Alanine-scanning Mutagenesis of a C-terminal Ligand Binding Domain of the Insulin Receptor α Subunit

Abstract

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...

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

“…Insulin Binding-We have previously characterized the functional epitopes of the secreted recombinant insulin receptor extracellular domain by alanine-scanning mutagenesis (23)(24)(25). To determine significance of these epitopes for insulin binding in a physiological context, we subcloned the alanine mutations used to characterize them into the full-length B isoform receptor cDNA-tagged at its 3Ј end with the coding sequence for a triple repeat of the AU5 epitope tag.…”

“…Expression of these receptor mutants was subsequently confirmed and shown to be comparable of that of the wild-type receptor by Western blotting with anti-AU5 antibody (data not shown). We have shown previously that these mutants are also expressed normally as the recombinant secreted extracellular domain of the receptor (23)(24)(25).…”

“…Construction of Receptor Mutants-Alanine mutations of the cDNA encoding the recombinant secreted receptor extracellular domain have been described previously (23)(24)(25). These were used to generate fulllength epitope-tagged mutants of the full-length receptor by standard subcloning techniques.…”

“…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.…”

Functional Reconstitution of Insulin Receptor Binding Site from Non-binding Receptor Fragments

“…4B). Of these four positions, the insulin receptor phenylalanine 714, which is conserved as phenylalanine 701 in the IGFIR, is particularly interesting, as it has been shown by alanine scanning mutagenesis that alanine in this position is disruptive for ligand binding in both receptors (15,16). Accordingly, we synthesized a new CT peptide comprising residues 703-719 of IR, in which the phenylalanine in position 714 had been replaced by alanine, and we tested this peptide for its ability to reconstitute binding in the PEG binding assay.…”