G protein–coupled (GPC) receptors are phosphorylated in response to ligand binding, a modification that promotes receptor desensitization or downregulation. The α-factor pheromone receptor (Ste2p) of Saccharomyces cerevisiae is a GPC receptor that is hyperphosphorylated and ubiquitinated upon binding α-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 α-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.
Cytoplasmic Tail Phosphorylation of the α-Factor Receptor Is Required for Its Ubiquitination and Internalization
H. Riezman was funded by the Swiss National Science Foundation and the Canton Basel Stadt. L. Hicke was funded by a New Investigator Award in the Basic Pharmacological Sciences from the Burroughs Wellcome Foundation, a Lurie Cancer Center American Cancer Society Institutional Research grant (IRG-93-037-04-IRG), and the National Institutes of Health (1 R01 DK53257-01).
Address all correspondence to Linda Hicke, Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, 2153 Sheridan Road, Evanston, IL 60208. Tel.: (847) 467-4490. Fax: (847) 467-1380. E-mail: [email protected]
Linda Hicke, Bettina Zanolari, Howard Riezman; Cytoplasmic Tail Phosphorylation of the α-Factor Receptor Is Required for Its Ubiquitination and Internalization . J Cell Biol 20 April 1998; 141 (2): 349–358. doi: https://doi.org/10.1083/jcb.141.2.349
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