Isolation and genetic analysis of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones.

Chan, Russell K.; Otte, Christian

doi:10.1128/mcb.2.1.11

Cited by 303 publications

(213 citation statements)

References 12 publications

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“…There are several pieces of evidence that argue that much of the control of this system occurs at the front end in the G protein cycle. First, mutations with the most dramatic pheromone supersensitive͞resistant phenotypes affect genes involved in the G protein dynamics (4,15,(19)(20)(21). Here, we demonstrated quantitatively that three of these mutations (sst2⌬, ste2 300⌬ , and bar1⌬) did indeed affect G protein activation͞ deactivation in a direct and substantial manner.…”

Section: Discussionmentioning

confidence: 70%

A quantitative characterization of the yeast heterotrimeric G protein cycle

Kitano

2003

Proc. Natl. Acad. Sci. U.S.A.

171

188

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The yeast mating response is one of the best understood heterotrimeric G protein signaling pathways. Yet, most descriptions of this system have been qualitative. We have quantitatively characterized the heterotrimeric G protein cycle in yeast based on direct in vivo measurements. We used fluorescence resonance energy transfer to monitor the association state of cyan fluorescent protein (CFP)-G␣ and G␤␥-yellow fluorescent protein (YFP), and we found that receptor-mediated G protein activation produced a loss of fluorescence resonance energy transfer. Quantitative time course and dose-response data were obtained for both wild-type and mutant cells possessing an altered pheromone response. These results paint a quantitative portrait of how regulators such as Sst2p and the C-terminal tail of ␣-factor receptor modulate the kinetics and sensitivity of G protein signaling. We have explored critical features of the dynamics including the rapid rise and subsequent decline of active G proteins during the early response, and the relationship between the G protein activation dose-response curve and the downstream dose-response curves for cell-cycle arrest and transcriptional induction. Fitting the data to a mathematical model produced estimates of the in vivo rates of heterotrimeric G protein activation and deactivation in yeast. Living organisms detect and respond to a variety of environmental cues through heterotrimeric G protein signal transduction pathways. Genetic or pharmacologic perturbation of these systems in humans can have significant medical consequences (1). Many pharmaceutical agents are directed against components of the G protein activation͞deactivation cycle (1). Achieving a quantitative understanding of this cycle and of the relationship between G protein activation and downstream physiology can aid in drug development.One of the best studied heterotrimeric G protein signaling systems is the yeast mating response in Saccharomyces cerevisiae (2, 3). Haploid yeast cells respond to a peptide pheromone from their partner by undergoing a series of events (e.g., cell-cycle arrest, synthesis of new proteins) in preparation for mating. In a cells, the pheromone ␣-factor, secreted by ␣ cells, activates the G protein coupled ␣-factor receptor (Ste2p). Most of the components of this pathway have been identified, including the receptors Ste2p and Ste3p (a-factor receptor), the G protein subunits Gpa1p (G␣), Ste4p (G␤), and Ste18p (G␥), and the G protein regulator (RGS) Sst2p.Yeast geneticists have isolated mutants possessing altered sensitivity to the pheromone ␣-factor (4). In particular, the following all confer an ␣-factor supersensitive phenotype: the deletion of BAR1 (bar1⌬), which encodes a secreted protease that degrades ␣-factor, the deletion of SST2 (sst2⌬), and the removal of the C-terminal tail of ␣-factor receptor STE2 (ste2 300⌬). It is important to provide a quantitative description of how these mutations affect the G protein cycle to complement the existing qualitative understanding.Until recently, the ...

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Section: Discussionmentioning

confidence: 70%

A quantitative characterization of the yeast heterotrimeric G protein cycle

Kitano

2003

Proc. Natl. Acad. Sci. U.S.A.

171

188

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“…RGS proteins share an RGS domain (Ϸ120 amino acids) capable of accelerating G␣-dependent GTP hydrolysis and the conversion of active GTP-bound G␣ to its inactive GDP-bound form (2-7). The first RGS identified was the yeast protein Sst2, isolated in a screen for mutants that failed to down-regulate the GPCR-mediated response to mating pheromone (8)(9). Over 20 mammalian RGS proteins have been identified (7,10).…”

mentioning

confidence: 99%

Regulation of T cell activation, anxiety, and male aggression by RGS2

Oliveira-dos-Santos

Matsumoto

Snow

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

261

201

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Regulators of G protein signaling (RGS) proteins accelerate theGTPase activity of G␣ protein subunits in vitro, negatively regulating G protein-coupled receptor signaling. The physiological role of mammalian RGS proteins is largely unknown. The RGS family member rgs2 was cloned as an immediate early response gene up-regulated in T lymphocytes after activation. To investigate the role of RGS2 in vivo, we generated rgs2-deficient mice. We show that targeted mutation of rgs2 in mice leads to reduced T cell proliferation and IL-2 production, which translates in an impaired antiviral immunity in vivo. Interestingly, rgs2 ؊/؊ mice also display increased anxiety responses and decreased male aggression in the absence of cognitive or motor deficits. RGS2 also controls synaptic development and basal electrical activity in hippocampal CA1 neurons. Thus, RGS2 plays an important role in T cell activation, synapse development in the hippocampus, and emotive behaviors.

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“…We have shown previously that Sst2 interacts genetically (10, 11) and physically (12) with Gpa1 and can accelerate its GTPase activity more than 40-fold (13). Mutants lacking SST2 are ϳ100-fold more sensitive to pheromone (12,14,15). This pattern of induction and regulation of pheromone signaling suggests that Sst2 is part of a feedback inhibition loop that promotes desensitization.…”

mentioning

confidence: 93%

Regulators of G Protein Signaling and Transient Activation of Signaling

Hao

Yıldırım

Wang

et al. 2003

Journal of Biological Chemistry

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Cellular responses to hormones and neurotransmitters are necessarily transient. The mating pheromone signal in yeast is typical. Signal initiation requires cell surface receptors, a G protein heterotrimer, and downstream effectors. Signal inactivation requires Sst2, a regulator of G protein signaling (RGS) protein that accelerates GTPase activity. We conducted a quantitative analysis of RGS and G protein expression and devised computational models that describe their activity in vivo. These results indicated that pheromone-dependent transcriptional induction of the RGS protein constitutes a negative feedback loop that leads to desensitization. Modeling also suggested the presence of a positive feedback loop leading to resensitization of the pathway. In confirmation of the model, we found that the RGS protein is ubiquitinated and degraded in response to pheromone stimulation. We identified and quantitated these positive and negative feedback loops, which account for the transient response to external signals observed in vivo.One measure of our understanding of biological systems is our ability to predict their behavior in detail. One aspect of this endeavor is to model signal transduction events, defined here as the dynamic changes that occur within a cell in response to an external stimulus (1-3). Such models can help us to understand how small changes outside a cell produce strongly amplified changes within a cell, how graded signals are converted to all-or-none responses (4), or how activators of one pathway influence the function of a second pathway (5). A second goal, and the focus of this work, is to understand how transient external signals are prevented from being propagated indefinitely within the cell. Here we describe the molecular basis for signal activation, desensitization, and eventual resensitization of G proteins by receptors and RGS 1 proteins. The experimentally observed behavior is described mechanistically by computational modeling of the pathway.For these studies we investigated the mating pheromone signaling pathway in yeast Saccharomyces cerevisiae. The yeast mating response is arguably the best characterized signal transduction pathway of any eukaryote, and it has long served as a prototype for hormone, neurotransmitter, and sensory response systems in humans (6). Disruption or activation of pathway components leads to highly specific changes that can be easily quantified. Finally, because it is a unicellular eukaryote, every cell in a population is genetically and phenotypically identical (all cells are "typical").Mating in yeast is the fusion of a and ␣ haploid cell types to form an a/␣ diploid. The events leading to fusion are initiated by specific pheromones: ␣-type cells secrete ␣-factor pheromone, which binds to a specific receptor (Ste2) on a-cells, while a-cells secrete a-factor that binds to receptors (Ste3) on ␣-cells. Upon pheromone binding to its receptor, the G protein ␣ subunit (Gpa1) releases GDP, binds to GTP, and liberates the G protein ␤␥ subunits (Ste4/Ste18). Sustained...

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Isolation and genetic analysis of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones.

Cited by 303 publications

References 12 publications

A quantitative characterization of the yeast heterotrimeric G protein cycle

A quantitative characterization of the yeast heterotrimeric G protein cycle

Regulation of T cell activation, anxiety, and male aggression by RGS2

Regulators of G Protein Signaling and Transient Activation of Signaling

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