Molecular cloning and functional expression of the rat 175-kDa hyaluronan receptor for endocytosis.
Molecular cloning and functional expression of the rat 175-kDa hyaluronan receptor for endocytosis.
复制标题
大鼠 175-kDa 透明质酸受体内吞作用的分子克隆和功能表达。
DOI：
10.1091/mbc.02-03-0048
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发表时间：
2002
影响因子：
3.3
通讯作者：
Weigel,PaulH
中科院分区：
文献类型：
--
作者：
Zhou,Bin;Weigel,JanetA;Saxena,Amit;Weigel,PaulH
最近，我们纯化了大鼠肝脏透明质酸内吞受体（HARE），并发现在肝、脾和淋巴结的窦状内皮细胞中大量表达175-kDa和300-kDa HARE。我们在此报告的第一个克隆和功能表达的大鼠175 kDa的HARE。从纯化的175-kDa HARE中获得肽序列，并设计简并寡核苷酸引物用于逆转录-聚合酶链反应和cDNA克隆。cDNA末端5′-快速扩增、北方分析、N-末端测序和抗体反应性分析的结果表明，不存在直接编码175-kDa HARE的mRNA。这种蛋白质很可能来源于更大的前体。因此，我们构建了一个人工的4.7-kb的cDNA编码1431个氨基酸的175-kDa的HARE。预测的I型膜蛋白的质量为156，393 Da，pI为7.86。将与IG κ链N端前导序列融合的175-kDa HARE cDNA分别瞬时转染COS-7细胞和稳定转染SK-Hep-1细胞，以评估透明质酸或透明质酸（HA）结合活性和内吞作用。在这两种情况下，HARE表达和HA结合活性进行检测。此外，稳定的SK-175 HARE细胞表现出125 I-HA的特异性内吞和受体再循环。荧光激活细胞分选分析证实，重组HARE在细胞表面上表达，并且荧光HA摄取被针对HARE的特异性阻断单克隆抗体抑制。此外，HARE基本上与网格蛋白共定位，但不与递送至溶酶体的内化HA共定位。结果证实，重组175-kDa HARE是一个真正的内吞受体的HA，这种受体可以独立的功能的175-kDa HARE。HARE是第一个功能鉴定的蛋白质家族成员，该蛋白质家族具有Fasciclin、表皮生长因子样、Xlink和跨膜结构域的相似组织。
We recently purified the rat liver hyaluronan receptor for endocytosis (HARE) and found abundant expression of 175- and ∼300-kDa HARE species in sinusoidal endothelial cells of the liver, spleen, and lymph nodes. We report herein the first cloning and functional expression of the rat 175-kDa HARE. Peptide sequences were obtained from the purified 175-kDa HARE, and degenerate oligonucleotide primers were designed for reverse transcription-polymerase chain reaction and cDNA cloning. Results of 5′-rapid amplification of cDNA ends, Northern analysis, N-terminal sequence, and antibody reactivity analyses indicated the absence of mRNA directly encoding the 175-kDa HARE. This protein is most likely derived from a larger precursor. Accordingly, we constructed an artificial 4.7-kb cDNA encoding the 1431 amino acid 175-kDa HARE. The predicted type I membrane protein has a mass of 156,393 Da and a pI of 7.86. The 175-kDa HARE cDNA, fused to the N-terminal leader sequence of the Ig κ-chain, was transfected transiently into COS-7 cells and stably into SK-Hep-1 cells, respectively, to assess hyaluronan or hyaluronic acid (HA)-binding activity and endocytosis. In both cases, HARE expression and HA-binding activity were detected. Furthermore, stable SK-175HARE cells demonstrated specific endocytosis of125I-HA and receptor recycling. Fluorescence-activated cell sorting analysis confirmed that recombinant HARE was expressed on the cell surface and that fluorescent HA uptake was inhibited by a specific blocking monoclonal antibody against HARE. Additionally, HARE was substantially colocalized with clathrin, but not with internalized HA that was delivered to lysosomes. The results confirm that recombinant 175-kDa HARE is an authentic endocytic receptor for HA and that this receptor can function independently of the ∼300-kDa HARE. HARE is the first functionally identified member of a protein family that shares a similar organization of Fasciclin, epidermal growth factor-like, Xlink, and transmembrane domains.