Probing the hormone-binding site of the insulin receptor with photoreactive derivatives

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...

Section: Insulin-like Growth Factor 1 Receptor Binding Sitementioning

confidence: 99%

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confidence: 95%

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

Mynarcik¹,

Williams²,

Schäffer³

et al. 1997

“…FnIII1 is atypical because it contains a 120-amino acid insert domain of indeterminate structure (15). Affinity labeling studies, studies with chimeric receptors, and the epitopes of anti-receptor antibodies, which strongly inhibit insulin binding, suggest that each receptor monomer contains two topologically discrete ligand binding sites (17)(18)(19)(20)(21)(22).…”

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confidence: 99%

“…We have characterized a binding site of the secreted recombinant insulin receptor using alanine-scanning mutagenesis (17,18,(23)(24)(25). This is composed of two functional epitopes, one located on the base of the L1 domain and the other at the C terminus of the ␣ subunit.…”

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confidence: 99%

Characterization of the Functional Insulin Binding Epitopes of the Full-length Insulin Receptor

Whittaker

Whittaker²

2005

Mutational analyses of the secreted recombinant insulin receptor extracellular domain have identified a ligand binding site composed of residues located in the L1 domain (amino acids 1-470) and at the C terminus of the ␣ subunit (amino acids [705][706][707][708][709][710][711][712][713][714][715] , and Phe 89 on insulin-induced receptor autophosphorylation. They had no effect on the maximal response to insulin but produced an increase in the EC 50 commensurate with their effect on the affinity of the receptor for insulin.The initiating event in the insulin-signaling cascade is the binding of insulin to a specific plasma membrane receptor. This interaction has been studied extensively and was found to be extremely complex (see Ref. 1 for review). Scatchard plots (2) of equilibrium binding data are concave and curvilinear, suggesting heterogeneity of ligand binding sites, negatively cooperative site-site interactions, or a combination of both (1). These properties and high affinity interactions with insulin are dependent on the dimeric structure of the receptor; insulin binds non-cooperatively to a single population of binding sites in the monomeric receptor (3-5). The stoichiometry of binding to the native receptor appears to be one insulin molecule to one receptor dimer (6). The secreted recombinant extracellular domain of the receptor exhibits properties similar to those of the receptor monomer and has a stoichiometry of two insulin molecules to one receptor dimer (6, 7).A number of hypothetical models of insulin-receptor interactions have been proposed to explain these findings (7-9). The model that best explains the experimental findings is that of De Meyts (1). This proposes that insulin has two topographically distinct receptor binding sites and that the receptor has two topographically distinct insulin binding sites/monomer. In this model, insulin binds asymmetrically to the insulin dimer, with each of its binding sites contacting its cognate receptor site on a separate monomer. Although the detailed structural basis of this model has yet to be elucidated, the structure-function relationships of the insulin molecule and the receptor have been studied extensively and provide support for the essentials of the model.Insulin (1, 10). However, hagfish insulin, in which these residues and the tertiary structure of the molecule are conserved (11), displays anomalous receptor binding behavior (11,12), suggesting that other residues outside this region might also be involved in receptor interactions. This is supported by the finding that two recombinant insulin analogues with substitutions in residues located on the opposite side of the molecule, Leu A13 to Ser and Leu B17 to Gln, exhibited similar receptor binding behavior to hagfish insulin (7,8).The structure and structure-function relationships of the insulin receptor are not as well documented. It is a dimeric transmembrane protein (see Ref. 1 for review). Each monomer consists of disulfide-linked ␣ and ␤ subunits. The ␣ subunits are wholly extracellular and c...

“…Although site-directed mutagenesis, photoaffinity labeling studies, and the construction of an IR/insulin-like growth factor-1 receptor chimera have identified the presumptive insulin binding domains (11)(12)(13)(14)(15), the exact contact sites of insulin in the IR are still unknown. In addition, crystallization of the IR has not been done, and its three-dimensional structure is not clear.…”

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confidence: 99%

Replacements of Leucine 87 in Human Insulin Receptor Alter Affinity for Insulin

Nakae

Morioka

Ohtsuka

et al. 1995

In a previous analysis, we identified a point mutation that substituted Pro (CCG) for Leu (CTG) at amino acid 87 in the α-subunit of the insulin receptor (IR) in a Japanese patient with leprechaunism. In the present study, we transfected either the wild type (Leu-87) or the mutant (Pro-87) IR cDNA into NIH3T3 cells. Pulsechase in nonreducing conditions revealed that the dimerization of Pro-87 IR was slightly impaired. However, cell surface biotinylation showed that Pro-87 IR was transported to the cell surface. The Pro-87 IR reduced the insulin binding affinity to about 15% of Leu-87 IR, and the dissociation of insulin in Pro-87 IR was more rapid than in Leu-87 IR. The autophosphorylation of Pro-87 IR was less sensitive to insulin than that of Leu-87 IR, suggesting the reduced insulin binding affinity. Site-directed mutagenesis at amino acid 87 was performed to substitute Ile or Ala for Leu. Both mutant IRs were transported to the cell surface and labeled by cell surface biotinylation. The Ile-87 IR enhanced the insulin binding affinity about 4-fold. The insulin binding affinity of Ala-87 IR was reduced by 85% relative to that of Leu-87 IR. In addition, the dissociation of insulin in Ile-87 IR was slower than in Leu-87 IR, but in Ala-87 IR it was more rapid. These results provide the first direct evidence for a critical role of Leu-87 in binding insulin.