Truncated forms of the insulin-like growth factor II (IGF-II)/mannose 6-phosphate receptor encompassing the IGF-II binding site: characterization of a point mutation that abolishes IGF-II binding.
Complete understanding of the functional significance of insulin-like growth factor II (IGF-II) binding by the IGF-II/mannose-6-phosphate (Man-6-P) receptor requires mapping and ultimately mutational analysis of the receptor's IGF-II binding domain. Recent advances have localized the IGF-II binding site to extracytoplasmic repeats 10-11. To improve resolution of the binding site map, a nested set of epitope-tagged, truncated forms of the human IGF-II/Man-6-P receptor were transiently expressed in COS-7 cells. The IGF-II binding properties of truncated receptors immunoprecipitated from cell lysates and conditioned media were determined by affinity cross-linking. From the largest truncated receptor, encompassing extracytoplasmic repeats 8-11 (M(r) 68 K), through the smallest, comprised primarily of repeat 11 (M(r) 23 K), all were able to bind and cross-link to IGF-II. As a group, the truncated receptors had similar affinities for IGF-II, but with relative binding affinities 5-to 10-fold lower than those of full-length receptors. A point mutation substituting threonine for isoleucine at residue 1572, located in the NH2-terminal half of repeat 11, completely abolished IGF-II binding. We conclude that repeat 11 of the IGF-II/Man-6-P receptor's extracytoplasmic domain contains the minimal elements required for binding and cross-linking to IGF-II, and that lle1572 and other residues within the NH2-terminal half of repeat 11 are particularly important for IGF-II interaction.
Garmroudi
F
F
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha 68198-4525, USA.
Devi
G
G
Slentz
D H
DH
Schaffer
B S
BS
MacDonald
R G
RG
D000818:Animals; D001665:Binding Sites; D003432:Cross-Linking Reagents; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000235:genetics* / Q000378:metabolism*; D007457:Iodine Radioisotopes; D009154:Mutation; D011233:Precipitin Tests; D017527:Receptor, IGF Type 2 / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D013379:Substrate Specificity
D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D017527:Receptor, IGF Type 2; D011994:Recombinant Proteins; D007335:Insulin-Like Growth Factor II