Hormone binding site of the insulin receptor: analysis using photoaffinity-mediated avidin complexing.
A trifunctional reagent was designed which allows derivatization of ligands, particularly peptides and proteins, for subsequent photoaffinity labelling of receptors and specific isolation of the covalent complex or its fragments. B29-(2-nitro-4-azidophenyl)-biocytinyl-insulin (NB-insulin) was synthesized, radioiodinated, and the B26-mono-iodo derivative isolated by HPLC. It was used to photoaffinity label human placental membranes and the purified insulin receptor. Extensive digestion of the covalent insulin-receptor complex with trypsin (EC 3.4.21.4) led to the generation of a fragment of Mr 14,000. Specific complexing with avidin, derivatized avidin or streptavidin could be demonstrated for the photoaffinity labelled alpha-subunit and the 14,000 core fragment. The latter was isolated (approx. 100 pmol from 3-4 placentae) by streptavidin affinity chromatography and HPLC. According to microsequencing based on the known primary structure of the insulin receptor, the N-terminus of the core peptide appears to be Leu20-His21-Glu22-Leu23. We thus conclude: a part of the insulin-binding region of the receptor is located close to the N-terminus of its alpha-subunit in a remarkably stable domain of the sequence 20--(approx.) 120.
Wedekind
F
F
Deutsches Wollforschungsinstitut, RWTH Aachen.
Baer-Pontzen
K
K
Bala-Mohan
S
S
Choli
D
D
Zahn
H
H
Brandenburg
D
D
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001360:Avidin; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007202:Indicators and Reagents; D007328:Insulin / Q000031:analogs & derivatives; D010449:Peptide Mapping; D010777:Photochemistry; D011972:Receptor, Insulin / Q000302:isolation & purification / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013552:Swine; D014357:Trypsin
D000345:Affinity Labels; D007202:Indicators and Reagents; D007328:Insulin; D011956:Receptors, Cell Surface; D001360:Avidin; D011972:Receptor, Insulin; D014357:Trypsin