RECOMBINANT DOMAIN-III OF PERLECAN PROMOTES CELL ATTACHMENT THROUGH ITS RGDS SEQUENCE
RECOMBINANT DOMAIN-III OF PERLECAN PROMOTES CELL ATTACHMENT THROUGH ITS RGDS SEQUENCE
复制标题
DOI：
10.1074/jbc.270.1.404
复制
发表时间：
1995-01-06
影响因子：
4.8
通讯作者：
HASSELL, JR
中科院分区：
文献类型：
--
作者：
CHAKRAVARTI, S;HORCHAR, T;HASSELL, JR
Perlecan先前已被证明通过其核心蛋白与细胞表面的相互作用支持多种细胞的附着。然而，参与细胞粘附的核心蛋白结构域是未知的。鼠perlecan的层粘连蛋白样结构域III可以包含RGDS序列，并且可能是支持整合素介导的细胞附着的候选者。我们利用cDNA克隆技术构建了一个与结构域III对应的cDNA结构体，该结构体在5‘端含有帧内信号肽，在3’端含有帧内终止密码子。将构建体插入pRC/CMV载体，转染HT1080细胞，从培养基中纯化出130 kda的重组结构域III蛋白。V8蛋白酶酶切产生的蛋白水解片段的大小以及重组蛋白的旋转阴影图像分析表明，重组蛋白是以天然构象产生的。重组结构域III包被在组织培养皿上，以剂量依赖的方式支持上皮样小鼠乳腺肿瘤细胞系MMT 060562的粘附。这种相互作用被RGDS合成肽和完整的perlecan特异性抑制，而不是层粘连蛋白。这种结构域III依赖rgd的细胞附着活性表明perlecan在整合素介导的信号传导中起作用。
Perlecan has been previously been shown to support attachment of a wide variety of cells through interactions of its core protein with the cell surface. The core protein domains involved in cell adhesion are, however, unknown. The laminin-like domain III of murine perlecan can contains an RGDS sequence and is a likely candidate for supporting integrin-mediated cell attachment. We made a cDNA construct corresponding to domain III and containing an in frame signal peptide at the 5' end as well as in frame a stop codon at the 3' end by using cDNA clones to perlecan. The construct was inserted into the pRC/CMV vector and transfected into HT1080 cells, and the secreted recombinant domain III, a 130-kDa protein, was purified from the medium. The size of proteolytic fragments produced by digestion with V8 protease as well as analysis of the rotary shadowed image of the recombinant protein indicated it was produced in a native conformation. Recombinant domain III coated on tissue culture dishes, supports adhesion of an epithelial-like mouse mammary tumor cell line MMT 060562 in a dose-dependent manner. This interaction was inhibited specifically by the RGDS synthetic peptide and intact perlecan, but not laminin. This domain III RGD-dependent cell attachment activity indicates a role for perlecan in integrin-mediated signaling.