In vitro interaction of a polypeptide homologous to human Ro/SS-A antigen (calreticulin) with a highly conserved amino acid sequence in the cytoplasmic domain of integrin alpha subunits.
In vitro interaction of a polypeptide homologous to human Ro/SS-A antigen (calreticulin) with a highly conserved amino acid sequence in the cytoplasmic domain of integrin alpha subunits.
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DOI：
10.1021/bi00105a008
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发表时间：
1991-10
期刊：
影响因子：
2.9
通讯作者：
M. Rojiani;B. Finlay;V. Gray;S. Dedhar
中科院分区：
文献类型：
--
作者：
M. Rojiani;B. Finlay;V. Gray;S. Dedhar
我们endewitness确定蛋白质相互作用KLGFFKR，一个高度保守的基序在胞质结构域相邻的跨膜结构域的α亚基的整合素。我们发现，亲和层析的细胞提取物与此肽，然后用EDTA洗脱导致在60 kDa的蛋白质（p60）的分离。发现该60-kDa多肽的N-末端氨基酸序列与Ro/SS-A抗原高度同源，Ro/SS-A抗原是一种与钙网蛋白和失智症“记忆分子”同源的60-kDa蛋白。发现p60的结合对KLGFFKR序列是特异性的，因为该多肽不与具有乱序氨基酸序列（KLRFGFK）的肽结合，并且它也被可溶性KLGFFKR肽而不是乱序肽特异性地从KLGFFKR亲和基质洗脱。固相体外结合试验表明，p60与整合素α 3和α 5亚基，但不与β 1亚基的特异性相互作用。此外，p60可以与α 3 β 1在体外共孵育后共纯化。这些相互作用可被KLGFFKR肽和EDTA抑制，表明序列特异性和二价阳离子依赖性结合。尽管钙网蛋白被认为定位于内质网中，但发现Ro/SSA抗原同源60-kDa多肽库存在于可溶性细胞质中，表明p60与整联蛋白α亚基相互作用的可行性。我们的数据表明，p60（Ro/SS-A Ag）可以通过高度保守的KLGFFKR氨基酸序列特异性结合整合素α亚基。
We endeavored to identify proteins interacting with KLGFFKR, a highly conserved motif in the cytoplasmic domain adjacent to the transmembrane domain of the alpha subunit of integrins. We found that affinity chromatography of cell extracts with this peptide followed by elution with EDTA resulted in the isolation of a 60-kDa protein (p60). The N-terminal amino acid sequence of this 60-kDa polypeptide was found to be highly homologous to the Ro/SS-A antigen, a 60-kDa protein homologous to calreticulin and Aplysia "memory molecule". The binding of p60 was found to be specific for the KLGFFKR sequence since this polypeptide did not bind to a peptide with a scrambled amino acid sequence (KLRFGFK), and it was also specifically eluted from the KLGFFKR affinity matrix ith soluble KLGFFKR peptide but not with the scrambled peptide. Solid phase in vitro binding assays demonstrated specific interaction of p60 with integrin alpha 3 and alpha 5 subunits but not with the beta 1 subunit. Furthermore, p60 could be copurified with alpha 3 beta 1 following coincubation in vitro. These interactions could be inhibited by KLGFFKR peptide and also by EDTA, indicating sequence-specific and divalent cation dependent binding. Despite the fact that calreticulin is thought to be localized in the endoplasmic reticulum, a pool of Ro/SS A antigen homologous 60-kDa polypeptide was found to be present in the soluble cytoplasm, indicating the feasibility of an interaction of p60 with the integrin alpha subunits. Our data suggest that p60 (Ro/SS-A Ag) can specifically bind to integrin alpha subunits via the highly conserved KLGFFKR amino acid sequence.