Abstract
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.
Highlights
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
We wanted to characterize the domains of the insulin receptor (IR) that are essential for ligand binding
This construct (IRwt) has been described in detail by Bass et al [29], and we have previously reported that this receptor fusion construct binds insulin with biphasic binding curves [31], and the high affinity component of this receptor has affinity that is similar to what is found for the holoreceptor
Summary
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. 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. Predictions of the tertiary structure of the IR ectodomain have been based on alignment with epidermal growth factor receptor sequences [11, 12]. The consensus from these alignments is that the insulin receptor ␣-subunits have two large homologous domains, L1 and L2, separated by a cysteine-rich region. The first 468 amino acids of the ␣-subunit are predicted to be well defined domains with extensive homologies to the epidermal growth factor receptor. The corresponding domain of the IGFI receptor [1– 486] was expressed in C6 rat glioblastoma cells resulting in inhibited IGFI receptor signaling and inhibition of growth [14], and recently McKern et al [15] have reported crystallization of these first three domains of the IGFI receptor (residues 1– 462)
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