Affinity labeling studies and mutational analyses have implicated the involvement of a predicted domain of the insulin receptor (L1, amino acids 1-119) in ligand binding. In order to obtain a higher resolution localization of this ligand binding site, we have performed alanine scanning mutagenesis of this domain. Alanine mutant cDNAs encoding a secreted recombinant insulin receptor extracellular domain were expressed transiently in adenovirus transformed human embryonic kidney cells and the affinity of the expressed receptor for insulin was determined. Mutation of 14 amino acids located in four discontinuous peptide segments to alanine was disruptive of insulin binding: Segment 1, amino acids 12-15; Segment 2, amino acids 34-44; Segment 3, amino acids 64-67; and Segment 4, amino acids 89-91. The quantitative contribution of the four segments to the free energy of insulin binding was 1 > 3 > 2 > 4. Of the 14 amino acids whose mutation compromised insulin binding, 3 are charged, 3 hydrophobic, 5 aromatic, and 3 are amides.

Insulin initiates signal transduction in target cells by binding to a specific cell surface receptor, which is a member of the growth factor receptor tyrosine kinase superfamily of proteins (1). Ligand binding leads to the activation of the receptor's tyrosine kinase activity and the initiation of intracellular signaling. Mutational studies of receptor signaling and the elucidation of the structure of the receptor's tyrosine kinase catalytic domain suggest that kinase activation is effected by intramolecular transphosphorylation of the constituent tyrosine kinase catalytic domains of the receptor heterotetramer (2, 3). The molecular details of the mechanism by which insulin binding initiates the transphosphorylation event remain obscure and will require an understanding of the molecular organization of the extracellular domain of the receptor and the molecular basis of insulin binding.The insulin receptor is composed of two disulfide-linked heterodimers, each of which is composed in turn of a 135-kDa ␣ subunit (entirely extracellular) linked by a disulfide bond to a 95-kDa ␤ subunit, which has an extracellular domain, a single ␣ helical transmembrane domain, and an intracellular domain containing the tyrosine kinase catalytic activity (1). While the tertiary structure of the extracellular domain has not been elucidated, the presence of several characteristic structural motifs can be predicted from inspection of the deduced amino acid sequence (4, 5). The ␣ subunit contains a cysteine-rich domain homologous to that of the epidermal growth factor receptor (4, 5), and there are also two fibronectin type III repeats; the first is composed of the C terminus of the ␣ subunit and the N terminus of the ␤ subunit, and the second is composed of the C terminus of the extracellular region of the ␤ subunit (6).Bajaj et al. (7) have proposed a hypothetical model of the tertiary structure of the receptor extracellular domain based on homologies between the primary structures of the epidermal growth factor and insulin receptor families of tyrosine kinases. This model predicts that there are two homologous globular domains flanking the cysteine-rich domains: domain L1 containing amino acids 1-119 and domain L2 containing amino acids 311-428. Each contains repeating structural motifs (I-V) composed of ␣ helix, ␤-turn-␤, and hypervariable structures. Since all deletions and insertions occur in the hypervariable structures in the sequence alignments obtained for these proteins with this model, it was suggested that these may represent components of ligand binding domains.This proposal has received support from recent experimental observations (8 -12). We have recently performed alanine-scanning mutagenesis of the L1 domain of the insulin receptor and have shown it it to contain a ligand binding domain composed of 14 amino acids organized in four discontinuous peptide segments (13). However, it is unlikely that this is the complete insulin binding site, as secreted recombinant receptors with deletions N-terminal to the C terminus of...

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...