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

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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). 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.