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Papers In Press, published online ahead of print March 18, 2002
Molecular biology 528, Insulin Research, 2880 Bagsvaerd, - DK-2880
Corresponding Author: jakb{at}novonordisk.com
We have previously shown that a minimized insulin receptor (IR) consisting of the first 468 amino acids of the insulin receptor fused to 16 amino acids from the C-terminal of the alpha-subunit (CT domain) bound insulin with nano-molar affinity (Kristensen, C., Wiberg, F. C., Schäffer, L., and Andersen, A. S. (1998) J. Biol. Chem. 273, 17780-17786). In the present study, we show that a smaller construct which has the first 308 residues fused to the CT domain also binds insulin. Insulin receptor fragments consisting of the first 468 or 308 residues did not bind insulin. However, when these fragments were mixed with a synthetic peptide corresponding to the CT domain insulin binding was detectable. At concentrations of 10 µM of CT peptide insulin binding was fully reconstituted yielding apparent affinities of 9-11 nM. To further investigate the minimum requirement for the length of the N-terminal of IR, we tested smaller receptor fragments for insulin binding in the presence of the CT-peptide, and found that a fragment consisting of the first 255 amino acids of IR was able to fully reconstitute the insulin binding site, yielding an apparent affinity of 11 +/- 4 nM for insulin.
J. Biol. Chem, 10.1074/jbc.M112249200
Submitted on December 21, 2001
Revised on March 6, 2002
Accepted on March 15, 2002
Functional reconstitution of insulin receptor binding site from non-binding receptor fragments
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