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

Abstract

Although well documented for mammalian G-proteincoupled receptors, alternate functionalities and associated alternate signalling remain to be unequivocally established for the Saccharomyces cerevisiae pheromone Ste2p receptor. Here, evidence supporting alternate functionalities for Ste2p is re-evaluated, extended and quantified. In particular, strong mating and constitutive signalling mutations, focusing on residues S254, P258 and S259 in TM6 of Ste2p, are stacked and investigated in terms of their effects on classical G-protein-mediated signal transduction associated with cell cycle arrest, and alternatively, their impact on downstream mating projection and zygote formation events. In relative dose response experiments, accounting for systemic and observational bias, mutational-derived functional differences were observed, validating the S254L-derived bias for downstream mating responses and highlighting complex relationships between TM6-mutation derived constitutive signalling and ligandinduced functionalities. Mechanistically, localization studies suggest that alterations to receptor trafficking may contribute to mutational bias, in addition to expected receptor conformational stabilization effects. Overall, these results extend previous observations and quantify the contributions of Ste2p variants to mediating cell cycle arrest versus downstream mating functionalities.

Regulation of G protein‐coupled receptor signaling by plasma membrane organization and endocytosis

“…Homodimerization is evolutionarily conserved. The yeast α‐factor receptor (Ste2p) shows a significant tendency to dimerize, and dimerization of functional receptors might be required for receptor signaling . Dimeric receptor complexes can even couple to a single G protein or arrestin molecule, as demonstrated for the light‐activated GPCR rhodopsin .…”

“…The yeast α-factor receptor (Ste2p) shows a significant tendency to dimerize, 28,29 and dimerization of functional receptors might be required for receptor signaling. 30 Dimeric receptor complexes can even couple to a single G protein or arrestin molecule, as demonstrated for the light-activated GPCR rhodopsin. 31,32 Heteromers such as the μ-/κ-opioid receptor (μOR, κOR) dimer might couple to different effectors and induce functional effects distinct from their monomers.…”

Ste2 receptor-mediated chemotropism of Fusarium graminearum contributes to its pathogenicity against wheat

“…This phenomenon has been termed 'biased GPCR signaling' (reviewed extensively [9][10][11] ). While such research has largely been limited to mammalian systems, biased GPCR signaling has been observed in other phyla with the fungal pheromone receptor, Ste2p, in the model organism Saccharomyces cerevisiae serving as canonical example 12 .…”

“…However, multiple studies have suggested the existence of alternate functionalities for S. cerevisiae Ste2p (ScSte2p), specifically in the mating events that occur downstream of cell cycle arrest, which are influenced by factors such as pheromone gradients [17][18][19][20] as well as localization of the ScSte2p receptor to the mating projection 21 . Furthermore, specific mutations in ligand-interacting residues of ScSte2p resulted in different effects on G-protein-mediated MAPK signaling and diploid zygote formation 12,22,23 . In contrast, much less is known about the counterparts of ScSte2p in multicellular fungi where the mating type of an organism is governed by more complex mechanisms, or where mating is not relevant to the fungal life cycle.…”

A peroxidase-derived ligand that induces Fusarium graminearum Ste2 receptor-dependent chemotropism

“…Sc Ste2p has also been reported to exhibit alternate functionalities, dictated by factors such as pheromone gradients ( Segall, 1993 ; Brizzio et al., 1996 ; Elia, 1996 ; Barkai et al., 1998 ) and localization of the Sc Ste2p receptor to the mating projection ( Jackson et al., 1991 ). Mutations in the ligand-binding residues of the receptor alter G protein-mediated MAPK signaling and lead to different impacts on diploid zygote formation ( Dube and Konopka, 1998 ; Shi et al., 2007 ; Choudhary and Loewen, 2016a ).…”