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Genetics. 2016 May;203(1):299-317. doi: 10.1534/genetics.115.186122. Epub 2016 Feb 26.

Differential Phosphorylation Provides a Switch to Control How α-Arrestin Rod1 Down-regulates Mating Pheromone Response in Saccharomyces cerevisiae.

Author information

1
Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202.
2
Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202 jthorner@berkeley.edu.

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.

KEYWORDS:

adaptation; desensitization; down-regulation; endocytosis; mating pheromone response

PMID:
26920760
PMCID:
PMC4858781
DOI:
10.1534/genetics.115.186122
[Indexed for MEDLINE]
Free PMC Article

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