(820c) Human G Protein-Coupled Receptor Signaling – Engineering Yeast As Reporters of Interacting Pathways

G Protein-Coupled Receptors (GPCRs) act as members of a large signal transduction protein family, and are targets of approximately 40% of all drug discovery efforts. GPCRs bind ligands at the cell surface and transmit that signal to the cell via dissociation of the heterotrimeric G-proteins and initiation of a downstream signaling cascade that can result in a change in protein production or cell metabolism. Multiple receptor subtypes, receptor promiscuity, and common signaling relays following GPCR activation complicate the signaling process in the native mammalian tissue, and convolute our understanding of the action of potential therapeutic ligands. In contrast, the relative simplicity of the yeast proteome allows the study of human GPCR signaling in the yeast cellular environment, providing a eukaryotic context with reduced crosstalk. In contrast to human cells, yeast posses very few GPCRs, yet the signaling pathways are analogous to those of humans. By engineering the native yeast GPCR-modulated mating pheromone response, it is possible to deconstruct human receptor behavior in this less complex environment.

In this study, we have expressed recombinant human GPCRs involved in neurological and cardiological functions, the adenosine A_2A and A_2B receptors, in an engineered strain of Saccharomyces cerevisiae. To facilitate signal measurement, the endogenous mating pheromone promoter was fused to mCherry to enable quantitation of GPCR signaling via fluorescence measurement. In addition, GPCRs were fluorescently tagged to enable studies of co-localization, and effect of A_2A and A_2B receptor-specific ligands on pathway activation. These results will be presented in light of the growing discussion of heteroligomerization in signal activation.