Probing intercellular GPCR-GPCR ineractions in fungal cell fusion events

Ste2p和Ste3p是D类酵母信息素（分别为α因子和α因子）g蛋白偶联受体（gpcr），参与导致细胞融合的交配反应信号传导。它们与下游信号转导机制的相互作用已经很好地建立起来，并且与哺乳动物GPCR系统中的相互作用同源，这些系统构成了目前绝大多数的药物靶点。我们最近通过串联亲和纯化和表面等离子体共振方法检测并表征了酵母配对g蛋白偶联受体Ste2p和Ste3p之间的配体依赖性细胞间相互作用。细胞下拉实验证明了相互作用的胞外域介导性质，体内分析表明细胞-细胞相互作用的受体依赖性增加。考虑到Ste2p和Ste3p在相反交配细胞表面的自然定位，这些结果表明，这些受体作为融合原可能有额外的作用，参与将MATa和matα细胞膜物理地结合在一起。细胞-细胞融合是一个广泛存在的事件，发生在各种生理相关过程中，包括表皮细胞融合、成肌细胞融合、受精、胎盘滋养层融合、巨噬细胞融合和干细胞融合。尽管这种类型的细胞间异质GPCR相互作用从未被检测到，但基于GPCR的广泛存在，它可能会影响许多其他领域的细胞融合研究，其中融合原尚未确定。它也可能与其他GPCR系统有关，如神经元信号，其中无数的GPCR已知在神经连接处表达。GPCR-GPCR细胞间的相互作用可能代表了一个全新的水平的信号调节横跨重要的GPCR超家族。本研究项目关注的是对这一重要相互作用的持续表征。

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.