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Smith et al., (2009). Dual positive and negative regulation of GPCR signaling by GTP hydrolysis.

October 2016, model of the month by Shukanto Das
Original model: BIOMD0000000439


Introduction

Transmembrane G protein-coupled receptors (GPCRs) are of central importance to multiple intracellular signalling cascades. Activated GPCRs enhance the binding of GTP nucleotides to receptor-coupled heterotrimeric G proteins, stimulating and causing the trimer to split into two signaling subunits Gα and Gβγ, each capable of relaying signals downstream [1]. Regulator of G protein signalling (RGS) proteins, on the other hand, promote the intrinsic GTPase activity of Gα subunits to reduce spontaneous activation and help restore desensitization. After transducing signals, the two G protein subunits associate and reassemble with GPCR and are ready to participate in the next cycle of signalling.

Pheromone-response pathway in yeast is a well-characterized GPCR pathway [2]. In Saccharomyces pombe, in response to mating factors (P-factor or M-factor) binding to pheromone GPCRs, the Gα subunits of Gpa1 (G proteins) propagate signals by activating Ras1 initiatied MAPK signalling and resulting in expression of transcription factor Ste11. RGS proteins, such as Rgs1, are conventionally regarded to be negative regulators of the process.

But does RGS play only a negative regulatory role on GPCR signalling? Smith et al., 2009 [3] describes another role of RGS protiens in G-protein signalling, which is described in this "Model of the Month" article.


Model

The model presented here by Smith et al (2009) [3, BIOMD0000000439] is developed on evidence-based assumption that one GαGTP activates only one effector molecule per round of Guanosine nucleotide exchange and hydrolysis. It proposes that for maximal response, rather than prolonged retention of a GTP-bound active state, hydrolysis of GαGTP occurs followed by its entry into an inactive (or inert) GTP-bound state. The model suggests that by formation of this inert complex, instead of having the same GαGTP molecules promote effector activation repeatedly, they are removed from pool of molecules available for signalling. However, RGS catalyzed hydrolysis of GTP to GDP can deem them fit to carry out another cycle of signalling. In vivo and in silico approaches to this dual negative and positive character of RGS, exhibits a non-monotone relationship between pathway output and quantity of RGS proteins.


Results

It is proposed that RGS aids signalling by promoting the conversion of GαGTP to GαGDP and thereby returning inactive Gα-subunits to ligand-bound receptors to enable their reactivation. Experimental analysis, including β-galactosidase assays as described previously by Didmon et al. (2002) [4], of pheromone pathway in Saccharomyces pombe led to the following findings: (1) On stimulation with high ligand concentration, GTPase enhancing activity of Rgs1 is required for maximum output. (2) GTP hydrolysis is a must for maximal signalling in Gpa1 mutants deficient in GTPase activity. (3) An increased intracellular Gpa1 concentration led to an enhanced effector output in presence or absence of Rgs1. This confirms that absence of RGS proteins decreases effector output. All in vivo experimental methodologies as described in [3].

Since with removal of RGS activity, rate of GTP hydrolysis appears to have slowed down, the paper suggests that after encountering an effector, Gα subunits must enter a GTP-bound post-signalling state (ĜαGTP). Here, RGS ensures that this bound-GTP is hydrolyzed, so that GαGTP can re-enter the pool of available signalling molecules. Keeping all this in mind, a new reaction scheme for GPCR signalling is proposed (See Figure 1).

Using this new reaction scheme and building on experimental data that strengthen this hypothesis, a model simulating the negative and positive role of RGS was constructed (See Figure 2).


Figure 2

Figure 2. Computational modeling simulates dual positive and negative effects of RGS activity on effector output. Initial reactant concentrations and reaction parameters for all steps (k1-k17) are provided by model developers. Ligand concentration was carried over 0-100μM and simulated from 0-16 hour induction in the presence (-) and absence (-) of functional RGS activity. Figure taken from [3, BIOMD0000000439].

Further, the relationship between effector output and RGS concentration is investigated. This is necessary because in a system, where quantity of GαGTP dictates the signalling strength, amount of the regulator RGS proteins present in the system also has a major impact on signalling sensitivity. Doubling or trebling the RGS concentrations in model simulations alters ligand sensitivity and reduces the predicted EC50 value for ligand-stimulated effector activation by 4-fold and 8-fold respectively. These results are consistent with a role for RGS proteins as negative regulators. Also, a 1.5-fold increase in maximal effector output at doubled RGS concentrations reaffirms its role as positive regulators. However, effector output is reduced to half at tripled concentrations of RGS. Model simulations are confirmed by in vivo experiments, suggesting that transition between positive and negative roles of RGS within a GPCR pathway is not only dependent on concentration of GαGTP activated by ligand-bound receptors, but also the concentration of RGS in the system (See Figure 3).

Figure 1

Figure 1. New reaction scheme suggested by model developers describing GPCR signalling regulation involving RGS activity at two separate stages (GαGTP and ĜαGTP). Ligand-receptor binding (LR) is followed by association of heterotrimeric G protein (LRGα(βγ)). This dissociates into GTP bound Gα subunits (GαGTP), free βγ-subunits (Gβγ) and ligand bound receptor. GαGTP can either hydrolyze to GDP-bound Gα subunit and iP, which can be accelerated by RGS (via formation of RGSGαGTP) or encounter an effector, forming Gα*GTP effector complex. After effector activation, Gα*GTP enters inert state ĜαGTP and is not able to stimulate any further effectors, prior to RGS assisted conversion to GαGDP + P. GαGDP bind to Gβγ for another cycle. Colour schemes for the boxes are as follows: red; state with inactive G proteins, green; activated pre-signalling state, cyan; inert post signalling state, orange; pathway regulators; purple; regulators bound to pre-signalling state and dark blue; regulators bound to post-signalling state. Figure taken from [3, BIOMD0000000439].

Figure 3

Figure 3. Experimental and computational investigations of relationship between output and concentration of RGS. The concentration of RGS in the model or in yeast was varied over the range 0–3 fold for varying concentrations (0–100 μM) of ligand (model) or pheromone (in vivo) following 16 h induction. (A) Simulations in presence of 1x(-), 2x(-) 3x(-) RGS or no RGS (-). Output from model shows accumulation of Gα*GTP effector complex over simulated time duration. (B) Output from in vivo β-galactosidase assay (C) Simulation of accumulation of Gα*GTP effector complexes over a range of RGS (0–300 nM) and ligand concentrations (0–100 μM). (D) Plotting the mean (±S.E.M) β-galactosidase expression levels against number of copies of the rgs1 gene expressed within yeast. (C) and (D) demonstrate that the relationship between response and concentration of RGS, both in the model and in vivo, is non-monotone. Figure taken from [3, BIOMD0000000439]

The paper also derives and offers a simplified network motif, which captures the fundamental structure of the more complex model of G protein GTPase cycle.


Conclusions

Model suggests that GTP-dependent switches, particularly in GPCR signalling cascade, can be more complex than simply G proteinGDP(inactive) and G proteinGTP(active). A new reaction scheme for GPCR signalling includes an additional GTP-bound inert state, which allows the cell to dynamically adjust and fine-tune the sensitivity and size of response to any stimulus with assistance of RGS. RGS proteins help achieve this by accelerating GTP hydrolysis and thereby increasing the supply of GαGDP to GPCRs for the next signalling cycle. Built on strong experimental evidences, this non-monotone, unimodal relationship between RGS concentration and maximum signal transmission by GPCRs provides new perspectives in the G protein cycle.

References

  1. Ladds et al. Functional analysis of heterologous GPCR signalling pathways in yeast. Trends Biotechnol. 2005 Jul;23(7):367-73.
  2. Johnston et al. Receptor-mediated activation of heterotrimeric G-proteins: current structural insights. Mol. Pharmacol. 2007 Aug;72(2):219-30.
  3. Smith et al. Dual positive and negative regulation of GPCR signaling by GTP hydrolysis. Cell. Signal. 2009 Jul;21(7):1151-60.
  4. Didmon et al. Identifying regulators of pheromone signalling in the fission yeast Schizosaccharomyces pombe. Curr. Genet. 2002 Jul;41(4):241-53.
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