Specific α-Arrestins Negatively Regulate Saccharomyces cerevisiae Pheromone Response by Down-Modulating the G-Protein-Coupled Receptor Ste2

Abstract

e G-protein-coupled receptors (GPCRs) are integral membrane proteins that initiate responses to extracellular stimuli by mediating ligand-dependent activation of cognate heterotrimeric G proteins. In yeast, occupancy of GPCR Ste2 by peptide pheromone ␣-factor initiates signaling by releasing a stimulatory G␤␥ complex (Ste4-Ste18) from its inhibitory G␣ subunit (Gpa1). Prolonged pathway stimulation is detrimental, and feedback mechanisms have evolved that act at the receptor level to limit the duration of signaling and stimulate recovery from pheromone-induced G 1 arrest, including upregulation of the expression of an ␣-factor-degrading protease (Bar1), a regulator of G-protein signaling protein (Sst2) that stimulates Gpa1-GTP hydrolysis, and Gpa1 itself. Ste2 is also downregulated by endocytosis, both constitutive and ligand induced. Ste2 internalization requires its phosphorylation and subsequent ubiquitinylation by membrane-localized protein kinases (Yck1 and Yck2) and a ubiquitin ligase (Rsp5). Here, we demonstrate that three different members of the ␣-arrestin family (Ldb19/Art1, Rod1/Art4, and Rog3/ Art7) contribute to Ste2 desensitization and internalization, and they do so by discrete mechanisms. We provide genetic and biochemical evidence that Ldb19 and Rod1 recruit Rsp5 to Ste2 via PPXY motifs in their C-terminal regions; in contrast, the arrestin fold domain at the N terminus of Rog3 is sufficient to promote adaptation. Finally, we show that Rod1 function requires calcineurin-dependent dephosphorylation.

Substrate-Induced Ubiquitylation and Endocytosis of Yeast Amino Acid Permeases

“…In the yeast Saccharomyces cerevisiae, ubiquitylation of cell surface proteins is mediated by the HECT family Rsp5 Ub ligase. More recent analysis of several yeast plasma membrane permeases and receptors has shown that arrestin-like adaptors named Art (for arrestin-related trafficking) are essential to recruiting Rsp5 to these proteins (3)(4)(5).…”

Quantification of mutation-derived bias for alternate mating functionalities of theSaccharomyces cerevisiaeSte2p pheromone receptor

“…Will yeast arrestins be linked to alternate Ste2p receptor functionalites, as is the case in mammalian systems? Although this remains to be tested, a recent report has highlighted roles for arrestin-like proteins in Ste2p desensitization (17), setting the stage for possible roles for arrestins in mediating Ste2p-linked signalling pathways downstream of classical MAPK signalling. Overall, possible continued relevance of the yeast pheromone-mating pathway as a simplified model for GPCR research, in the broader context of alternate functionalities for GPCRs, is supported based on validation of alternate functionalities for Ste2p in modulation of mating events.…”

“…However, now, the possibility of biased signalling for the yeast pheromone receptors cannot be ignored. This is particularly true in light of the recent demonstration of a direct interaction between a-arrestins and Ste2p in internalization events (17).…”

Evidence of a role for S. cerevisiae α‐arrestin Art1 (Ldb19) in mating projection and zygote formations

“…Most recently, this was demonstrated for Ste2p, with Art1 (Ldb19), Art4 (Rod1) and Art7 (Rog3) all being implicated in modulation of receptor internalization (Alvaro et al, 2014).…”

“…While direct evidence is still lacking due to difficulties addressing the redundancy associated with a single receptor mediating two temporally distinct steps in the same process, the possibility of alternate GPCR-linked signaling in yeast can no longer be ignored. This is particularly true in light of the recent demonstration of a direct interaction between yeast arrestin-like proteins and Ste2p in internalization events (Alvaro et al, 2014). A family of 10 arrestin-like proteins, termed -arrestins is known to be expressed in S. cerevisiae.…”