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

To identify a site within the insulin receptor ectodomain which forms a binding pocket for B25 Phe and is responsible for initiating conform ational changes required for high affinity binding of insulin we have used a novel photoreactive insulin, despentapeptide-(B26-B30) [B25 p-a zidophenylalanine-alpha-carboxamide] insulin (APC insulin). This deriv ative has a highly photoreactive azido group incorporated into the aro matic 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 B 1 Phe amino group to form despentapeptide-(B26-B30) [B1 (6-biotinylami docaproyl)phenylalanine B25 p-azidophenylalanine-alpha-carboxamide] in sulin derivative (Bio APC insulin) did not adversely affect receptor-b inding affinity and provided a convenient ligand for purification of c ross-linked complexes. The efficiency of receptor cross-linking with t hese reagents was high (70%). To identify the site(s) of cross-linking , the insulin receptor in P3-Acells was first metabolically labeled wi th various individual H-3-labeled amino acids and then pho to affinity labeled with I-125-Bio-APC insulin, isolated, and digested with Lys-C endoproteinase. The resulting crosslinked peptide fragments were sepa rated by streptavidin-affinity chromatography and sequenced. The small est identified fragment comprised residues 704-718 of the COOH terminu s of the alpha-subunit of the insulin receptor. This B25 Phe cross-lin ked region of the alpha-subunit lies just upstream of the Exon 11-enco ded 12-amino acid COOH-terminal region. Aromatic residues in this pred icted alpha-helical region may form a binding pocket for B25 Phe to in itiate conformational changes required for stabilizing the high affini ty binding state.