Requirements for recruitment of a G protein-coupled receptor to clathrin-coated pits in budding yeast

Junko Y. Toshima, Jun Ichi Nakanishi, Kensaku Mizuno, Jiro Toshima, David G. Drubin

    Research output: Contribution to journalArticle

    28 Citations (Scopus)

    Abstract

    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 spatiotemporal features of GPCR endocytosis in budding yeast. Here, we identify recruitment of Ste2p to preexisting 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. In addition, 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.

    Original languageEnglish
    Pages (from-to)5039-5050
    Number of pages12
    JournalMolecular Biology of the Cell
    Volume20
    Issue number24
    DOIs
    Publication statusPublished - 2009

    Fingerprint

    Clathrin
    Saccharomycetales
    G-Protein-Coupled Receptors
    Ligands
    Ubiquitination
    Endocytosis
    Serine
    Pheromone Receptors
    Yeasts
    Casein Kinase I
    Vesicular Transport Adaptor Proteins
    Pheromones
    Alanine
    Signal Transduction
    Down-Regulation
    Phosphorylation
    Mutation

    ASJC Scopus subject areas

    • Molecular Biology
    • Cell Biology

    Cite this

    Requirements for recruitment of a G protein-coupled receptor to clathrin-coated pits in budding yeast. / Toshima, Junko Y.; Nakanishi, Jun Ichi; Mizuno, Kensaku; Toshima, Jiro; Drubin, David G.

    In: Molecular Biology of the Cell, Vol. 20, No. 24, 2009, p. 5039-5050.

    Research output: Contribution to journalArticle

    Toshima, Junko Y. ; Nakanishi, Jun Ichi ; Mizuno, Kensaku ; Toshima, Jiro ; Drubin, David G. / Requirements for recruitment of a G protein-coupled receptor to clathrin-coated pits in budding yeast. In: Molecular Biology of the Cell. 2009 ; Vol. 20, No. 24. pp. 5039-5050.
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