Identification of a novel protein-protein interaction motif mediating interaction of GPCR-associated sorting proteins with G protein-coupled receptors.
Identification of a novel protein-protein interaction motif mediating interaction of GPCR-associated sorting proteins with G protein-coupled receptors.
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DOI：
10.1371/journal.pone.0056336
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Simonin F
中科院分区：
文献类型：
--
作者：
Bornert O;Møller TC;Boeuf J;Candusso MP;Wagner R;Martinez KL;Simonin F
GPCR脱敏和下调被认为是体内耐受性形成的关键分子事件。在参与这些复杂过程的许多调节蛋白中，GASP-1已被证明参与了几种受体向降解途径的分选。该蛋白属于最近发现的GPCR相关分类蛋白(GAP)家族，该家族由10个成员组成，其结构和功能细节尚不清楚。我们对GAP和一组GPCRs之间的分子相互作用进行了详细的结构-功能关系分析。在第一步中，GST下拉实验显示，所有测试的GAP都与广泛的GPCRs显示出显著的相互作用。重要的是，不同的GAP成员表现出最强的相互作用特性，其特征也是存在一个由15个氨基酸组成的小的、高度保守的和重复的“GAP基序”。我们进一步通过GST-Down、表面等离子体共振和免疫共沉淀实验表明，GAP-1的中心结构域包含22个GAP基序，对于与GPCRs的相互作用是必不可少的。然后，我们用合成肽的定点突变和竞争实验证明了GAP基序，特别是其高度保守的核心序列SWFW，在与GPCRs的相互作用中起着至关重要的作用。总体而言，我们的数据显示GAP家族中的几个成员与GPCRs相互作用，并突出了GAP中存在一个新的蛋白质-蛋白质相互作用基序，该基序可能代表了研究GAP参与GPCRs活性调控的新靶点。
GPCR desensitization and down-regulation are considered key molecular events underlying the development of tolerance in vivo. Among the many regulatory proteins that are involved in these complex processes, GASP-1 have been shown to participate to the sorting of several receptors toward the degradation pathway. This protein belongs to the recently identified GPCR-associated sorting proteins (GASPs) family that comprises ten members for which structural and functional details are poorly documented. We present here a detailed structure–function relationship analysis of the molecular interaction between GASPs and a panel of GPCRs. In a first step, GST-pull down experiments revealed that all the tested GASPs display significant interactions with a wide range of GPCRs. Importantly, the different GASP members exhibiting the strongest interaction properties were also characterized by the presence of a small, highly conserved and repeated “GASP motif” of 15 amino acids. We further showed using GST-pull down, surface plasmon resonance and co-immunoprecipitation experiments that the central domain of GASP-1, which contains 22 GASP motifs, is essential for the interaction with GPCRs. We then used site directed mutagenesis and competition experiments with synthetic peptides to demonstrate that the GASP motif, and particularly its highly conserved core sequence SWFW, is critically involved in the interaction with GPCRs. Overall, our data show that several members of the GASP family interact with GPCRs and highlight the presence within GASPs of a novel protein-protein interaction motif that might represent a new target to investigate the involvement of GASPs in the modulation of the activity of GPCRs.
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