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J. Biol. Chem., Vol. 277, Issue 21, 18340-18345, May 24, 2002

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Functional Reconstitution of Insulin Receptor Binding Site from Non-binding Receptor Fragments^*

Claus Kristensen, Asser Sloth Andersen^§, Søren Østergaard^§, Per Hertz Hansen^§, and Jakob Brandt^§¶

From the Departments of  Protein Expression and ^§ Insulin Research, Novo Nordisk A/S, 2880 Bagsvaerd, Denmark

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 terminus of the -subunit (CT domain) bound insulin with nanomolar 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 that 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 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 terminus 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.

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