Abstract Mammalian G protein coupled receptors (GPCRs) mediate a vast array of biological responses and have been implicated in numerous diseases. GPCRs are highly druggable and the target of about one-third of all FDA approved drugs. Currently, all drugs targeting GPCRs have been developed to modulate signals transduced at the plasma membrane. However, we and others have shown that GPCRs remain active inside the cell and signal from endosomes. The orchestration of GPCR signaling from the plasma membrane and endosomes is essential for achieving proper cellular responses, dysregulation of these pathways, through either aberrant increases or decreases in signaling drives disease progression. Our laboratory has long focused on understanding the regulatory mechanisms that control GPCR signaling. In recent projects funded by MIRA, we discovered that ubiquitination of a subset of GPCRs drives p38 mitogen-activated protein kinase (MAPK) endosomal signaling and vascular inflammation. The molecular mechanisms by which key regulators and mediators of ubiquitination regulate GPCR-p38 endosomal signaling is not known and a gap in knowledge. In the next 5 years, our laboratory will focus on understanding how two key deubiquitinases regulate GPCR-stimulated p38 signaling by identifying key substrates and elucidating the mechanisms of regulation and function in vascular inflammation. We also discovered that the α-arrestin arrestin-related domain containing protein-3 (ARRDC3) is an endosomal multi-functional adaptor protein that controls GPCR signaling and trafficking via distinct mechanisms in projects funded by MIRA. Unlike classical arrestins, virtually nothing is known about the mechanisms that regulate α- arrestin activity and how α-arrestins govern mammalian GPCR function and a major gap in knowledge. In the next 5 years, we will define the molecular mechanisms that control GPCR-stimulated ARRDC3 activity and elucidate the mechanisms of regulation and function in cancer progression. We will integrate hypothesis-driven and unbiased systems approaches to interrogate the mechanisms that control ubiquitin-driven GPCR endosomal signaling and ARRDC3 activity and function utilizing innovative and cutting-edge technologies. A thorough understanding of the spatial-temporal regulatory mechanisms that control GPCR signaling is critical for improving the development of novel drugs targeting GPCRs.