Probing intercellular GPCR-GPCR ineractions in fungal cell fusion events

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Ste2p and Ste3p are well characterized Class D yeast pheromone (alpha- and a- factor respectively) G-protein coupled receptors (GPCRs) involved in signaling of mating responses that lead to cell fusion.  Their interactions with downstream signal transduction machinery are well established and homologous to those in mammalian GPCR systems, which comprise the vast majority of pharmaceutical targets at this time. We recently detected and characterized a ligand-dependent, intercellular interaction between the yeast mating G-protein coupled receptors Ste2p and Ste3p through applications of tandem affinity purification and surface plasmon resonance methods. Cellular pull-down assays demonstrated the extracellular domain-mediated nature of the interaction and in vivo analyses indicated receptor-dependent increases in cell-cell interactions.  Consideration of these results in the context of the natural localization of Ste2p and Ste3p on the surfaces of opposite mating cell types suggests the possibility of an additional role for these receptors as the fusogens, involved in physically bringing the MATa and MATalpha cell membranes together. Cell-cell fusion is a widespread event which occurs during a variety of physiologically relevant processes including epidermal cell fusion, myoblast fusion, fertilization, trophoblast fusion in the placenta, macrophage fusion and stem cell fusion.  Although this type of intercellular heteromeric GPCR interaction has never before been detected, based on the wide spread occurrence of GPCRs it may impact on many of these other areas of cell fusion research for which fusogens remain as yet unidentified.  It may also be relevant to other GPCR systems such as neuronal signaling where a myriad of GPCRs are known to be expressed at neural junctions.  GPCR-GPCR intercellular interactions may represent an entirely new level of signaling regulation across the pharmaceutically important GPCR superfamily. This research program is concerned with the continued characterization of this important interaction.