric subunit interactions.
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.
Swanson
M L
ML
Department of Physiology & Biophysics, University of Iowa, Iowa City 52242.
Pessin
J E
JE
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*
D007328:Insulin; D011972:Receptor, Insulin; D004229:Dithiothreitol