Investigating the molecular details of assembly, disassembly and trafficking of GPCR-arrestin complexes

Project Summary: Mis-regulation of G protein-coupled receptor (GPCR) trafficking and signaling is implicated in causing several diseases and the development of drug tolerance, having a major impact on human health. GPCRs evolved to be the most important means for communication between cells and tissues in higher organisms. They are responsive to a wide range of stimuli including light, odorants, peptides, neurotransmitters, and hormones, making GPCRs critical players in regulating human physiology. Owing to their importance, they are the targets for a third of all FDA-approved drugs. For signaling to be temporally regulated, after agonist stimulation, GPCRs are desensitized. This desensitization occurs as a two-step process: first by phosphorylation, then by binding to proteins called -arrestins. -arrestin binding promotes acute desensitization by blocking access of G proteins to receptors. In addition, -arrestins act as adapters to proteins involved in clathrin-mediated endocytosis, facilitating internalization of the GPCR. Once internalized, the fate of a GPCR can differ dramatically, from being rapidly recycled back to the plasma membrane to being degraded. While classically GPCR signaling was thought to be confined to the plasma membrane, it is now appreciated that GPCRs can also signal from various intracellular compartments. Though our understanding of G protein-mediated signaling has matured over years of study, our understanding of how GPCRs are recognized as endocytic cargo remains limited. An important protein complex for this process is retromer, which sorts cargo at endosomes for recycling. A key component of retromer, vps26, is structurally similar to -arrestins, and is important for cargo recognition. I hypothesize that arrestin domain proteins are a privileged scaffold for recognition and trafficking of membrane proteins. As a result, understanding the molecular mechanisms that determine how GPCR--arrestins assemble and disassemble, and how they are trafficked in a cell, will have a profound impact on our understanding of signaling from GPCRs and the action of drugs. Using the 2AR together with V2R and NTSR1 as model receptors, I will (1) characterize how GPCR--arrestin complexes assemble and disassemble, and how this is affected by membrane lipids, GPCR phosphorylation, and the presence of other binding partners. I will also (2) identify protein interaction partners of GPCR--arrestin complexes in cells to understand which factors regulate the rapid or slow recycling behavior of these receptors. Finally, (3) I will characterize the engagement of a GPCR by retromer. These aims will be addressed using single-molecule fluorescence spectroscopy, state-of-the-art mass spectrometry, and in-cell photo-crosslinking. These aims will answer long-standing questions pertaining to arrestin function, and open new lines of investigation into regulation of GPCRs at endosomes. My Mentor, Dr. Kobilka, co-mentor Dr. von Zastrow and expert advisors in proteomics and protein-protein interactions (Drs. Hüttenhain, Krogan, Ting) and arrestin proteins (Dr. Benovic), will provide me with the training necessary to complete these aims and launch my independent research career.