High-affinity ligand binding by wild-type/mutant heteromeric complexes of the mannose 6-phosphate/insulin-like growth factor II receptor
High-affinity ligand binding by wild-type/mutant heteromeric complexes of the mannose 6-phosphate/insulin-like growth factor II receptor
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DOI：
10.1111/j.1742-4658.2009.06917.x
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发表时间：
2009-04-01
期刊：
影响因子：
5.4
通讯作者：
MacDonald, Richard G.
中科院分区：
文献类型：
--
作者：
Hartman, Michelle A.;Kreiling, Jodi L.;MacDonald, Richard G.
甘露糖6-磷酸/胰岛素样生长因子II受体具有多种配体结合特性，有助于其在溶酶体生物发生和生长抑制中发挥作用。甘露糖6-磷酸（Man-6-P）配体的最佳受体结合和内化需要二聚体结构，导致二价高亲和力结合，可能是由两个亚基上的位点之间的合作介导的。胰岛素样生长因子II （IGF-II）与每个单体上的单个位点结合。据推测，IGF-II与每个单体上的同源位点的结合是独立发生的，但二价Man-6-P配体的结合需要两个单体上的位点的合作贡献。为了验证这一假设，我们共同免疫沉淀差异表位标记的可溶性微受体并评估配体结合。野生型和点突变的IGF-II结合位点在两个二聚体微型受体之间配对对侧结合位点的功能没有影响，这表明IGF-II与二聚体两侧的结合是独立的，没有亚基间效应。正如预期的那样，由野生型单体和携带两个man -6- p结合敲除突变的突变体组成的异二聚体受体形成了功能性的IGF-II结合位点。与预测相反，这种异二聚体受体也能以高亲和力结合man -6- p基配体，其结合量完全归因于免疫沉淀的野生型受体。锚定异二聚体的两个c端产生IGF-II和Man-6-P配体的最佳结合。因此，IGF-II独立结合二聚体甘露糖6-磷酸/胰岛素样生长因子II受体的两个亚基。虽然野生型/突变型异聚物在混合时很容易形成，但似乎多价Man-6-P配体优先结合野生型位点，可能是通过固定受体簇内的交叉桥接受体。
The mannose 6-phosphate/insulin-like growth factor II receptor has diverse ligand-binding properties contributing to its roles in lysosome biogenesis and growth suppression. Optimal receptor binding and internalization of mannose 6-phosphate (Man-6-P)-bearing ligands requires a dimeric structure leading to bivalent high-affinity binding, presumably mediated by cooperation between sites on both subunits. Insulin-like growth factor II (IGF-II) binds to a single site on each monomer. It is hypothesized that IGF-II binding to cognate sites on each monomer occurs independently, but bivalent Man-6-P ligand binding requires cooperative contributions from sites on both monomers. To test this hypothesis, we co-immunoprecipitated differentially epitope-tagged soluble mini-receptors and assessed ligand binding. Pairing of wild-type and point-mutated IGF-II binding sites between two dimerized mini-receptors had no effect on the function of the contralateral binding site, indicating IGF-II binding to each side of the dimer is independent and manifests no intersubunit effects. As expected, heterodimeric receptors composed of a wild-type monomer and a mutant bearing two Man-6-P-binding knockout mutations form functional IGF-II binding sites. By contrast to prediction, such heterodimeric receptors also bind Man-6-P-based ligands with high affinity, and the amount of binding can be attributed entirely to the immunoprecipitated wild-type receptors. Anchoring of both C-terminal ends of the heterodimer produces optimal binding of both IGF-II and Man-6-P ligands. Thus, IGF-II binds independently to both subunits of the dimeric mannose 6-phosphate/insulin-like growth factor II receptor. Although wild-type/mutant hetero-oligomers form readily when mixed, it appears that multivalent Man-6-P ligands bind preferentially to wild-type sites, possibly by cross-bridging receptors within clusters of immobilized receptors.