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

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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.