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High affinity insulin binding in the human placenta insulin receptor requires αβ heterodimeric subunit interactions

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Summary

Insulin binding to human placenta membranes treated at pH 7.6 or 8.5 in the presence or absence of 2.0mm 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 αβ heterodimeric insulin receptor complex from the α2β2 heterotetrameric disulfide-linked state. The ability of alkaline pH and DTT to produce a functional αβ 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 αβ heterodimeric insulin receptor complex. (ii) the αβ heterodimeric complex displays homogeneous insulin binding. (iii) the insulin receptor membrane environment maintains the α2β2 association state, which displays heterogeneous insulin binding, despite reduction of the critical domains that are responsible for the covalent interaction between the αβ heterodimers.

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Abbreviations

ATP:

adenosine 5′-triphosphate

DTT:

dithiothreitol

SDS:

sodium dodecyl sulfate

DSS:

disuccinimidyl suberate

NEM:

N-ethylmaleimide

IGF-I:

insulin-like growth factor-I

EDTA:

ethylenediaminetetraacetic acid

HEPES:

4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid

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  1. Department of Physiology & Biophysics, The University of Iowa, 52242, Iowa City, Iowa

    Michael L. Swanson & Jeffrey E. Pessin

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  1. Michael L. Swanson
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  2. Jeffrey E. Pessin
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Swanson, M.L., Pessin, J.E. High affinity insulin binding in the human placenta insulin receptor requires αβ heterodimeric subunit interactions. J. Membrain Biol. 108, 217–225 (1989). https://doi.org/10.1007/BF01871736

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  • DOI: https://doi.org/10.1007/BF01871736

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