title: Role of insulin receptor dimerization domains in ligand binding, cooperativity, and modulation by anti-receptor antibodies.
abstract: To define the structures within the insulin receptor (IR) that are required for high affinity ligand binding, we have used IR fragments consisting of four amino-terminal domains (L1, cysteine-rich, L2, first fibronectin type III domain) fused to sequences encoded by exon 10 (including the carboxyl terminus of the alpha-subunit). The fragments contained one or both cysteine residues (amino acids 524 and 682) that form disulfides between alpha-subunits in native IR. A dimeric fragment designated IR593.CT (amino acids 1-593 and 704-719) bound (125)I-insulin with high affinity comparable to detergent-solubilized wild type IR and mIR.Fn0/Ex10 (amino acids 1-601 and 650-719) and greater than that of dimeric mIR.Fn0 (amino acids 1-601 and 704-719) and monomeric IR473.CT (amino acids 1-473 and 704-719). However, neither IR593.CT nor mIR.Fn0 exhibited negative cooperativity (a feature characteristic of the native insulin receptor and mIR.Fn0/Ex10), as shown by failure of unlabeled insulin to accelerate dissociation of bound (125)I-insulin. Anti-receptor monoclonal antibodies that recognize epitopes in the first fibronectin type III domain (amino acids 471-593) and inhibit insulin binding to wild type IR inhibited insulin binding to mIR.Fn0/Ex10 but not IR593.CT or mIR.Fn0. We conclude the following: 1) precise positioning of the carboxyl-terminal sequence can be a critical determinant of binding affinity; 2) dimerization via the first fibronectin domain alone can contribute to high affinity ligand binding; and 3) the second dimerization domain encoded by exon 10 is required for ligand cooperativity and modulation by antibodies.
authors.affiliation: University of Cambridge, Department of Clinical Biochemistry, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QR, United Kingdom.
authors.forename: Katharina Helen
authors.initials: KH
authors.lastname: Surinya
authors.forename: Laurence
authors.initials: L
authors.lastname: Molina
authors.forename: Maria A
authors.initials: MA
authors.lastname: Soos
authors.forename: Jakob
authors.initials: J
authors.lastname: Brandt
authors.forename: Claus
authors.initials: C
authors.lastname: Kristensen
authors.forename: Kenneth
authors.initials: K
authors.lastname: Siddle
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D000906:Antibodies / Q000737:chemistry; D001665:Binding Sites; D001667:Binding, Competitive; D015153:Blotting, Western; D002417:Cattle; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D005091:Exons; D005353:Fibronectins / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D020128:Inhibitory Concentration 50; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D008024:Ligands; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011972:Receptor, Insulin / Q000737:chemistry* / Q000276:immunology / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors
chemical_list: D000596:Amino Acids; D000906:Antibodies; D018076:DNA, Complementary; D005353:Fibronectins; D008024:Ligands; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D011972:Receptor, Insulin