Summary GPR133 (ADGRD1) is an adhesion G protein-coupled receptor that signals through Gαs and is required for growth of glioblastoma (GBM), an aggressive brain malignancy. The regulation of GPR133 signaling is incompletely understood. Here, we use proximity biotinylation proteomics to identify ESYT1, a Ca 2+ -dependent mediator of endoplasmic reticulum-plasma membrane bridge formation, as an intracellular interactor of GPR133. ESYT1 knockdown or knockout increases GPR133 signaling, while its overexpression has the opposite effect, without altering GPR133 levels in the plasma membrane. The GPR133-ESYT1 interaction requires the Ca 2+ -sensing C2C domain of ESYT1. Thapsigargin-mediated increases in cytosolic Ca 2+ relieve signaling-suppressive effects of ESYT1 by promoting ESYT1-GPR133 dissociation. ESYT1 knockdown or knockout in GBM impairs tumor growth in vitro , suggesting functions of ESYT1 beyond the interaction with GPR133. Our findings suggest a novel mechanism for modulation of GPR133 signaling by increased cytosolic Ca 2+ , which reduces the signaling-suppressive interaction between GPR133 and ESYT1 to raise cAMP levels.

Modulation of GPR133 (ADGRD1) Signaling by its Intracellular Interaction Partner Extended Synaptotagmin 1 (ESYT1)