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. Synopsis Receptor abundance varies both from cell to cell and over time. Mathematical modeling and experimentation show that a yeast GPCR pathway compensates for these variations by responding to the fraction, and not to the number, of occupied receptors. Mathematical modeling indicates that if unbound receptors actively inactivate downstream effectors, the system may respond to fractional, rather than absolute, receptor occupancy. Fractional occupancy measurement makes the system robust to changes in receptor abundance. In the yeast pheromone response system, the GPCR Ste2 binds the inhibitory RGS Sst2, thus forming a complex with antagonistic functions. The GTP/GDP state of the regulated G protein is determined by the ligand occupation state of the last GPCR–RGS complex it visited, independently of receptor abundance. Experimentally uncoupling the inhibitory RGS activity from the receptor abolished fractional occupancy measurement. Graphical Abstract Receptor abundance varies both from cell to cell and over time. Mathematical modeling and experimentation show that a yeast GPCR pathway compensate