Regulators of G Protein Signaling and Transient Activation of Signaling

Abstract

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...

Computational modeling reveals how interplay between components of a GTPase-cycle module regulates signal transduction

“…The model is distinct from previous models (27)(28)(29)(30)(31)(32)(33) in that (i) it includes GAP, which many previous models have excluded; (ii) it is based on a general mechanism that allows several modes of coupling of G proteins with active receptors and GAPs, including a ternary complex of receptor, G protein, and GAP; and (iii) it is based on data pertaining to a single mammalian GTPase cycle module; i.e., the m1 muscarinic acetylcholine receptor (m1 MAchR), Gq, and RGS4 (34)(35)(36)(37). The predictions of the model elucidate how changes in concentrations of molecules can result in a variety of G protein signaling properties, both in terms of dynamics as well as steady-state behavior, and how these properties are governed by the kinetics of the GTPase-cycle module.…”

Pheromone- and RSP5-dependent Ubiquitination of the G Protein β Subunit Ste4 in Yeast

“…Earlier studies indicate that Ste4 represents a crucial component in the pathway, and its activity must be tightly regulated (30,40). Growing evidence suggests that ubiquitination is involved in the regulation of G protein signaling (17,18).…”

“…Thus, although Ste4 is a critical determinant of the intensity and duration of downstream MAP kinase signaling (30,40), ubiquitination does not appear to alter the abundance of Ste4 and thereby the intensity of downstream MAP kinase signaling. Rather, ubiquitination of Ste4 appears to have a very specific role in regulating the timing of polarized cell growth in response to pheromone stimulation.…”

“…Specificity of detection was established using fus3⌬ and kss1⌬ cell extracts as negative controls. The pheromone-dependent transcription assay using the green fluorescent protein reporter (FUS1-GFP) has been described previously (30,37). To monitor the loss of Ste4 over time, early log cell cultures were treated with 3 M ␣-factor for 60 min, followed by cycloheximide (10 g/ml in 0.1% ethanol, final concentrations) for up to 2 h. Growth was stopped by the addition of 10 mM NaN 3 and transfer to an ice bath.…”

“…Estimates from a large scale quantitative immunoblotting study indicated that the number of Ste4 molecules (ϳ2050/cell) is much lower than either G␣ Gpa1 (ϳ9920/cell) or G␥ Ste18 (ϳ5550/cell) (29). Moreover, as little as 2-fold overexpression of Ste4 (but not Gpa1 and Ste18) is sufficient to yield full activation of the pathway (30). Given the limiting abundance of Ste4 and its crucial roles in pheromone signaling, it is likely that a battery of mechanisms may exist to regulate its activity to ensure accurate cellular responses to pheromone treatment.…”