Role of insulin receptor dimerization domains in ligand binding, cooperativity, and modulation by anti-receptor antibodies.
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.
Surinya
Katharina Helen
KH
University of Cambridge, Department of Clinical Biochemistry, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QR, United Kingdom.
Molina
Laurence
L
Soos
Maria A
MA
Brandt
Jakob
J
Kristensen
Claus
C
Siddle
Kenneth
K
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
D000596:Amino Acids; D000906:Antibodies; D018076:DNA, Complementary; D005353:Fibronectins; D008024:Ligands; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D011972:Receptor, Insulin