Comparison of Experimental Approaches Used to Determine the Structure and Function of the Class D G Protein-Coupled Yeast α-Factor Receptor.

The Saccharomyces cerevisiae α-factor mating pheromone receptor (Ste2p) has been studied as a model for the large medically important family of G protein-coupled receptors. Diverse yeast genetic screens and high-throughput mutagenesis of STE2 identified a large number of loss-of-function, constitutively-active, dominant-negative, and intragenic second-site suppressor mutants as well as mutations that specifically affect pheromone binding. Facile genetic manipulation of Ste2p also aided in targeted biochemical approaches, such as probing the aqueous accessibility of substituted cysteine residues in order to identify the boundaries of the seven transmembrane segments, and the use of cysteine disulfide crosslinking to identify sites of intramolecular contacts in the transmembrane helix bundle of Ste2p and sites of contacts between the monomers in a Ste2p dimer. Recent publication of a series of high-resolution cryo-EM structures of Ste2p in ligand-free, agonist-bound and antagonist-bound states now makes it possible to evaluate the results of these genetic and biochemical strategies, in comparison to three-dimensional structures showing activation-related conformational changes. The results indicate that the genetic and biochemical strategies were generally effective, and provide guidance as to how best to apply these experimental strategies to other proteins. These strategies continue to be useful in defining mechanisms of signal transduction in the context of the available structures and suggest aspects of receptor function beyond what can be discerned from the available structures.

Activation mechanism of the class D fungal GPCR dimer Ste2.

The fungal class D1 G-protein-coupled receptor (GPCR) Ste2 has a different arrangement of transmembrane helices compared with mammalian GPCRs and a distinct mode of coupling to the heterotrimeric G protein Gpa1-Ste2-Ste181. In addition, Ste2 lacks conserved sequence motifs such as DRY, PIF and NPXXY, which are associated with the activation of class A GPCRs2. This suggested that the activation mechanism of Ste2 may also differ. Here we determined structures of Saccharomyces cerevisiae Ste2 in the absence of G protein in two different conformations bound to the native agonist α-factor, bound to an antagonist and without ligand. These structures revealed that Ste2 is indeed activated differently from other GPCRs. In the inactive state, the cytoplasmic end of transmembrane helix H7 is unstructured and packs between helices H1-H6, blocking the G protein coupling site. Agonist binding results in the outward movement of the extracellular ends of H6 and H7 by 6 Å. On the intracellular surface, the G protein coupling site is formed by a 20 Å outward movement of the unstructured region in H7 that unblocks the site, and a 12 Å inward movement of H6. This is a distinct mechanism in GPCRs, in which the movement of H6 and H7 upon agonist binding facilitates G protein coupling.

Oligomerization of yeast α-factor receptor detected by fluorescent energy transfer between ligands.

G-protein-coupled receptors (GPCRs) comprise a large superfamily of transmembrane receptors responsible for transducing responses to the binding of a wide variety of hormones, neurotransmitters, ions, and other small molecules. There is extensive evidence that GPCRs exist as homo-and hetero-oligomeric complexes; however, in many cases, the role of oligomerization and the extent to which it occurs at low physiological levels of receptor expression in cells remain unclear. We report here the use of flow cytometry to detect receptor-receptor interactions based on fluorescence resonance energy transfer between fluorescently labeled cell-impermeant ligands bound to yeast α-mating pheromone receptors that are members of the GPCR superfamily. A novel, to our knowledge, procedure was used to analyze energy transfer as a function of receptor occupancy by donor and acceptor ligands. Measurements of loss of donor fluorescence due to energy transfer in cells expressing high levels of receptors were used to calibrate measurements of enhanced acceptor emission due to energy transfer in cells expressing low levels of receptors. The procedure allows determination of energy transfer efficiencies over a 50-fold range of expression of full-length receptors at the surface of living cells without the need to create fluorescent or bioluminescent fusion proteins. Energy transfer efficiencies for fluorescently labeled derivatives of the receptor agonist α-factor do not depend on receptor expression level and are unaffected by C-terminal truncation of receptors. Fluorescently labeled derivatives of α-factor that act as receptor antagonists exhibit higher transfer efficiencies than those for labeled agonists. Although the approach cannot determine the number of receptors per oligomer, these results demonstrate that ligand-bound, native α-factor receptors exist as stable oligomers in the cell membranes of intact yeast cells at normal physiological expression levels and that the extent of oligomer formation is not dependent on the concentration of receptors in the membrane.

Ste2p Under the Microscope: the Investigation of Oligomeric States of a Yeast G Protein-Coupled Receptor.

Oligomerization of G protein-coupled receptors (GPCRs) may play important roles in maturation, internalization, signaling, and pharmacology of these receptors. However, the nature and extent of their oligomerization is still under debate. In our study, Ste2p, a yeast mating pheromone GPCR, was tagged with enhanced green fluorescent protein (EGFP), mCherry, and with split florescent protein fragments at the receptor C-terminus. The Förster resonance energy transfer (FRET) technique was used to detect receptors' oligomerization by calculating the energy transfer from EGFP to mCherry. Stimulation of Ste2p oligomers with the receptor ligand did not result in any significant change on observed FRET values. The bimolecular fluorescence complementation (BiFC) assay was combined with FRET to further investigate the tetrameric complexes of Ste2p. Our results suggest that in its quiescent (nonligand-activated) state, Ste2p is found at least as a tetrameric complex on the plasma membrane. Intriguingly, receptor tetramers in their active form showed a significant increase in FRET. This study provides a direct in vivo visualization of Ste2p tetramers and the pheromone effect on the extent of the receptor oligomerization.

Dielectric Spectroscopy Based Detection of Specific and Nonspecific Cellular Mechanisms.

Using radiofrequency dielectric spectroscopy, we have investigated the impact of the interaction between a G protein-coupled receptor (GPCR), the sterile2 α-factor receptor protein (Ste2), and its cognate agonist ligand, the α-factor pheromone, on the dielectric properties of the plasma membrane in living yeast cells (Saccharomyces cerevisiae). The dielectric properties of a cell suspension containing a saturating concentration of α-factor were measured over the frequency range 40Hz-110 MHz and compared to the behavior of a similarly prepared suspension of cells in the absence of α-factor. A spherical three-shell model was used to determine the electrical phase parameters for the yeast cells in both types of suspensions. The relative permittivity of the plasma membrane showed a significant increase after exposure to α-factor (by 0.06 ± 0.05). The equivalent experiment performed on yeast cells lacking the ability to express Ste2 showed no change in plasma membrane permittivity. Interestingly, a large change also occurred to the electrical properties of the cellular interior after the addition of α-factor to the cell suspending medium, whether or not the cells were expressing Ste2. We present a number of different complementary experiments performed on the yeast to support these dielectric data and interpret the results in terms of specific cellular reactions to the presence of α-factor.

Structure of the class D GPCR Ste2 dimer coupled to two G proteins.

G-protein-coupled receptors (GPCRs) are divided phylogenetically into six classes1,2, denoted A to F. More than 370 structures of vertebrate GPCRs (belonging to classes A, B, C and F) have been determined, leading to a substantial understanding of their function3. By contrast, there are no structures of class D GPCRs, which are found exclusively in fungi where they regulate survival and reproduction. Here we determine the structure of a class D GPCR, the Saccharomyces cerevisiae pheromone receptor Ste2, in an active state coupled to the heterotrimeric G protein Gpa1-Ste4-Ste18. Ste2 was purified as a homodimer coupled to two G proteins. The dimer interface of Ste2 is formed by the N terminus, the transmembrane helices H1, H2 and H7, and the first extracellular loop ECL1. We establish a class D1 generic residue numbering system (CD1) to enable comparisons with orthologues and with other GPCR classes. The structure of Ste2 bears similarities in overall topology to class A GPCRs, but the transmembrane helix H4 is shifted by more than 20 Å and the G-protein-binding site is a shallow groove rather than a cleft. The structure provides a template for the design of novel drugs to target fungal GPCRs, which could be used to treat numerous intractable fungal diseases4.

In-Cell Detection of Conformational Substates of a G Protein-Coupled Receptor Quaternary Structure: Modulation of Substate Probability by Cognate Ligand Binding.

While the notion that G protein-coupled receptors (GPCRs) associate into homo- and hetero-oligomers has gained more recognition in recent years, a lack of consensus remains among researchers regarding the functional relevance of GPCR oligomerization. A technique, Förster resonance energy transfer (FRET) spectrometry, allows for the determination of the oligomeric (or quaternary) structure of proteins in living cells via analysis of efficiency distributions of energy transferred from optically excited fluorescent tags acting as donors of energy to fluorescent tags acting as acceptors of energy and residing within the same oligomer. In this study, we significantly improved the resolution of FRET spectrometry to detect subtle differences in quaternary structures of GPCR oligomers within living cells. We then used this approach to study the conformational substates of oligomers of the sterile 2 α-factor receptor (Ste2), a class D GPCR found in the yeast Saccharomyces cerevisiae of mating type a. Ste2 has previously been shown to form tetramers at relatively low expression levels (11 to 140 molecules/µm2) in the absence of its cognate ligand, the α-factor pheromone. The significantly improved FRET spectrometry technique allowed us to detect multiple distinct quaternary conformational substates of Ste2 oligomers, and to assess how the α-factor ligand altered the proportion of such substates. The ability to determine quaternary structure substates of GPCRs provides exquisite means to elucidate functional relevance of GPCR oligomerization.

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

Fusarium Head Blight of wheat, caused by the filamentous fungus Fusarium graminearum, leads to devastating global food shortages and economic losses. While many studies have addressed the responses of both wheat and F. graminearum during their interaction, the possibility of fungal chemotropic sensing enabling pathogenicity remains unexplored. Based on recent findings linking the pheromone-sensing G-protein-coupled receptor Ste2 to host-directed chemotropism in Fusarium oxysporum, we investigated the role of the Ste2 receptor and its downstream signaling pathways in mediating chemotropism of F. graminearum. Interestingly, a chemotropic response of growing hyphae towards catalytically active Triticum aestivum 'Roblin' cultivar secreted peroxidases was detected, with deletion of STE2 in F. graminearum leading to loss of the observed response. At the same time, deletion of STE2 significantly decreased infection on germinating wheat coleoptiles, highlighting an association between Ste2, chemotropism and infection by F. graminearum. Further characterization revealed that the peroxidase-directed chemotropism is associated with stimulation of the fungal cell wall integrity mitogen-activated protein kinase signaling cascade. Altogether, this study demonstrates conservation of Ste2-mediated chemotropism by Fusarium species, and its important role in mediating pathogenicity.

CRISPR-addressable yeast strains with applications in human G protein-coupled receptor profiling and synthetic biology.

Genome stability is essential for engineering cell-based devices and reporter systems. With the advent of CRISPR technology, it is now possible to build such systems by installing the necessary genetic parts directly into an organism's genome. Here, we used this approach to build a set of 10 versatile yeast-based reporter strains for studying human G protein-coupled receptors (GPCRs), the largest class of membrane receptors in humans. These reporter strains contain the necessary genetically encoded parts for studying human GPCR signaling in yeast, as well as four CRISPR-addressable expression cassettes, i.e. landing pads, installed at known safe-harbor sites in the yeast genome. We showcase the utility of these strains in two applications. First, we demonstrate that increasing GPCR expression by incrementally increasing GPCR gene copy number potentiates Gα coupling of the pharmacologically dark receptor GPR68. Second, we used two CRISPR-addressable landing pads for autocrine activation of a GPCR (the somatostatin receptor SSTR5) with its peptide agonist SRIF-14. The utility of these reporter strains can be extended far beyond these select examples to include applications such as nanobody development, mutational analysis, drug discovery, and studies of GPCR chaperoning. Additionally, we present a BY4741 yeast strain created for broad applications in the yeast and synthetic biology communities that contains only the four CRISPR-addressable landing pads. The general utility of these yeast strains provides an inexpensive, scalable, and easy means of installing and expressing genes directly from the yeast genome to build genome-barcoded sensors, reporter systems, and cell-based factories.

Ratiometric GPCR signaling enables directional sensing in yeast.

Accurate detection of extracellular chemical gradients is essential for many cellular behaviors. Gradient sensing is challenging for small cells, which can experience little difference in ligand concentrations on the up-gradient and down-gradient sides of the cell. Nevertheless, the tiny cells of the yeast Saccharomyces cerevisiae reliably decode gradients of extracellular pheromones to find their mates. By imaging the behavior of polarity factors and pheromone receptors, we quantified the accuracy of initial polarization during mating encounters. We found that cells bias the orientation of initial polarity up-gradient, even though they have unevenly distributed receptors. Uneven receptor density means that the gradient of ligand-bound receptors does not accurately reflect the external pheromone gradient. Nevertheless, yeast cells appear to avoid being misled by responding to the fraction of occupied receptors rather than simply the concentration of ligand-bound receptors. Such ratiometric sensing also serves to amplify the gradient of active G protein. However, this process is quite error-prone, and initial errors are corrected during a subsequent indecisive phase in which polarity clusters exhibit erratic mobile behavior.

Development of a Highly Active Fluorescence-Based Detector for Yeast G Protein-Coupled Receptor Ste2p.

Twenty analogs of [Orn6,D-Ala9]α-factor were synthesized and assayed for their biological activities: seven analogs of [Orn6,X9]α-factor, seven analogs of [X6,D-Ala9]α-factor, five analogs of [X5,X6,D-Ala9]α-factor, and native α-factor (X = amino acids). Their biological activities (halo, gene induction, and affinity) were measured using S. cerevisiae Y7925 and LM102 and compared with those of native α-factor (100%). G protein-coupled receptor was expressed in strain LM102 containing pESC-LEU-STE2 vector. [Dap6,D-Ala9]α-factor with weak halo activity (10%) showed the highest receptor affinity (＞ 230%) and the highest gene induction activity (167%). [Arg6,D-Ala9]α-factor showed the highest halo activity (2,000%). The number of active binding sites per cell (about 20,000 for strain LM102) was determined using a newly-designed fluorescence-based detector, [Arg6,D-Ala9]α-factor-Edan, with high sensitivity (12,500-fold higher than the absorption-based detector [Orn6]α-factor-[Cys]3).

Tracking yeast pheromone receptor Ste2 endocytosis using fluorogen-activating protein tagging.

To observe internalization of the yeast pheromone receptor Ste2 by fluorescence microscopy in live cells in real time, we visualized only those molecules present at the cell surface at the time of agonist engagement (rather than the total cellular pool) by tagging this receptor at its N-terminus with an exocellular fluorogen-activating protein (FAP). A FAP is a single-chain antibody engineered to bind tightly a nonfluorescent, cell-impermeable dye (fluorogen), thereby generating a fluorescent complex. The utility of FAP tagging to study trafficking of integral membrane proteins in yeast, which possesses a cell wall, had not been examined previously. A diverse set of signal peptides and propeptide sequences were explored to maximize expression. Maintenance of the optimal FAP-Ste2 chimera intact required deletion of two, paralogous, glycosylphosphatidylinositol (GPI)-anchored extracellular aspartyl proteases (Yps1 and Mkc7). FAP-Ste2 exhibited a much brighter and distinct plasma membrane signal than Ste2-GFP or Ste2-mCherry yet behaved quite similarly. Using FAP-Ste2, new information was obtained about the mechanism of its internalization, including novel insights about the roles of the cargo-selective endocytic adaptors Ldb19/Art1, Rod1/Art4, and Rog3/Art7.

Detection of a Peptide Biomarker by Engineered Yeast Receptors.

Directed evolution of membrane receptors is challenging as the evolved receptor must not only accommodate a non-native ligand, but also maintain the ability to transduce the detection of the new ligand to any associated intracellular components. The G-protein coupled receptor (GPCR) superfamily is the largest group of membrane receptors. As members of the GPCR family detect a wide range of ligands, GPCRs are an incredibly useful starting point for directed evolution of user-defined analytical tools and diagnostics. The aim of this study was to determine if directed evolution of the yeast Ste2p GPCR, which natively detects the α-factor peptide, could yield a GPCR that detects Cystatin C, a human peptide biomarker. We demonstrate a generalizable approach for evolving Ste2p to detect peptide sequences. Because the target peptide differs significantly from α-factor, a single evolutionary step was infeasible. We turned to a substrate walking approach and evolved receptors for a series of chimeric intermediates with increasing similarity to the biomarker. We validate our previous model as a tool for designing optimal chimeric peptide steps. Finally, we demonstrate the clinical utility of yeast-based biosensors by showing specific activation by a C-terminally amidated Cystatin C peptide in commercially sourced human urine. To our knowledge, this is the first directed evolution of a peptide GPCR.

Distinct roles for plasma membrane PtdIns(4)P and PtdIns(4,5)P2 during receptor-mediated endocytosis in yeast.

Clathrin-mediated endocytosis requires the coordinated assembly of various endocytic proteins and lipids at the plasma membrane. Accumulating evidence demonstrates a crucial role for phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] in endocytosis but specific roles for phosphatidylinositol-4-phosphate [PtdIns(4)P], other than as the biosynthetic precursor of PtdIns(4,5)P2, have not been clarified. In this study we investigated the roles of PtdIns(4)P and PtdIns(4,5)P2 in receptor-mediated endocytosis through the construction of temperature-sensitive (ts) mutants for the phosphatidylinositol 4-kinases (PI4-kinases) Stt4p and Pik1p and the 1-phosphatidylinositol-4-phosphate 5-kinase [PtdIns(4) 5-kinase] Mss4p. Quantitative analyses of endocytosis revealed that both the stt4tspik1ts and mss4ts mutants have a severe defect in endocytic internalization. Live-cell imaging of endocytic protein dynamics in stt4tspik1ts and mss4ts mutants revealed that PtdIns(4)P is required for the recruitment of the α-factor receptor Ste2p to clathrin-coated pits, whereas PtdIns(4,5)P2 is required for membrane internalization. We also found that the localization to endocytic sites of the ENTH/ANTH domain-bearing clathrin adaptors, Ent1p, Ent2p, Yap1801p and Yap1802p, is significantly impaired in the stt4tspik1ts mutant but not in the mss4ts mutant. These results suggest distinct roles in successive steps for PtdIns(4)P and PtdIns(4,5)P2 during receptor-mediated endocytosis.

GPCR-Gα protein precoupling: Interaction between Ste2p, a yeast GPCR, and Gpa1p, its Gα protein, is formed before ligand binding via the Ste2p C-terminal domain and the Gpa1p N-terminal domain.

G protein coupled receptors bind ligands that initiate intracellular signaling cascades via heterotrimeric G proteins. In this study, involvement of the N-terminal residues of yeast G-alpha (Gpa1p) with the C-terminal residues of a full-length or C-terminally truncated Ste2p were investigated using bioluminescence resonance energy transfer (BRET), a non-radiative energy transfer phenomenon where protein-protein interactions can be quantified between a donor bioluminescent molecule and a suitable acceptor fluorophore. Constitutive and position-dependent BRET signal was observed in the absence of agonist (α-factor). Upon the activation of the receptors with α-factor, no significant change in BRET signal was observed. The location of Ste2p-Gpa1p heterodimer was investigated using confocal fluorescence microscopy and bimolecular fluorescence complementation (BiFC) assay, a technique where two non-fluorescent fragments of a fluorescent protein reassemble in vivo to restore fluorescence property thereby directly reporting a protein-protein interaction. BiFC experiments resulted in a dimerization signal intracellularly during biosynthesis on the endoplasmic reticulum (ER) and on the plasma membrane (PM). The constitutive BRET and BiFC signals observed on ER between Ste2p and Gpa1p in their quiescent and activated states are indicative of pre-coupling between these two proteins. This study is the first to show that the extreme N-terminus of yeast G protein alpha subunit is in close proximity to its receptor. The data suggests a pre-coupled heterodimer prior to receptor activation. The images presented in this study are the first direct in vivo evidence showing the localization of receptor - G protein heterodimers during biosynthesis and before reaching the plasma membrane.

Medium-Throughput Screen of Microbially Produced Serotonin via a G-Protein-Coupled Receptor-Based Sensor.

Chemical biosensors, for which chemical detection triggers a fluorescent signal, have the potential to accelerate the screening of noncolorimetric chemicals produced by microbes, enabling the high-throughput engineering of enzymes and metabolic pathways. Here, we engineer a G-protein-coupled receptor (GPCR)-based sensor to detect serotonin produced by a producer microbe in the producer microbe's supernatant. Detecting a chemical in the producer microbe's supernatant is nontrivial because of the number of other metabolites and proteins present that could interfere with sensor performance. We validate the two-cell screening system for medium-throughput applications, opening the door to the rapid engineering of microbes for the increased production of serotonin. We focus on serotonin detection as serotonin levels limit the microbial production of hydroxystrictosidine, a modified alkaloid that could accelerate the semisynthesis of camptothecin-derived anticancer pharmaceuticals. This work shows the ease of generating GPCR-based chemical sensors and their ability to detect specific chemicals in complex aqueous solutions, such as microbial spent medium. In addition, this work sets the stage for the rapid engineering of serotonin-producing microbes.

Modulating and evaluating receptor promiscuity through directed evolution and modeling.

The promiscuity of G-protein-coupled receptors (GPCRs) has broad implications in disease, pharmacology and biosensing. Promiscuity is a particularly crucial consideration for protein engineering, where the ability to modulate and model promiscuity is essential for developing desirable proteins. Here, we present methodologies for (i) modifying GPCR promiscuity using directed evolution and (ii) predicting receptor response and identifying important peptide features using quantitative structure-activity relationship models and grouping-exhaustive feature selection. We apply these methodologies to the yeast pheromone receptor Ste2 and its native ligand α-factor. Using directed evolution, we created Ste2 mutants with altered specificity toward a library of α-factor variants. We then used the  Vectors of Hydrophobic, Steric, and Electronic properties and partial least squares regression to characterize receptor-ligand interactions, identify important ligand positions and properties, and predict receptor response to novel ligands. Together, directed evolution and computational analysis enable the control and evaluation of GPCR promiscuity. These approaches should be broadly useful for the study and engineering of GPCRs and other protein-small molecule interactions.

The yeast Ste2p G protein-coupled receptor dimerizes on the cell plasma membrane.

Dimerization of G protein-coupled receptors (GPCR) may play an important role in maturation, internalization, signaling and/or pharmacology of these receptors. However, the location where dimerization occurs is still under debate. In our study, variants of Ste2p, a yeast mating pheromone GPCR, were tagged with split EGFP (enhanced green fluorescent protein) fragments inserted between transmembrane domain seven and the C-terminus or appended to the C-terminus. Bimolecular Fluorescence Complementation (BiFC) assay was used to determine where receptor dimerization occurred during protein trafficking by monitoring generation of EGFP fluorescence, which occurred upon GPCR dimerization. Our results suggest that these tagged receptors traffic to the membrane as monomers, undergo dimerization or higher ordered oligomerization predominantly on the plasma membrane, and are internalized as dimers/oligomers. This study is the first to provide direct in vivo visualization of GPCR dimerization/oligomerization, during trafficking to and from the plasma membrane.

Quaternary structure of the yeast pheromone receptor Ste2 in living cells.

Transmembrane proteins known as G protein-coupled receptors (GPCRs) have been shown to form functional homo- or hetero-oligomeric complexes, although agreement has been slow to emerge on whether homo-oligomerization plays functional roles. Here we introduce a platform to determine the identity and abundance of differing quaternary structures formed by GPCRs in living cells following changes in environmental conditions, such as changes in concentrations. The method capitalizes on the intrinsic capability of FRET spectrometry to extract oligomer geometrical information from distributions of FRET efficiencies (or FRET spectrograms) determined from pixel-level imaging of cells, combined with the ability of the statistical ensemble approaches to FRET to probe the proportion of different quaternary structures (such as dimers, rhombus or parallelogram shaped tetramers, etc.) from averages over entire cells. Our approach revealed that the yeast pheromone receptor Ste2 forms predominantly tetramers at average expression levels of 2 to 25 molecules per pixel (2.8·10-6 to 3.5·10-5molecules/nm2), and a mixture of tetramers and octamers at expression levels of 25-100 molecules per pixel (3.5·10-5 to 1.4·10-4molecules/nm2). Ste2 is a class D GPCR found in the yeast Saccharomyces cerevisiae of the mating type a, and binds the pheromone α-factor secreted by cells of the mating type α. Such investigations may inform development of antifungal therapies targeting oligomers of pheromone receptors. The proposed FRET imaging platform may be used to determine the quaternary structure sub-states and stoichiometry of any GPCR and, indeed, any membrane protein in living cells. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova.

Yeast GPCR signaling reflects the fraction of occupied receptors, not the number.

According to receptor theory, the effect of a ligand depends on the amount of agonist-receptor complex. Therefore, changes in receptor abundance should have quantitative effects. However, the response to pheromone in Saccharomyces cerevisiae is robust (unaltered) to increases or reductions in the abundance of the G-protein-coupled receptor (GPCR), Ste2, responding instead to the fraction of occupied receptor. We found experimentally that this robustness originates during G-protein activation. We developed a complete mathematical model of this step, which suggested the ability to compute fractional occupancy depends on the physical interaction between the inhibitory regulator of G-protein signaling (RGS), Sst2, and the receptor. Accordingly, replacing Sst2 by the heterologous hsRGS4, incapable of interacting with the receptor, abolished robustness. Conversely, forcing hsRGS4:Ste2 interaction restored robustness. Taken together with other results of our work, we conclude that this GPCR pathway computes fractional occupancy because ligand-bound GPCR-RGS complexes stimulate signaling while unoccupied complexes actively inhibit it. In eukaryotes, many RGSs bind to specific GPCRs, suggesting these complexes with opposing activities also detect fraction occupancy by a ratiometric measurement. Such complexes operate as push-pull devices, which we have recently described.