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(GPCRs)，参与细胞融合的交配反应信号传递。它们与下游信号转导机制的相互作用已得到很好的证实，并与哺乳动物GPCR系统中的作用同源，后者构成了目前绝大多数药物靶标。我们最近通过串联亲和纯化和表面等离子共振方法检测和表征了酵母交配G蛋白偶联受体Ste2p和Ste3p之间的配体依赖的细胞间相互作用。细胞下拉分析证明了细胞外结构域介导的相互作用的性质，体内分析表明细胞-细胞相互作用依赖于受体。考虑到这些结果在相对交配细胞类型的表面上的自然定位的背景下，这些受体作为融合原，参与物理地将MATA和MATα细胞膜结合在一起，表明这些受体可能具有额外的作用。细胞-细胞融合是一种广泛发生的事件，发生在各种生理相关的过程中，包括表皮细胞融合、成肌细胞融合、受精、胎盘滋养细胞融合、巨噬细胞融合和干细胞融合。尽管这种细胞间异构体GPCR相互作用以前从未被检测到，基于GPCRs的广泛发生，它可能会影响许多其他领域的细胞融合研究，其中融合原仍未确定。它也可能与其他GPCRs系统相关，如神经元信号转导系统，其中已知的无数GPCRs在神经连接处表达。GPCRGPCR超家族的细胞间相互作用可能代表着整个具有重要药学意义的GPCR超家族信号调节的一个全新水平。这项研究计划关注对这种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.