Mapping of the binding of platelet-derived growth factor to distinct domains of the basement membrane proteins BM-40 and perlecan and distinction from the BM-40 collagen-binding epitope
Mapping of the binding of platelet-derived growth factor to distinct domains of the basement membrane proteins BM-40 and perlecan and distinction from the BM-40 collagen-binding epitope
复制标题
DOI：
10.1046/j.1432-1327.1998.2550060.x
复制
发表时间：
1998-07-01
期刊：
影响因子：
--
通讯作者：
Timpl, R
中科院分区：
文献类型：
--
作者：
Göhring, W;Sasaki, T;Timpl, R
使用表面等离子体共振测定来分析血小板衍生生长因子（PDGF）-AA和PDGF-BB与细胞外基质的各种蛋白质的结合。这鉴定了几种胶原蛋白类型;层粘连蛋白-1、巢蛋白、串珠素和BM-40作为PDGF的潜在配体，K-d值在2-3200 nM范围内。Perlecan和BM-40用于检查相互作用的结构域特异性和其他参数。重组人和小鼠BM-40显示以相似的方式结合两种PDGF，Kd为约5-10 nM。对人BM-40缺失突变体的研究表明，与其C-末端细胞外钙结合（EC）模块结合，但相互作用不需要钙。这将其与EC模块与各种胶原类型的结合区分开来，后者是严格依赖于钙的。此外，螺旋α C的缺失或EC模块的螺旋α A中的两个点突变增强或消除了与胶原IV的结合。由于这些突变对与PDGF的结合没有影响，因此证明存在两种不同的结合表位。PDGF-BB与串珠素核心蛋白的结合可以定位到其结构域III-2（K-d = 8 nM），对于结构域I、IV-1和V显示出较低的亲和力（K-d = 34-64 nM）。其它串珠蛋白聚糖结构域（II、III-1、III-3、IV-2）是无活性的。PDGF-AA也显示结合结构域III-2，但不结合III-1。巢蛋白、BM-40或串珠蛋白聚糖结构域III-2均不干扰PDGF与其α和β受体的结合，然而，这表明这些相互作用可能主要用于PDGF在细胞外基质中的储存。
A surface plasmon resonance assay was used to analyze the binding of platelet-derived growth factor (PDGF)-AA and PDGF-BB to various proteins of the extracellular matrix. This identified several collagen types; laminin-l, nidogen, perlecan and BM-40 as potential ligands for PDGF with K-d values in the range 2-3200 nM. Perlecan and BM-40 were used to examine the domain specificity and other parameters of the interactions. Recombinant human and mouse BM-40 were shown to bind both PDGFs in a similar manner with a K-d of about 5-10 nM. Studies with deletion mutants of human BM-40 demonstrated binding to its C-terminal extracellular calcium-binding (EC) module, yet the interaction did not require calcium. This distinguishes this from the binding of the EC module to various collagen types, which is strictly calcium dependent. Furthermore, deletion of helix alpha C or two point mutations in helix alpha A of the EC module either enhanced or abolished binding to collagen IV. Since these mutations had no effects on binding to PDGF it demonstrated the presence of two different binding epitopes. Binding of PDGF-BB to the perlecan core protein could be mapped to its domain III-2 (K-d = 8 nM) with lower affinities shown for domains I, IV-1 and V (K-d = 34-64 nM). Other perlecan domains (II, III-1, III-3, IV-2) were inactive. PDGF-AA was also shown to bind domain III-2 but not III-1. Neither nidogen, BM-40 or perlecan domain III-2 interfered with the binding of PDGF to its alpha and beta receptors, however, suggesting that these interactions may be mainly used for storage of PDGF in the extracellular matrix.