THE DISULFIDE BONDS IN THE C-TERMINAL DOMAINS OF THE HUMAN INSULIN-RECEPTOR ECTODOMAIN

The human insulin receptor is a homodimer consisting of two monomers l inked by disulfide bonds, Each monomer comprises an alpha-chain that i s entirely extracellular and a beta-chain that spans the cell membrane , The alpha-chain has a total of 37 cysteine residues, most of which f orm intrachain disulfide bonds, whereas the beta-chain contains 10 cys teine residues, four of which are in the extracellular region, There a re two classes of disulfide bonds in the insulin receptor, those that can be reduced under mild reducing conditions to give alpha-beta monom ers (class I) and those that require stronger reducing conditions (cla ss II), The number of class I disulfides is small and includes the alp ha-alpha dimer bond Cys(524), In this report we describe the use of cy anogen bromide and protease digestion of the exon 11 plus form of the receptor ectodomain to identify disulfide linkages between the beta-ch ain residues Cys(798) and Cys(807) and between the alpha-chain Cys(647 ) and the beta-chain Cys(872), The latter bond is the sole alpha-beta link in the molecule and implies a side-by-side alignment of the two f ibronectin III domains of the receptor, Also presented is evidence for additional alpha-alpha dimer bond(s) involving at least one of the cy steine residues of the triplet at positions 682, 683, and 685, Evidenc e is also presented to show that Cys(884) exists as a buried thiol in the soluble ectodomain.