Craft C M, Xu J, Slepak V Z, Zhan-Poe X, Zhu X, Brown B, Lolley R N
Mary D. Allen Laboratory for Vision Research, Doheny Eye Institute, University of Southern California School of Medicine, Los Angeles 90033, USA.
Biochemistry. 1998 Nov 10;37(45):15758-72. doi: 10.1021/bi980921a.
In this study, we identify new isoforms of the retinal phosducin and investigate the expression of the phosducin family, showing that an isoform, PhLP1, has sequence homology with Phd and Gbeta gamma binding capability, whereas two isoforms (phosducin-like orphan proteins, PhLOPs) share sequence homology with Phd but fail to bind Gbeta gamma. Original identification of PhLP1 and the PhLOPs was from a human retina cDNA library, using a PCR product for library hybridization screening that contained a predicted functional epitope domain. The screen identified Phd and three related, but distinct, recombinants (PhLP1, PhLOP1, and PhLOP2). By RT-PCR, all isoforms are expressed in either retina or forskolin-stimulated Y79 retinoblastoma cells; however, the new isoforms are below the level of detection on Northern blot analysis. The predicted amino acid translation of each homologue revealed major differences, arising from either splice variants or gene duplication of Phd. To test the functional interaction of all phosducin isoforms with Gbeta gamma in vitro, a glutathione S-transferase (GST) fusion protein was developed for each member. Biochemical interaction with purified retinal transducin Gbeta gamma was verified for GST-Phd and demonstrated for GST-PhLP1; however, neither GST-PhLOP1 nor GST-PhLOP2 bound Gbeta gamma. Comparable results were observed when the GST-phosducin fusion proteins selectively sequestered Gbeta gammas from retinal extracts or when functional Gbeta gamma interactions were assessed using surface plasmon resonance technology. Phosducin and its isoforms are widely distributed in body tissues where they may participate in signal transduction pathways. Phd and PhLP1 possess an 11-amino acid conserved epitope domain (TGPKGVINDWR) that controls the high-affinity binding of Gbeta gamma; these isoforms are implicated in the G-protein signaling pathway. The phosducin-like orphan proteins (PhLOPs) fail to bind Gbeta gamma, suggesting that the PhLOP isoforms may participate in still unidentified signaling pathways.
在本研究中，我们鉴定出视网膜磷蛋白的新亚型，并研究了磷蛋白家族的表达情况，结果表明一种亚型PhLP1与磷蛋白具有序列同源性且具备结合Gβγ的能力，而另外两种亚型（类磷蛋白孤儿蛋白，PhLOPs）与磷蛋白有序列同源性，但无法结合Gβγ。PhLP1和PhLOPs最初是从人视网膜cDNA文库中鉴定出来的，使用了一个包含预测功能表位结构域的PCR产物进行文库杂交筛选。该筛选鉴定出了磷蛋白以及三个相关但不同的重组体（PhLP1、PhLOP1和PhLOP2）。通过逆转录聚合酶链反应（RT-PCR），所有亚型均在视网膜或福斯高林刺激的Y79视网膜母细胞瘤细胞中表达；然而，在Northern印迹分析中，新亚型的表达水平低于检测限。每个同源物的预测氨基酸翻译显示出主要差异，这些差异源于磷蛋白的剪接变体或基因复制。为了在体外测试所有磷蛋白亚型与Gβγ的功能相互作用，为每个成员开发了一种谷胱甘肽S-转移酶（GST）融合蛋白。已证实GST-磷蛋白与纯化的视网膜转导蛋白Gβγ存在生化相互作用，并且GST-PhLP1也有此作用；然而，GST-PhLOP1和GST-PhLOP2均不与Gβγ结合。当GST-磷蛋白融合蛋白从视网膜提取物中选择性隔离Gβγ时，或者使用表面等离子体共振技术评估功能性Gβγ相互作用时，观察到了类似的结果。磷蛋白及其亚型广泛分布于身体组织中，它们可能参与信号转导途径。磷蛋白和PhLP1拥有一个11个氨基酸的保守表位结构域（TGPKGVINDWR），该结构域控制Gβγ的高亲和力结合；这些亚型与G蛋白信号通路有关。类磷蛋白孤儿蛋白（PhLOPs）无法结合Gβγ，这表明PhLOP亚型可能参与仍未明确的信号通路。
