Localization of the insulin-like growth factor II (IGF-II) binding/cross-linking site of the IGF-II/mannose 6-phosphate receptor to extracellular repeats 10-11.
The insulin-like growth factor II (IGF-II) binding/cross-linking domain of the IGF-II/Man-6-P receptor was mapped by sequencing receptor fragments covalently attached to IGF-II. Rat placental or bovine liver receptors were purified by pentamannosyl-6-phosphate-Sepharose chromatography, affinity-labeled with 125I-IGF-II using disuccinimidyl tartrate, and digested with endoproteinase Glu-C. Analysis of small scale digests by gel electrophoresis revealed radiolabeled bands of approximately 17 kDa (rat) or approximately 18 kDa (bovine). For purification and sequencing of these radiolabeled receptor fragments, three receptor preparations were analyzed. The initial digests were fractionated by gel filtration followed by reverse-phase high performance liquid chromatography (HPLC), but the final purification steps differed somewhat in the three studies, using combinations of two-dimensional HPLC, gel electrophoresis, and electroblotting. Multiple sequences detected in each of these samples were unscrambled by computer-assisted and manual methods and by comparison with the quantity of labeled IGF-II present to identify sequences corresponding to fragments of the receptor covalently attached to IGF-II. The sequence, S(H)VNSXPMF, located in the COOH-terminal end of extracellular repeat 10 and beginning with serine 1488 of the bovine receptor, was the only receptor sequence common to all the samples and was the best candidate for the IGF-II cross-linked peptide by quantitative analysis. These data indicate residues within repeats 10-11 are likely to be important for IGF-II binding. We conclude that cross-linking between IGF-II and its receptor involves one or more of the 4 lysine residues located within extracellular repeat 11.
Garmroudi
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F
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha 68198.
MacDonald
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RG
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003432:Cross-Linking Reagents; D005260:Female; D007335:Insulin-Like Growth Factor II / Q000737:chemistry / Q000378:metabolism*; D007457:Iodine Radioisotopes; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010920:Placenta; D011247:Pregnancy; D011485:Protein Binding; D051381:Rats; D017527:Receptor, IGF Type 2 / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000378:metabolism; D013045:Species Specificity; D013388:Succinimides / Q000737:chemistry
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D017527:Receptor, IGF Type 2; D013388:Succinimides; C018052:disuccinimidyl tartarate; D007335:Insulin-Like Growth Factor II; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase