Probing intercellular GPCR-GPCR ineractions in fungal cell fusion events

Ste 2 p和Ste 3 p是充分表征的D类酵母信息素（分别为α-和α-因子）G蛋白偶联受体（GPCR），其参与导致细胞融合的交配反应的信号传导。 它们与下游信号转导机制的相互作用已充分确立，并且与哺乳动物GPCR系统中的相互作用同源，哺乳动物GPCR系统目前包含绝大多数药物靶标。我们最近检测和表征的配体依赖性，酵母交配G-蛋白偶联受体之间的相互作用的STE 2 P和STE 3 P通过串联亲和纯化和表面等离子体共振方法的应用。细胞下拉试验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-细胞相互作用的受体依赖性增加。 考虑到这些结果的背景下的自然本地化的Ste 2 p和Ste 3 p的表面上的相反的交配细胞类型的建议的可能性，额外的作用，这些受体作为融合，参与物理上带来的MATa和MAT α细胞膜在一起。细胞-细胞融合是一种广泛的事件，其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、胎盘中的滋养层融合、巨噬细胞融合和干细胞融合。 尽管这种类型的细胞间异聚GPCR相互作用以前从未被检测到，但基于GPCR的广泛存在，它可能会影响细胞融合研究的许多其他领域，其中融合子仍然未被鉴定。 它也可能与其他GPCR系统相关，例如神经元信号传导，其中已知无数GPCR在神经连接处表达。 GPCR-GPCR细胞间相互作用可能代表了整个药物重要的GPCR超家族的信号调节的全新水平。本研究计划关注的是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.