Abstract
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 alpha-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 microm 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.
Highlights
Insulin mediates its effects by binding to tyrosine kinase receptors in the plasma membrane of targets cells
The nanomolar binding affinity obtained with IR468.CT is believed to reflect the interaction between binding site 1 on the receptor and the classical binding site on the insulin molecule described in the binding models proposed by Schaffer [17] and DeMeyts [11]
The corresponding constructs without the CT domain, IR468 and IR308, did not bind insulin. This supports previous findings that the presence of the CT domain is essential for insulin binding but that it can be effective from different positions in the primary sequence [8, 18]
Summary
Miscellaneous Materials—Insulin and 125I-labeled Tyr-A14-substituted insulin were from Novo Nordisk. IR468.CT is the minimized ␣-subunit construct (mIR) comprising the first 3 domains of IR (residues 1– 468) fused to a 16-amino acid peptide from the C terminus of the ␣-subunit (residues 704 –719) followed by the FLAG epitope (DYKDDDDK) This receptor has been described previously [7]. Insulin Binding Assays—For insulin binding studies, a polyethylene glycol (PEG) precipitation assay was used For this assay, receptor samples were incubated for 16 h at 4 °C in a total volume of 100 l with 20 –50 pM 125I-labeled Tyr-A14-substituted insulin together with unlabeled insulin and/or CT peptides at various concentrations in binding buffer (100 mM Hepes, pH 8.0, 100 mM NaCl, 10 mM MgCl2, 0.25% (w/v) BSA, 0.025% (w/v) Triton X-100). The peptides were cleaved from the resin with trifluoroacetic acid containing 5% phenol, 3% triisopropylsilane, and 2% thioanisole for 1–2 h, and the peptides were precipitated in diethylether and lyophilized after suspension in 10% AcOH or 1% NH4HCO3
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