Hicke L, Zanolari B, Riezman H
Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208, USA.
J Cell Biol. 1998 Apr 20;141(2):349-58. doi: 10.1083/jcb.141.2.349.
G protein-coupled (GPC) receptors are phosphorylated in response to ligand binding, a modification that promotes receptor desensitization or downregulation. The alpha-factor pheromone receptor (Ste2p) of Saccharomyces cerevisiae is a GPC receptor that is hyperphosphorylated and ubiquitinated upon binding alpha-factor. Ubiquitination triggers Ste2p internalization into the endocytic pathway. Here we demonstrate that phosphorylation of Ste2p promotes downregulation by positively regulating ubiquitination and internalization. Serines and a lysine are essential elements of the Ste2p SINNDAKSS internalization signal that can mediate both constitutive and ligand-stimulated endocytosis. The SINNDAKSS serines are required for receptor phosphorylation which, in turn, facilitates ubiquitination of the neighboring lysine. Constitutive phosphorylation is required to promote constitutive internalization, and is also a prerequisite for ligand-induced phosphorylation at or near the SINNDAKSS sequence. Mutants defective in yeast casein kinase I homologues are unable to internalize alpha-factor, and do not phosphorylate or ubiquitinate the receptor, indicating that these kinases play a direct or indirect role in phosphorylating the receptor. Finally, we provide evidence that the primary function of phosphorylation controlled by the SINNDAKSS sequence is to trigger receptor internalization, demonstrating that phosphorylation-dependent endocytosis is an important mechanism for the downregulation of GPC receptor activity.
G蛋白偶联（GPC）受体在配体结合后会发生磷酸化，这种修饰可促进受体脱敏或下调。酿酒酵母的α因子信息素受体（Ste2p）是一种GPC受体，在结合α因子后会发生过度磷酸化和泛素化。泛素化会触发Ste2p内化进入内吞途径。在此我们证明，Ste2p的磷酸化通过正向调节泛素化和内化来促进下调。丝氨酸和一个赖氨酸是Ste2p的SINNDAKSS内化信号的关键元件，该信号可介导组成型和配体刺激的内吞作用。SINNDAKSS丝氨酸是受体磷酸化所必需的，而受体磷酸化反过来又促进邻近赖氨酸的泛素化。组成型磷酸化是促进组成型内化所必需的，也是在SINNDAKSS序列处或其附近进行配体诱导磷酸化的先决条件。酵母酪蛋白激酶I同源物缺陷的突变体无法内化α因子，也不会使受体磷酸化或泛素化，这表明这些激酶在受体磷酸化过程中起直接或间接作用。最后，我们提供证据表明，由SINNDAKSS序列控制的磷酸化的主要功能是触发受体内化，这表明磷酸化依赖性内吞作用是GPC受体活性下调的重要机制。
