BME PhD Dissertation Defense: Ben Fitzsimmons

Title: "Noncanonical structure and mechanism of organelle-dedicated signaling by GPR143"

Advisory Committee: Ahmad Khalil, PhD – BME (Co-research Advisor) Mikel Garcia-Marcos, PhD – Biochemistry & Cell Biology, Biology (Co-research Advisor) Wilson Wong, PhD – BME (Chair) John Ngo, PhD – BME Ruth Huttenhain, PhD – Stanford Molecular and Cellular Physiology

Abstract: GPCRs are one of the most widely studied groups of proteins. There are over 800 unique GPCRs in the human genome, interacting with many types of extracellular signals including photons, protons, lipids, peptides, and hormones. With such a great number of GPCRs interacting with so many signals, they are involved in most physiological processes in the body, and thus also associated with disease if they are dysregulated. The large body of research into the GPCR family of receptors has established what defines a GPCR—their structure, their localization, and their signaling mechanisms. Yet, in the GPCR family tree, there exist outliers, understudied because standard assays for measuring GPCR activity are not suitable for one of many reasons: they may lack conventional G protein coupling, signal through non-canonical mediators, or function primarily as intracellular receptors rather than at the plasma membrane. Here, we resolve the structure and mechanism of one of these dogma-defying GPCRs in GPR143. Mutation of this receptor causes the disease ocular albinism, and dysregulation of its expression is also linked to several cancers. There are many peculiarities about GPR143—it localizes internally to melanosomes, many functional motifs found in GPCRs show a marked divergence in GPR143, and the nature of its native ligand and signaling mechanism remain controversial. To dissect these peculiarities, we combined cryo-electron microscopy with high-throughput cell signaling BRET assays and unbiased interactome analysis to comprehensively examine the structure, mechanism, function, and molecular pathogenesis of GPR143. We reveal the unique seven-transmembrane structure of GPR143, in which its own N-terminus occupies the canonical orthosteric binding pocket found in GPCRs. We demonstrate that the N-terminus auto-activates the receptor, allowing for constitutive β-arrestin recruitment rather than G protein activation. Next, using APEX proximity labeling, we discover a noncanonical protein that pairs with GPR143 in HPS1, which is also implicated in albinism disorders. Finally, we begin to dissect the mechanism of action of GPR143, looking at the interplay between β-arrestins and HPS1. This characterization of GPR143 challenges not only previous findings on the receptor, but also canonical knowledge about GPCRs as a whole.