Mechanisms of spatiotemporal signaling by GPCRs

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Project Summary G protein-coupled receptors (GPCRs) comprise the largest family of signaling receptors in animals, and as such, they represent an important class of therapeutic targets. Following ligand binding and G protein activation at the plasma membrane, GPCRs undergo regulated endocytosis and sorting for recycling or degradation. Traditionally, studies of ligand-dependent GPCR signaling have focused on receptor-G protein coupling at the plasma membrane (PM). However, it is now clear that GPCRs can continue to signal from internal membrane locations and that the downstream responses elicited from intracellular signaling are distinct from those elicited from PM signaling. While the biochemistry of GPCR activation has been studied in detail, the organization and regulation of GPCR signaling in living cells remains an underexplored frontier. The proposed studies will investigate fundamental mechanisms underpinning spatiotemporal regulation of GPCR signaling and will provide key training to enable Dr. Blythe to become an independent leader in this emerging area of molecular and cellular physiology. The mentored phase of this project will be carried out at the University of California, San Francisco under the primary mentorship of Dr. Mark von Zastrow, a leader in the field of GPCR trafficking and signaling. The long-term goals of this work are to understand (1) how the subcellular localization of GPCRs and their associated proteins change in response to signaling and (2) how this cellular reorganization regulates their distinct downstream responses. The first two Aims will focus on receptor trafficking and signaling, defining the mechanisms by which unique endocytic (Aim 1) and recycling (Aim 2) pathways sculpt signaling by endogenously expressed GPCRs in a HEK293 cell model. In carrying out these experiments, Dr. Blythe will gain new experience in advanced imaging techniques, as well as in integral membrane protein biochemistry with the help of Dr. Aashish Manglik (collaborator). Aim 3 frames the biology in a broader perspective by asking how the dynamic subcellular localization of other proteins contributes to the spatiotemporal regulation of GPCR signaling. Using a novel proximity labeling approach under the mentorship of Dr. Nevan Krogan (co-Mentor) and Dr. Ruth Hüttenhain (collaborator), Dr. Blythe will map the changes in the proteomes of specific cellular compartments during the activation of the same model GPCRs and explore how these changes dictate signaling. The proposed work will enable a systems-level analysis of GPCR signaling that was not feasible with current approaches and provide an invaluable training opportunity for Dr. Blythe in mass spectrometry-based proteomics. In summary, this project will take advantage of the expertise of a diverse mentorship team and the world-class resources and facilities at UCSF to tackle fundamental questions in GPCR biology.