CYTOPLASMIC TAIL PHOSPHORYLATION OF THE ALPHA-FACTOR RECEPTOR IS REQUIRED FOR ITS UBIQUITINATION AND INTERNALIZATION

G protein-coupled (GPC) receptors are phosphorylated in response to li gand binding, a modification that promotes receptor desensitization or down-regulation. The alpha-factor pheromone receptor (Ste2p) of Sacch aromyces 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 regulat ing ubiquitination and internalization. Serines and a lysine are essen tial elements of the Ste2p SINNDAKSS internalization signal that can m ediate both constitutive and ligand-stimulated endocytosis. The SINNDA KSS serines are required for receptor phosphorylation which, in turn, facilitates ubiquitination of the neighboring lysine. Constitutive pho sphorylation 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 hom ologues are unable to internalize alpha-factor, and do not phosphoryla te or ubiquitinate the receptor, indicating that these kinases play a direct or indirect role in phosphorylating the receptor. Finally, we p rovide evidence that the primary function of phosphorylation controlle d by the SINNDAKSS sequence is to trigger receptor internalization, de monstrating that phosphorylation-dependent endocytosis is an important mechanism for the downregulation of GPC receptor activity.