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Reference: Toshima JY, et al. (2009) Requirements for recruitment of a G protein-coupled receptor to clathrin-coated pits in budding yeast. Mol Biol Cell 20(24):5039-50

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Abstract


Monitoring Editor: Howard Riezman Endocytic internalization of G protein-coupled receptors (GPCRs) plays a critical role in down-regulation of GPCR signaling. The yeast mating pheromone receptor, Ste2p, has been used as a model to investigate mechanisms of signal transduction, modification, and endocytic internalization of GPCRs. We previously used a fluorescently labeled mating pheromone derivative to reveal unappreciated molecular and spatio-temporal features of GPCR endocytosis in budding yeast. Here we identify recruitment of Ste2p to pre-existing clathrin-coated pits (CCPs) as a key step regulated by receptor phosphorylation and subsequent ubiquitination upon ligand binding. The yeast casein kinase I homologue, Yck2p, directly phosphorylates six serine residues located in the C-terminal tail of Ste2p, and mutation of these serine residues to alanine significantly decreased recruitment of Ste2p to CCPs. We also found that the clathrin adaptors Ent1p, Ent2p and Ede1p work cooperatively to recruit ubiquitinated Ste2p to CCPs. Additionally, ubiquitination has a role in ligand-independent constitutive recruitment of Ste2p to CCPs, although this process is much slower than ligand-induced recruitment. These results suggest that ubiquitination of Ste2p is indispensable for recruiting Ste2p to CCPs in both ligand-dependent and ligand-independent endocytosis.

Reference Type
Journal Article
Authors
Toshima JY, Nakanishi JI, Mizuno K, Toshima J, Drubin DG
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