Identification of Common Ligand Binding Determinants of the Insulin and Insulin-like Growth Factor 1 Receptors

Abstract

Insulin and insulin-like growth factor 1 (IGF-1) are peptides that share nearly 50% sequence homology. However, although their cognate receptors also exhibit significant overall sequence homology, the affinity of each peptide for the non-cognate receptor is 2-3 orders of magnitude lower than for the cognate receptor. The molecular basis for this discrimination is unclear, as are the molecular mechanisms underlying ligand binding. We have recently identified a major ligand binding site of the insulin receptor by alanine scannning mutagenesis. These studies revealed that a number of amino acids critical for insulin binding are conserved in the IGF-1 receptor, suggesting that they may play a role in ligand binding. We therefore performed alanine mutagenesis of these amino acids to determine whether this is the case. Insulin and insulin-like growth factor 1 are circulating serum peptides that share nearly 50% sequence homology (for a review, see Ref. 1). Conservation of the predicted major secondary structural elements of both peptides suggests that their tertiary structures are similar (2). Crystallographic and solution NMR structural studies have shown this to be the case (3, 4). Their cognate receptors also exhibit significant overall sequence homology (1). However, despite the overall homology of receptors and ligands, insulin and IGF-1 1 only bind weakly to each other's receptors; the affinity of each peptide for the noncognate receptor is at least 3 orders of magnitude lower than for the cognate receptor (5). The molecular basis for this discrimination is at present unclear, as are the molecular mechanisms underlying ligand binding.As a consequence of the availability of a large number of naturally occurring and chemically and biosynthetically modified analogs of insulin, an extensive body of information regarding its receptor binding determinants has accumulated. A current consensus is that A2 isoleucine, A3 valine, B12 valine, B24 and B25 phenylalanine, A19 tyrosine, A21 asparagine, and the partially buried residues A16 and B15 leucine are the major determinants of the receptor binding site, with A8 threonine, B9 serine, B10 histidine, B13 glutamate, and B16 tyrosine making minor contributions.2 This forms a patch on the surface of the molecule overlapping its dimerization surface. More recent studies also suggest that a small patch formed from the residues A13 and B17 on the hexamerization surface of insulin may represent a topologically distinct receptor binding site located on the opposite side of the molecule (6). Much less information is available with regard to the receptor binding determinants of IGF-1. Studies by Bayne and Cascieri (7,8) implicate a role of the C region of the molecule in receptor binding; specifically, tyrosine 31 appears to be essential for high affinity binding (8). In addition, tyrosines 24 and 60, corresponding to B25 phenylalanine and A19 tyrosine of insulin, respectively, both essential for high affinity binding of insulin, play important roles in the receptor binding of IGF-1...

Functionally significant insulin-like growth factor I receptor mutations in centenarians

“…Alignment of IGFIR with the IR shows that Ala-37 is just one residue away from IR Phe-39, a residue important for insulin ligand specificity (26). Ala-37 is also adjacent to a highly conserved stretch of residues beginning with Glu-44, which has important function for insulin/IR binding (27). Interestingly, IGFIR residues around Ala-37 are highly conserved among distant vertebrate species, indicating that sequence variants most probably affect function.…”

Correlations of receptor binding and metabolic and mitogenic potencies of insulin analogs designed for clinical use.

“…Binding experiments with insulin detemir were therefore done using albumin-free buffers and a modified procedure. The binding assays used were microtiter plate antibody capture assays, essentially as described in the literature (17). Microtiter plates were coated with affinity-purified goat anti-mouse IgG antibody (Pierce, Rockford, IL) (50 µl/well of 20 µg/ml solution in Tris-buffered saline [TBS]: 0.01 mol/l Tris, pH 7.5, 100 mmol/l NaCl).…”

Dimeric Fragment of the Insulin Receptor α-Subunit Binds Insulin with Full Holoreceptor Affinity

“…For the PEG assay a suitable dilution of receptor sample was incubated for 16 - The microtiter plate assay was performed essentially as described in the literature (30). For immobilization of receptor fragments, an insulin receptor specific antibody, F12, was used.…”