Devi G R, Byrd J C, Slentz D H, MacDonald R G
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha 68198-4525, USA.
Mol Endocrinol. 1998 Nov;12(11):1661-72. doi: 10.1210/mend.12.11.0192.
Insulin-like growth factor II (IGF-II) and phosphomannosylated glycoproteins bind to distinct sites on the same receptor, the IGF-II/mannose 6-phosphate receptor (IGF2R). Analysis of truncated receptors (minireceptors) has been used to map the IGF-II binding site within the receptor's extracytoplasmic domain, which consists of 15 homologous repeats. A minireceptor consisting of repeat 11 contained the minimal elements for binding IGF-II, but with 5- to 10-fold lower relative binding affinity than the full-length receptor. We hypothesized that the complete, high-affinity IGF-II binding site is formed by interaction between the primary site in repeat 11 and a putative affinity-enhancing domain. To determine the minimum portion of the IGF2R's extracytoplasmic domain needed for expression of high-affinity IGF-II binding, a nested set of FLAG epitope-tagged minireceptors encompassing repeats 11 through 15 was prepared and transiently expressed in 293T cells. Minireceptors containing repeats 11-13 or 11-15 exhibited high affinity, comparable to the full-length receptor (IC50 = 1-2 nM), whereas constructs containing repeat 11 only or repeats 11-12 did not (IC50 = 10-20 nM). These data suggested that the affinity-enhancing domain is located within repeat 13, which contains a unique 43-residue insert that has approximately 50% sequence identity to the type II repeat of fibronectin. Although a repeat 13 minireceptor did not bind IGF-II on its own, an 11-13 minireceptor containing a deletion of the 43-residue insert exhibited low IGF-II binding affinity (IC50 = 10-20 nM). Expression of mutant receptors from a full-length IGF2R construct bearing a deletion of the 43-residue insert was very low relative to wild type. Depletion assays using IGF-II-Sepharose showed that the mutant receptor had lower affinity for IGF-II than the wild-type receptor. This study reveals that two independent receptor domains are involved in the formation of a high-affinity binding site for IGF-II, and that a complete repeat 13 is required for high-affinity IGF-II binding.
胰岛素样生长因子II（IGF-II）和磷酸甘露糖基化糖蛋白与同一受体——IGF-II/甘露糖6-磷酸受体（IGF2R）上的不同位点结合。对截短受体（微型受体）的分析已用于绘制受体胞外结构域内的IGF-II结合位点，该结构域由15个同源重复序列组成。由重复序列11组成的微型受体包含结合IGF-II的最小元件，但相对结合亲和力比全长受体低5至10倍。我们推测完整的高亲和力IGF-II结合位点是由重复序列11中的主要位点与假定的亲和力增强结构域之间的相互作用形成的。为了确定表达高亲和力IGF-II结合所需的IGF2R胞外结构域的最小部分，制备了一组包含重复序列11至15的带FLAG表位标签的嵌套微型受体，并在293T细胞中瞬时表达。包含重复序列11-13或11-15的微型受体表现出高亲和力，与全长受体相当（IC50 = 1-2 nM），而仅包含重复序列11或重复序列11-12的构建体则没有（IC50 = 10-20 nM）。这些数据表明亲和力增强结构域位于重复序列13内，该序列包含一个独特的43个残基的插入片段，与纤连蛋白的II型重复序列具有约50%的序列同一性。尽管仅由重复序列13组成的微型受体自身不结合IGF-II，但包含43个残基插入片段缺失的11-13微型受体表现出低IGF-II结合亲和力（IC50 = 10-20 nM）。相对于野生型，来自带有43个残基插入片段缺失的全长IGF2R构建体的突变受体的表达非常低。使用IGF-II-琼脂糖进行的消耗试验表明，突变受体对IGF-II的亲和力低于野生型受体。这项研究表明，两个独立的受体结构域参与了IGF-II高亲和力结合位点的形成，并且完整的重复序列13是高亲和力IGF-II结合所必需的。
