Modulation of G protein-coupled receptor trafficking and activity by receptor-interacting proteins

G蛋白偶联受体(GPCRs)属于细胞表面受体的超家族，通过对不同的细胞外刺激做出反应来调节多种细胞功能。这些GPCRs的质膜表达是一个平衡顺行和逆行运输的动态过程。在这种情况下，新合成的GPCRs从细胞内隔间输出到细胞表面是控制质膜上可用功能受体水平和配体引发的细胞反应的关键检查点。尽管细胞表面GPCRs的输出通常被认为与结构性的非调控分泌途径有关，但现在越来越多的证据表明，这些跨膜受体在高尔基体后转运到细胞表面的过程受到分子伴侣/辅助护送蛋白的动态调节，也可能依赖于非规范调节的分泌途径，通过它们包装成大的致密核心囊泡(LDCV)。然而，与致力于了解内吞和循环途径中所涉及的事件的广泛努力相反，GPCR转运到质膜的分子机制仍然不清楚。我们先前已经证明，属于A类GPCRs的神经降压素(NT)受体亚型2(NTS2)主要与细胞内细胞器相关，持续的NT暴露促进了NTS2的细胞表面募集。此外，利用酵母双杂交系统筛选与NTS2细胞内第三环相互作用的蛋白质，我们最近发现分泌颗粒素III(SGIII)是一种新的与NTS2相互作用的蛋白质。被归类为颗粒蛋白家族的一员，SgIII确保将生物活性多肽分选为分泌颗粒。因此，我们的第一个目标是通过体外GST下拉试验以及免疫共沉淀和FRET研究，在细胞环境中使用标记的融合蛋白来表征SGIII-NTS2的分子相互作用。此外，我们将使用定点突变方法来进一步提炼NTS2与SGIII相互作用中涉及的分子决定因素。第二个目标将有助于确定SGIII是否通过促进NTS2的包装进入LDCV来调节NTS2的细胞表面表达，并调节NTS2的生理功能。为此，我们将研究27-聚DICER-底物siRNA(DsiRNA)下调SGIII对细胞表面生物可用NTS2受体数量的影响，以及它在激动剂刺激下与ERK1/2信号通路偶联的影响。最后，我们将确定NTS2通过调节的分泌途径分类是否对脊髓止痛重要。内质网/高尔基体后输出是新生GPCRs细胞表面运输的限速步骤。最近的研究表明，GPCRs在不同的分泌途径中的分类不仅取决于这些蛋白的内在性质，还取决于与这些受体相互作用的外在分子伴侣/辅助蛋白。因此，我们的长期目标是更好地了解GPCRs的ER-高尔基体细胞表面运输是如何通过识别和表征参与GPCRs分类的新分子护送蛋白来调节的，特别是通过调节的分泌途径。为了深入了解通过非规范途径调节这些GPCRs分选的机制，我们建议使用针对不同GPCRs的特异性抗体进行免疫沉淀，并结合基于功能质谱学(MS)的蛋白质组学方法来鉴定新的GPCRs相关护送蛋白。

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). 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.We previously demonstrated that the neurotensin (NT) receptor subtype 2 (NTS2), belonging to the class A of GPCRs is mainly associated with intracellular organelles and that sustained NT exposure promotes cell surface recruitment of NTS2. In addition, using the yeast two-hybrid system to screen for proteins that interact with the third intracellular loop of NTS2, we recently identified the secretogranin III (SgIII) as a new NTS2-interacting protein. Classified as a member of the granin protein family, SgIII ensures the sorting of bioactive peptides to secretory granules. Our FIRST OBJECTIVE is therefore to characterize the SgIII-NTS2 molecular interaction by using in vitro GST pull-down assays as well as co-immunoprecipitation and FRET studies in a cellular context using tagged fusion proteins. Additionally, we will use a site-directed mutagenesis method to further refine the molecular determinants of NTS2 involved in the interaction with SgIII. The SECOND OBJECTIVE will help to determine whether SgIII regulates the cell surface expression of NTS2 by promoting its packaging into LDCVs and modulates NTS2 physiological function. For this purpose, we will investigate the effects of the down-regulation of SgIII by the use of 27-mer Dicer-substrate siRNA (DsiRNA) on the number of bioavailable NTS2 receptors at the cell surface and on its coupling to the ERK1/2 signaling pathway in response to agonist stimulation. Finally, we will determine whether the sorting of NTS2 through the regulated secretory pathway is important for spinal analgesia.ER/post-Golgi export is a rate-limiting step for cell surface transport of nascent GPCRs. Recent progress suggest that the sorting of GPCRs into distinct secretory pathways is determined not only by the intrinsic properties of these proteins but also by the extrinsic molecular chaperones/accessory proteins that interact with these receptors. OUR LONG-TERM 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.