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

G蛋白偶联受体（GPCRs）属于细胞表面受体超家族，其通过响应多种细胞外刺激来调节多种细胞功能。这些GPCR的质膜表达是平衡顺行和逆行运输的动态过程。在这种情况下，新合成的GPCR从细胞内区室输出到细胞表面代表了控制质膜上可用的功能性受体水平和配体引起的细胞应答的大小的关键检查点。虽然细胞表面出口的GPCR通常被认为是牵连的组成性不受调节的分泌途径，现在有越来越多的证据表明，这些跨膜受体的高尔基体后运输到细胞表面的动态调节分子伴侣/辅助护送蛋白，也可能依赖于非经典的调节分泌途径，通过包装成大致密核心囊泡（LDCV）。然而，与致力于了解内吞和再循环途径中涉及的事件的广泛努力相反，GPCR转运至质膜的分子机制仍然不清楚。 我们以前证明，神经降压素（NT）受体亚型2（NTS 2），属于A类GPCRs主要与细胞内细胞器和持续的NT暴露促进细胞表面招募的NTS 2。此外，使用酵母双杂交系统筛选与NTS 2的第三胞内环相互作用的蛋白质，我们最近确定分泌颗粒蛋白III（Sg III）作为一个新的NTS 2相互作用蛋白。分类为颗粒蛋白家族的成员，SgIII确保将生物活性肽分选为分泌颗粒。因此，我们的第一个目标是通过使用体外GST下拉测定以及在细胞环境中使用标记的融合蛋白的免疫共沉淀和FRET研究来表征SgIII-NTS 2分子相互作用。此外，我们将使用定点诱变方法，以进一步完善参与与SgIII相互作用的NTS 2的分子决定簇。第二个目的将有助于确定SgIII是否通过促进NTS 2包装到LDCV中来调节NTS 2的细胞表面表达并调节NTS 2的生理功能。为此，我们将研究通过使用27-mer Dicer底物siRNA（DsiRNA）下调SgIII对细胞表面生物可利用的NTS 2受体数量及其与ERK 1/2信号通路的偶联的影响。最后，我们将确定NTS 2通过调节分泌途径的排序是否对脊髓镇痛很重要。 ER/post-Golgi输出是新生GPCR的细胞表面转运的限速步骤。最近的进展表明，GPCR的分选到不同的分泌途径不仅是由这些蛋白质的内在特性，但也由外部分子伴侣/辅助蛋白与这些受体相互作用。因此，我们的长期目标是通过鉴定和表征参与GPCR分选的新分子护送蛋白，特别是通过调节分泌途径，更好地了解GPCR的ER-高尔基体细胞表面转运是如何调节的。为了深入了解通过非经典途径调节这些GPCR分选的机制，我们建议使用针对与LDCV富集组分相关的不同GPCR的特异性抗体进行免疫沉淀，并结合基于功能性质谱（MS）的蛋白质组学方法来鉴定新的GPCR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.