Identification of G Protein-coupled receptor associated sorting proteins through a functional mass spectrometry (MS)-based proteomic approach.

G蛋白偶联受体（GPCRs）属于细胞表面受体的超家族，其调节多种细胞因子， 细胞功能通过响应不同的细胞外刺激。这些GPCR的质膜表达 是平衡顺行和逆行运输的动态过程。在这方面，出口 从细胞内隔室到细胞表面的新合成的GPCR代表了细胞内的关键检查点。 控制质膜上可用的功能性受体的水平和细胞内受体的大小， 由配体引起的反应。尽管GPCR的细胞表面输出通常被认为涉及细胞内的细胞毒性， 组成型不受调节的分泌途径，现在有越来越多的证据表明，后高尔基体 这些跨膜受体向细胞表面的运输受分子生物学的动态调节。 伴侣蛋白/辅助护送蛋白，也可能依赖于非经典的调节分泌途径， 它们包装成大的致密核心囊泡（LDCV）。ER/后高尔基体输出因此是一个限速步骤， 新生GPCR的细胞表面转运。然而，与致力于 了解内吞和再循环途径中涉及的事件，分子机制， GPCR向质膜的潜在转运仍然不清楚。因此，我们的目标是更好地 通过识别和表征GPCR的ER-高尔基体细胞表面转运， 新的分子护送蛋白参与GPCR的分选，特别是通过调节分泌 通路为了深入了解通过非经典的GPCR来调节这些GPCR的排序的机制， 途径，我们建议用针对不同GPCR的特异性抗体进行免疫沉淀 与LDCV富集级分结合基于功能质谱（MS）的 蛋白质组学方法鉴定新的GPCR相关的护送蛋白。

G protein-coupled receptors (GPCRs) belong to a superfamily of cell-surface receptors that regulate a variety of cellular functions by responding to diverse extracellular stimuli. Plasma membrane expression of these GPCRs is a dynamic process balancing anterograde and retrograde trafficking. In this context, export of neo-synthesized GPCRs from intracellular compartments to the cell surface represents a crucial checkpoint in controlling the level of functional receptors available at the plasma membrane and the magnitude of the cellular response elicited by a ligand. Although cell-surface export of GPCRs is commonly considered to implicate the constitutive unregulated secretory pathway, there is now growing evidence indicating that the post-Golgi trafficking of these transmembrane receptors to the cell surface is dynamically regulated by molecular chaperones/accessory escort proteins and may also rely on the non-canonical regulated secretory pathway via their packaging into large dense-core vesicles (LDCVs). ER/post-Golgi export is thus a rate-limiting step for cell surface transport of nascent GPCRs. However, in contrast to the extensive efforts dedicated to understanding the events involved in the endocytic and recycling pathways, the molecular mechanisms underlying GPCR transport to the plasma membrane remain poorly defined. Our goal is thus to better understand how this ER-Golgi-cell surface transport of GPCRs is regulated by identifying and characterizing new molecular escort proteins involved in the sorting of GPCRs, notably through the regulated secretory pathway. To gain insight into mechanisms that regulate the sorting of these GPCRs via the non-canonical pathway, we propose to perform immunoprecipitations with specific antibodies raised against different GPCRs associated with LDCV-enriched fractions combined with a functional mass spectrometry (MS)-based proteomic approach for the identification of new GPCR associated escort proteins.