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.
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.
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DOI：
10.1210/mend.10.6.8776724
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发表时间：
1996-06
影响因子：
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通讯作者：
Farideh Garmroudi;G. Devi;D. Slentz;B. Schaffer;R. Macdonald
中科院分区：
文献类型：
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作者：
Farideh Garmroudi;G. Devi;D. Slentz;B. Schaffer;R. Macdonald
要完全理解胰岛素样生长因子II（IGF-II）与IGF-II/甘露糖-6-磷酸（Man-6-P）受体结合的功能意义，需要对受体的IGF-II结合结构域进行作图和最终突变分析。最近的进展已经将IGF-II结合位点定位于胞质外重复序列10-11。为了提高结合位点图的分辨率，在COS-7细胞中瞬时表达了一组嵌套的表位标记的截短形式的人IGF-II/Man-6-P受体。从细胞裂解物和条件培养基中免疫沉淀的截短受体的IGF-II结合特性通过亲和交联测定。从最大的截短受体（包括胞质外重复序列8-11（M（r）68 K））到最小的截短受体（主要由重复序列11（M（r）23 K）组成），所有受体都能够与IGF-II结合并交联。作为一组，截短的受体对IGF-II具有相似的亲和力，但相对结合亲和力比全长受体低5- 10倍。一个点突变取代异亮氨酸苏氨酸在残基1572，位于NH 2-末端的一半重复11，完全废除IGF-II结合。我们的结论是重复11的IGF-II/Man-6-P受体的胞质外结构域包含的最小元素所需的结合和交联IGF-II，和Ile 1572和其他残基内的NH 2-末端的一半重复11是特别重要的IGF-II的相互作用。
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.