Localization of the insulin-like growth factor II binding site to amino acids 1508-1566 in repeat 11 of the mannose 6-phosphate/insulin-like growth factor II receptor.
The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) binds insulin-like growth factor II (IGF-II) with high affinity. To localize the IGF-II binding site within the 15 repeating units that form the extracytoplasmic domain of the receptor, purified human M6P/IGF-II receptor was digested with thermolysin, and the fragments were analyzed for their ability to bind 125I-IGF-II in a cross-linking assay. Two IGF-II-binding receptor fragments of 23 and 37 kDa were purified. Sequence analysis revealed that the fragments consist of disulfide connected peptides comprising amino acids 1331-1566 and 1331-1697 of the receptor repeats 9-12. In a second approach we expressed truncated forms of the M6P/IGF-II receptor fused to the C terminus of the extracytoplasmic domain of the 46-kDa mannose 6-phosphate receptor. Fusion proteins containing M6P/IGF-II receptor repeats 10-15, 10-11, or 11-15 bound IGF-II, whereas a fusion protein containing the single repeat 10 failed to bind. This result indicates that repeat 11 (amino acids 1508-1650) is sufficient for binding of IGF-II. Residues 1508-1566, which are shared by the 23-kDa IGF-II-binding fragment and repeat 11, are proposed to form the IGF-II binding site of the M6P/IGF-II receptor.
Schmidt
B
B
Georg-August-Universität, Zentrum für Biochemie und Molekulare Zellbiologie, Abteilung Biochemie II, Göttingen, Federal Republic of Germany.
Kiecke-Siemsen
C
C
Waheed
A
A
Braulke
T
T
von Figura
K
K
D000595:Amino Acid Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004220:Disulfides / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D017527:Receptor, IGF Type 2 / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013820:Thermolysin
D003432:Cross-Linking Reagents; D004220:Disulfides; D010446:Peptide Fragments; D017527:Receptor, IGF Type 2; D011994:Recombinant Proteins; D007335:Insulin-Like Growth Factor II; D013820:Thermolysin