Differential Phosphorylation Provides a Switch to Control How α-Arrestin Rod1 Down-regulates Mating Pheromone Response in Saccharomyces cerevisiae.
Differential Phosphorylation Provides a Switch to Control How α-Arrestin Rod1 Down-regulates Mating Pheromone Response in Saccharomyces cerevisiae.
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DOI：
10.1534/genetics.115.186122
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发表时间：
2016-05
期刊：
影响因子：
3.3
通讯作者：
Thorner J
中科院分区：
文献类型：
--
作者：
Alvaro CG;Aindow A;Thorner J
G蛋白偶联受体（GPCR）是一种膜蛋白，可激活同源异源三聚体G蛋白，引发下游细胞反应。需要严格调节GPCR诱发的途径，因为长时间的刺激可能对生物体有害。Ste 2是酿酒酵母中的一种GPCR，介导MATa单倍体对肽交配信息素α因子的反应，其通过组成性和激动剂诱导的内吞作用下调。有效的激动剂刺激的Ste 2内化需要其与衔接蛋白α-arrestin Rod 1/Art 4的结合，其募集HECT结构域泛素连接酶Rsp 5，允许受体C-末端尾部的泛素化及其通过网格蛋白依赖性内吞机制的接合。我们以前表明，钙调磷酸酶（磷蛋白磷酸酶2B）的Rod 1的去磷酸化是必需的最佳的Rod 1功能Ste 2下调。我们在这里表明，Rod 1的磷酸化负调控介导的两个不同的应激激活蛋白激酶，Snf 1/AMPK和Ypk 1/SGK 1，并证明在体外和体内，这种磷酸化调节阻碍能力的Rod 1，以促进交配途径脱敏。这些研究还表明，在缺乏其磷酸化的情况下，Rod 1可以独立于Rsp 5介导的受体泛素化促进适应，这与最近的证据一致，即α-arrestins可以通过网格蛋白依赖性和网格蛋白非依赖性机制促进货物识别。然而，在缺乏网格蛋白非依赖性进入所需的组分（Rbbni 1）的细胞中，大部分未磷酸化且不能与Rsp 5结合的Rod 1衍生物仍然促进有效的适应，表明这种α-抑制蛋白促进信息素反应途径脱敏的第三种机制。
G-protein-coupled receptors (GPCRs) are integral membrane proteins that initiate stimulus-dependent activation of cognate heterotrimeric G-proteins, triggering ensuing downstream cellular responses. Tight regulation of GPCR-evoked pathways is required because prolonged stimulation can be detrimental to an organism. Ste2, a GPCR in Saccharomyces cerevisiae that mediates response of MATa haploids to the peptide mating pheromone α-factor, is down-regulated by both constitutive and agonist-induced endocytosis. Efficient agonist-stimulated internalization of Ste2 requires its association with an adaptor protein, the α-arrestin Rod1/Art4, which recruits the HECT-domain ubiquitin ligase Rsp5, allowing for ubiquitinylation of the C-terminal tail of the receptor and its engagement by the clathrin-dependent endocytic machinery. We previously showed that dephosphorylation of Rod1 by calcineurin (phosphoprotein phosphatase 2B) is required for optimal Rod1 function in Ste2 down-regulation. We show here that negative regulation of Rod1 by phosphorylation is mediated by two distinct stress-activated protein kinases, Snf1/AMPK and Ypk1/SGK1, and demonstrate both in vitro and in vivo that this phospho-regulation impedes the ability of Rod1 to promote mating pathway desensitization. These studies also revealed that, in the absence of its phosphorylation, Rod1 can promote adaptation independently of Rsp5-mediated receptor ubiquitinylation, consistent with recent evidence that α-arrestins can contribute to cargo recognition by both clathrin-dependent and clathrin-independent mechanisms. However, in cells lacking a component (formin Bni1) required for clathrin-independent entry, Rod1 derivatives that are largely unphosphorylated and unable to associate with Rsp5 still promote efficient adaptation, indicating a third mechanism by which this α-arrestin promotes desensitization of the pheromone-response pathway.