Functional domains of the receptor-associated protein (RAP).
Functional domains of the receptor-associated protein (RAP).
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DOI：
10.1073/pnas.91.8.3161
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发表时间：
1994-04
影响因子：
11.1
通讯作者：
Robert A. Orlando;M. Farquhar
中科院分区：
文献类型：
--
作者：
Robert A. Orlando;M. Farquhar
受体相关蛋白（RAP）特异性地与低密度脂蛋白（LDL）受体基因家族的两个最新成员gp 330和低密度脂蛋白（LDL）受体相关蛋白（LRP）相关。通过配体印迹、亲和层析和密度梯度沉降获得的结果表明RAP以高亲和力与两种受体结合，并且该结合是Ca 2+依赖性的。RAP还结合肝素，并且与在畸胎瘤细胞系（F9）中鉴定的小鼠肝素结合蛋白（HBP-44）相同。虽然生物化学研究表明，RAP存在于细胞表面上，是一种有效的抑制剂的配体结合到gp 330和LRP，免疫细胞化学的研究结果表明，RAP是最丰富的内质网腔，并可能在受体折叠和/或运输的功能。为了便于RAP功能的表征，我们通过对代表RAP重叠结构域的融合蛋白进行直接结合研究，绘制了RAP的gp 330和肝素结合位点。发现gp 330结合RAP上的两个独立位点--即，在氨基酸85-148和178-248之间。用放射性标记的肝素进行的结合研究表明，肝素结合位点在氨基酸261和323之间，这与我们先前基于RAP的C末端的两亲性质提出的位点（残基287-306）一致。这些数据表明，先前证明的gp 330和肝素结合位点以及Heymann肾炎致病性表位（氨基酸1-86）由RAP多肽的不同结构域代表。
The receptor-associated protein (RAP) specifically associates with gp330 and the low density lipoprotein (LDL) receptor-related protein (LRP), the two newest members of the LDL receptor gene family. Results obtained by ligand blotting, affinity chromatography, and density-gradient sedimentation demonstrate that RAP binds to both receptors with high affinity and that the binding is Ca2+ dependent. RAP also binds heparin and is identical to a mouse heparin binding protein (HBP-44) identified in a teratocarcinoma cell line (F9). While biochemical studies have shown that RAP is present on the cell surface and is an effective inhibitor of ligand binding to gp330 and LRP, immunocytochemical findings indicate that RAP is most abundant in the endoplasmic reticulum lumen and may function in receptor folding and/or trafficking. To facilitate the characterization of RAP's function(s) we have mapped its gp330 and heparin binding sites by performing direct binding studies on fusion proteins representing overlapping domains of RAP. gp330 was found to bind to two separate sites on RAP--i.e., between amino acids 85-148 and 178-248. Binding studies with radiolabeled heparin indicate that the heparin binding site is between amino acids 261 and 323, which is consistent with our previously proposed site (residues 287-306) based on the amphipathic nature of the C terminus of RAP. These data demonstrate that the gp330 and heparin binding sites and the Heymann nephritis pathogenic epitope (amino acids 1-86) demonstrated earlier are represented by distinct domains of the RAP polypeptide.