Strategies for isolating constitutively active and dominant-negative pheromone receptor mutants in yeast.
Strategies for isolating constitutively active and dominant-negative pheromone receptor mutants in yeast.
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DOI：
10.1016/b978-0-12-381296-4.00019-1
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发表时间：
2010
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酿酒酵母交配信息素受体是研究G蛋白偶联受体的有用模型。交配信息素受体Ste2和Ste3对于生存能力不是必需的，因此它们可以容易地通过各种遗传方法进行靶向分析。本章将描述两种突变体的识别方法，这两种突变体对受体信号传导的机制非常有用：组成型活性突变体和显性阴性突变体。有趣的是，这些不同类型的突变体揭示了互补的信息。组成性信号传导是由突变引起的，这些突变被认为削弱了七个跨膜结构域之间的相互作用，而显性负突变体显然稳定了跨膜结构域之间的接触，并将受体锁定在关闭构象。为了支持这些结论，组成型活性突变体和显性阴性突变体的某些组合恢复了几乎正常的信号传导特性。
Mating pheromone receptors of the yeast Saccharomyces cerevisiae are useful models for the study of G protein-coupled receptors. The mating pheromone receptors, Ste2 and Ste3, are not essential for viability so they can be readily targeted for analysis by a variety of genetic approaches. This chapter will describe methods for identification of two kinds of mutants that have been very informative about the mechanisms of receptor signaling: constitutively-active mutants and dominant-negative mutants. Interestingly, these distinct types of mutants have revealed complementary information. Constitutive signaling is caused by mutations that are thought to weaken interactions between the seven transmembrane domains, whereas the dominant-negative mutants apparently stabilize contacts between transmembrane domains and lock receptors in the off conformation. In support of these conclusions, certain combinations of constitutively-active and dominant-negative mutants restore nearly normal signaling properties.