Togashi Masaru, Okuyama Shin, Wakui Hideki, Komatsuda Atsushi, Suzuki Daisuke, Toyoda Masao, Sawada Ken-ichi
Third Department of Internal Medicine, Akita University School of Medicine, Akita, Japan.
Nephron Exp Nephrol. 2007;107(2):e65-72. doi: 10.1159/000108644. Epub 2007 Sep 20.
The aim of this study was to identify novel binding partners for alpha-actinin-4 (actinin-4), an essential component of the glomerular filtration barrier.
We performed bacterial two-hybrid screenings of a human kidney cDNA library using as a bait human actinin-4 containing the spectrin-like repeat R1. The identified interactions were further verified by in vitro affinity assays. To investigate the expression of the identified molecules in the kidney and other tissues, a human tissue cDNA panel screening and in situ hybridization were performed.
One isolated cDNA from the library encoded OK/SW-CL.16 protein with a segment similar to known actinin-interacting regions. OK/SW-CL.16 protein also contained the class I PxxP motif, which can participate in binding to Src homology 3 (SH3) domain-containing signaling proteins. In vitro affinity assays showed interactions of recombinant actinin-4 R1 and full-length renal actinin-4 with recombinant OK/SW-CL.16 protein. A tissue cDNA panel screening revealed a ubiquitous expression of OK/SW-CL.16 mRNA. In situ hybridization showed glomerular expression of OK/SW-CL.16 mRNA, mainly in podocytes and mesangial cells.
Our results suggest that OK/SW-CL.16 protein is a novel binding partner for actinin-4. OK/SW-CL16 protein might act as a linker between actinin-4 and some SH3 domain-containing signaling proteins in podocytes.
本研究的目的是鉴定α-辅肌动蛋白-4（辅肌动蛋白-4）的新型结合伴侣，α-辅肌动蛋白-4是肾小球滤过屏障的重要组成部分。
我们使用包含血影蛋白样重复序列R1的人辅肌动蛋白-4作为诱饵，对人肾cDNA文库进行细菌双杂交筛选。通过体外亲和力测定进一步验证所鉴定的相互作用。为了研究所鉴定分子在肾脏和其他组织中的表达，进行了人组织cDNA面板筛选和原位杂交。
从文库中分离出的一个cDNA编码OK/SW-CL.16蛋白，其一段序列与已知的辅肌动蛋白相互作用区域相似。OK/SW-CL.16蛋白还包含I类PxxP基序，可参与与含Src同源3（SH3）结构域的信号蛋白结合。体外亲和力测定显示重组辅肌动蛋白-4 R1和全长肾辅肌动蛋白-4与重组OK/SW-CL.16蛋白之间存在相互作用。组织cDNA面板筛选显示OK/SW-CL.16 mRNA广泛表达。原位杂交显示OK/SW-CL.16 mRNA在肾小球表达，主要在足细胞和系膜细胞中。
我们的结果表明OK/SW-CL.16蛋白是辅肌动蛋白-4的新型结合伴侣。OK/SW-CL16蛋白可能在足细胞中作为辅肌动蛋白-4与一些含SH3结构域的信号蛋白之间的连接物发挥作用。
