Expression and characterization of a 70-kDa fragment of the insulin receptor that binds insulin. Minimizing ligand binding domain of the insulin receptor.
In order to characterize regions of the insulin receptor that are essential for ligand binding and possibly identify a smaller insulin-binding fragment of the receptor, we have used site-directed mutagenesis to construct a series of insulin receptor deletion mutants. From 112 to 246 amino acids were deleted from the alpha-subunit region comprising amino acids 469-729. The receptor constructs were expressed as soluble insulin receptor IgG fusion proteins in baby hamster kidney cells and were characterized in binding assays by immunoblotting and chemical cross-linking with radiolabeled insulin. The shortest receptor fragment identified was a free monomeric alpha-subunit deleted of amino acids 469-703 and 718-729 (exon 11); the mass of this receptor fragment was found by mass spectrometry to be 70 kDa. This small insulin receptor fragment bound insulin with an affinity (Kd) of 4.4 nM, which is similar to what was found for the full-length ectodomain of the insulin receptor (5.0 nM). Cross-linking experiments confirmed that the 70-kDa receptor fragment specifically bound insulin. In summary we have minimized the insulin binding domain of the insulin receptor by identifying a 70-kDa fragment of the ectodomain that retains insulin binding affinity making this an interesting candidate for detailed structural analysis.
Kristensen
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C
Department of Insulin Research, Health Care Discovery, Novo Nordisk, 2880 Bagsvaerd, Denmark. clak@novo.dk
Wiberg
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FC
Schäffer
L
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Andersen
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AS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers; D006031:Glycosylation; D007328:Insulin / Q000378:metabolism*; D007457:Iodine Radioisotopes / Q000737:chemistry; D008024:Ligands; D013058:Mass Spectrometry; D011485:Protein Binding; D011972:Receptor, Insulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion
D017931:DNA Primers; D007328:Insulin; D007457:Iodine Radioisotopes; D008024:Ligands; D011972:Receptor, Insulin