Internalization plays a crucial role in regulating the density of cell surface receptors and has been demonstrated to regulate intracellular signaling. Dysregulation of this process has been implicated in various diseases. The vast majority of GPCRs were considered to adopt one way for internalization. We challenged this conventional view by showing that multiple pathways converge to regulate the internalization of a specific receptor, based on an unparalleled characterization of 60 GPCR internalization profiles, both in the absence and presence of individual β-arrestins (βarrs). Furthermore, we revealed the internalization mechanism of the glucagon-like peptide-1 receptor (GLP-1R), a class B GPCR pivotal in promoting insulin secretion from pancreatic beta cells to maintain glucose homeostasis. GLP-1R can undergo agonist-induced internalization without βarrs, but can recruit and form stable complexes with βarrs. We found that GLP-1R recruits clathrin adaptor protein-2 for agonist-induced internalization in both βarr-dependent and -independent manners. These results provide a valuable resource for GPCR signaling and reveal the plasticity of different GPCRs to employ or not βarrs in the clathrin-mediated internalization.

Glioblastoma (GBM) is the most common and aggressive primary brain malignancy. Adhesion G protein-coupled receptors (aGPCRs) have attracted interest for their potential as treatment targets. Here, we show that CD97 (ADGRE5) is the most promising aGPCR target in GBM, by virtue of its de novo expression compared to healthy brain tissue. CD97 knockdown or knockout significantly reduces the tumor initiation capacity of patient-derived GBM cultures (PDGCs) in vitro and in vivo. We find that CD97 promotes glycolytic metabolism via the mitogen-activated protein kinase (MAPK) pathway, which depends on phosphorylation of its C terminus and recruitment of β-arrestin. We also demonstrate that THY1/CD90 is a likely CD97 ligand in GBM. Lastly, we show that an anti-CD97 antibody-drug conjugate selectively kills tumor cells in vitro. Our studies identify CD97 as a regulator of tumor metabolism, elucidate mechanisms of receptor activation and signaling, and provide strong scientific rationale for developing biologics to target it therapeutically in GBM.Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

Arrestins interact with G protein-coupled receptors (GPCRs) to stop G protein activation and to initiate key signaling pathways. Recent structural studies shed light on the molecular mechanisms involved in GPCR-arrestin coupling, but whether this process is conserved among GPCRs is poorly understood. Here, we report the cryo-electron microscopy active structure of the wild-type arginine-vasopressin V2 receptor (V2R) in complex with β-arrestin1. It reveals an atypical position of β-arrestin1 compared to previously described GPCR-arrestin assemblies, associated with an original V2R/β-arrestin1 interface involving all receptor intracellular loops. Phosphorylated sites of the V2R C-terminus are clearly identified and interact extensively with the β-arrestin1 N-lobe, in agreement with structural data obtained with chimeric or synthetic systems. Overall, these findings highlight a striking structural variability among GPCR-arrestin signaling complexes.