Abstract
We report here on the relationship between ligand binding and signaling responses in the yeast pheromone response pathway, a well characterized G protein-coupled receptor system. Responses to agonist (α-factor) by cells expressing widely varying numbers of receptors depend primarily on fractional occupancy, not the absolute number of agonist-bound receptors. Furthermore, the concentration of competitive antagonist required to inhibit α-factor-dependent signaling is more than 10-fold higher than predicted based on the known ligand affinities. Thus, responses to a particular number of agonist-bound receptors can vary greatly, depending on whether there are unoccupied or antagonist-bound receptors present on the same cell surface. This behavior does not appear to be due to pre-coupling of receptors to G protein or to the Sst2p regulator of G protein signaling. The results are consistent with a signaling response that is determined by the integration of positive signals from agonist-occupied receptors and inhibitory signals from unoccupied receptors, where the inhibitory signals can be diminished by antagonist binding.
Highlights
We report here on the relationship between ligand binding and signaling responses in the yeast pheromone response pathway, a well characterized G protein-coupled receptor system
Signaling in the yeast pheromone response pathway is characterized by a remarkable proportionality between receptor occupancy and pathway activation, measured either via G protein dissociation, MAPK activation, transcriptional induction of reporter genes, or cell cycle arrest [14, 20], indicating that signal output is a direct function of the number of agonist-occupied receptors at the cell surface
We examined the relationship between receptor occupancy and signaling in the G protein-coupled pheromone response pathway of the yeast Saccharomyces cerevisiae in the following three ways: 1) incubating cells expressing a set number of receptors with varying concentrations of ␣-factor; 2) varying the number of cell surface receptors expressed on each cell; and 3) using competitive antagonists of ␣-factor to vary the number of sites available for agonist binding
Summary
Effect of Varying Receptor Expression Levels on Pheromone Responses—To quantitatively examine the relationship between signaling output and numbers of receptors at the cell surface, we performed assays of ␣-factor-dependent induction of the pheromone-responsive FUS1-lacZ reporter in yeast strains expressing varying levels of full-length Ste2p receptors ranging from Ͻ0.2 times the level expressed from the normal chromosomal STE2 locus to ϳ9 times this level. In contrast to a previous report that deletion of SST2 results in constitutive signaling even in the absence of receptor [33], we find that cells lacking the RGS protein display only low levels of basal FUS1-lacZ expression (Fig. 7) The basis for this difference is not known, but it could involve differences in strain backgrounds. Relative maximal inductionc nm Chromosomal Repressed GAL1-STE2 CEN-STE2 Multicopy STE2
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