Isolation of a proteolytically derived domain of the insulin receptor containing the major site of cross-linking/binding.
Radiolabeled insulin was affinity cross-linked to purified insulin receptor with six separate bifunctional N-hydroxysuccinimide esters of different lengths. Results were qualitatively identical for each cross-linker in that insulin was predominantly cross-linked through its B chain to the receptor's alpha subunit. The maximum efficiencies of cross-linking were 10-15% for the most effective reagents, and this value was dependent upon the concentration and length of the cross-linker. In an effort to locate the cross-linking site, monoiodoinsulin was cross-linked to affinity-purified insulin receptor with disuccinimidyl suberate. Limited proteolysis of the hormone/receptor adduct with Staphylococcus aureus V8 protease, chymotrypsin, or thermolysin in an SDS-containing buffer rapidly generated a 55-kDa, insulin-labeled fragment as shown by SDS-polyacrylamide gel electrophoresis. We reported earlier that the 55-kDa chymotryptic fragment contained multiple internal disulfide bonds as evidenced by its shifting mobility on an SDS gel after dithiothreitol treatment [Boni-Schnetzler et al. (1987) J. Biol. Chem. 262, 8395-8401]. Here we show that the 55-kDa fragment is also formed by proteolysis of the receptor in the absence of prior insulin cross-linking. This fragment was prepared in amounts sufficient for sequence analysis and was purified by passage successively over gel permeation and reverse-phase HPLC columns. The sequence of the fragment's amino terminus corresponds to that of the amino terminus of the receptor's alpha subunit. This fragment also reacts with an antibody raised against a synthetic peptide corresponding to residues 242-253 of the receptor's alpha subunit.(ABSTRACT TRUNCATED AT 250 WORDS)
Waugh
S M
SM
Department of Biochemistry, School of Medicine, Boston University, Massachusetts 02118.
DiBella
E E
EE
Pilch
P F
PF
D000595:Amino Acid Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D011972:Receptor, Insulin / Q000302:isolation & purification* / Q000378:metabolism
D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D011972:Receptor, Insulin