title: Differential Phosphorylation Provides a Switch to Control How α-Arrestin Rod1 Down-regulates Mating Pheromone Response in Saccharomyces cerevisiae.
abstract: G-protein-coupled receptors (GPCRs) are integral membrane proteins that initiate stimulus-dependent activation of cognate heterotrimeric G-proteins, triggering ensuing downstream cellular responses. Tight regulation of GPCR-evoked pathways is required because prolonged stimulation can be detrimental to an organism. Ste2, a GPCR in Saccharomyces cerevisiae that mediates response of MATa haploids to the peptide mating pheromone α-factor, is down-regulated by both constitutive and agonist-induced endocytosis. Efficient agonist-stimulated internalization of Ste2 requires its association with an adaptor protein, the α-arrestin Rod1/Art4, which recruits the HECT-domain ubiquitin ligase Rsp5, allowing for ubiquitinylation of the C-terminal tail of the receptor and its engagement by the clathrin-dependent endocytic machinery. We previously showed that dephosphorylation of Rod1 by calcineurin (phosphoprotein phosphatase 2B) is required for optimal Rod1 function in Ste2 down-regulation. We show here that negative regulation of Rod1 by phosphorylation is mediated by two distinct stress-activated protein kinases, Snf1/AMPK and Ypk1/SGK1, and demonstrate both in vitro and in vivo that this phospho-regulation impedes the ability of Rod1 to promote mating pathway desensitization. These studies also revealed that, in the absence of its phosphorylation, Rod1 can promote adaptation independently of Rsp5-mediated receptor ubiquitinylation, consistent with recent evidence that α-arrestins can contribute to cargo recognition by both clathrin-dependent and clathrin-independent mechanisms. However, in cells lacking a component (formin Bni1) required for clathrin-independent entry, Rod1 derivatives that are largely unphosphorylated and unable to associate with Rsp5 still promote efficient adaptation, indicating a third mechanism by which this α-arrestin promotes desensitization of the pheromone-response pathway.
authors.affiliation: Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202.
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Alvaro
authors.affiliation: Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202.
authors.forename: Ann
authors.initials: A
authors.lastname: Aindow
authors.affiliation: Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202 jthorner@berkeley.edu.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Thorner
mesh_terms: D000220:Adaptation, Biological; D019703:Calcineurin / Q000378:metabolism; D015536:Down-Regulation; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051964:Receptors, Mating Factor / Q000737:chemistry / Q000378:metabolism; D012100:Reproduction, Asexual; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D008565:Membrane Proteins; C099318:ROD1 protein, S cerevisiae; D051964:Receptors, Mating Factor; C498031:STE2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes; C071570:SNF1-related protein kinases; D017346:Protein Serine-Threonine Kinases; D038362:Glycogen Synthase Kinase 3; C068124:MCK1 protein, S cerevisiae; D019703:Calcineurin; C469287:RSP5 protein, S cerevisiae
keywords: adaptation; desensitization; down-regulation; endocytosis; mating pheromone response