Cross-linking of a B25 azidophenylalanine insulin derivative to the carboxyl-terminal region of the alpha-subunit of the insulin receptor. Identification of a new insulin-binding domain in the insulin receptor

• Published in: The Journal of biological chemistry Vol. 269; no. 46; pp. 29190 - 29197
• Main Authors: Kurose, T, Pashmforoush, M, Yoshimasa, Y, Carroll, R, Schwartz, G P, Burke, G T, Katsoyannis, P G, Steiner, D F
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18.11.1994; American Society for Biochemistry and Molecular Biology
• Subjects: Affinity Labels; Amino Acid Sequence; Animals; Azides - chemistry; CHO Cells; Cricetinae; Humans; Insulin - metabolism; Molecular Sequence Data; Phenylalanine - analogs & derivatives; Phenylalanine - chemistry; Protein Binding; Rats; Receptor, Insulin - chemistry; Receptor, Insulin - metabolism; Swine; Ultraviolet Rays
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)62029-6

To identify a site within the insulin receptor ectodomain which forms a binding pocket for B25 Phe and is responsible for initiating conformational changes required for high affinity binding of insulin we have used a novel photoreactive insulin, despentapeptide-(B26-B30) [B25 p-azidophenylalanine-alpha-carboxamide] insulin (APC insulin). This derivative has a highly photoreactive azido group incorporated into the aromatic ring of the B25 phenylalanine amide. APC insulin bound to human insulin receptors overexpressed on a transfected Chinese hamster ovary cell line (P3-A) with an apparent potency of 9-fold relative to that of native insulin and stimulated lipogenesis in rat adipocytes with an average potency equal to porcine insulin. Addition of biotin to the B1 Phe amino group to form despentapeptide-(B26-B30) [B1 (6-biotinylamidocaproyl)phenylalanine B25 p-azidophenylalanine-alpha-carboxamide] insulin derivative (Bio-APC insulin) did not adversely affect receptor-binding affinity and provided a convenient ligand for purification of cross-linked complexes. The efficiency of receptor cross-linking with these reagents was high (70%). To identify the site(s) of cross-linking, the insulin receptor in P3-A cells was first metabolically labeled with various individual 3H-labeled amino acids and then photoaffinity labeled with 125I-Bio-APC insulin, isolated, and digested with Lys-C endoproteinase. The resulting cross-linked peptide fragments were separated by streptavidin-affinity chromatography and sequenced. The smallest identified fragment comprised residues 704-718 of the COOH terminus of the alpha-subunit of the insulin receptor. This B25 Phe cross-linked region of the alpha-subunit lies just upstream of the Exon 11-encoded 12-amino acid COOH-terminal region. Aromatic residues in this predicted alpha-helical region may form a binding pocket for B25 Phe to initiate conformational changes required for stabilizing the high affinity binding state.