Skip to main content
eScholarship
Open Access Publications from the University of California

Scholarly Works (4 results)

  • Article
  • Peer Reviewed
UC San Diego Previously Published Works (2023)
G protein-coupled receptors (GPCRs) are highly druggable and implicated in numerous diseases, including vascular inflammation. GPCR signals are transduced from the plasma membrane as well as from endosomes and controlled by posttranslational modifications. The thrombin-activated GPCR protease-activated receptor-1 is modified by ubiquitin. Ubiquitination of protease-activated receptor-1 drives recruitment of transforming growth factor-β-activated kinase-1-binding protein 2 (TAB2) and coassociation of TAB1 on endosomes, which triggers p38 mitogen-activated protein kinase-dependent inflammatory responses in endothelial cells. Other endothelial GPCRs also induce p38 activation via a noncanonical TAB1-TAB2-dependent pathway. However, the regulatory processes that control GPCR ubiquitin-driven p38 inflammatory signaling remains poorly understood. We discovered mechanisms that turn on GPCR ubiquitin-dependent p38 signaling, however, the mechanisms that turn off the pathway are not known. We hypothesize that deubiquitination is an important step in regulating ubiquitin-driven p38 signaling. To identify specific deubiquitinating enzymes (DUBs) that control GPCR-p38 mitogen-activated protein kinase signaling, we conducted a siRNA library screen targeting 96 DUBs in endothelial cells and HeLa cells. We identified nine DUBs and validated the function two DUBs including cylindromatosis and ubiquitin-specific protease-34 that specifically regulate thrombin-induced p38 phosphorylation. Depletion of cylindromatosis expression by siRNA enhanced thrombin-stimulated p38 signaling, endothelial barrier permeability, and increased interleukin-6 cytokine expression. Conversely, siRNA knockdown of ubiquitin-specific protease-34 expression decreased thrombin-promoted interleukin-6 expression and had no effect on thrombin-induced endothelial barrier permeability. These studies suggest that specific DUBs distinctly regulate GPCR-induced p38-mediated inflammatory responses.
    Cover page: An siRNA library screen identifies CYLD and USP34 as deubiquitinases that regulate GPCR-p38 MAPK signaling and distinct inflammatory responses
    • Multimedia
    • Peer Reviewed
    UC San Diego Electronic Theses and Dissertations (2024)

    Cardiovascular disease (CVD) is a leading cause of deaths in the United States, however the underlying inflammatory pathways that contribute to CVD are poorly understood. Endothelial dysfunction is a hallmark of CVD and induced by various inflammatory mediators, many of which signal through G-protein coupled receptor (GPCRs). Thrombin, an inflammatory mediator, is generated in response to vascular injury and disrupts endothelial barrier integrity and promotes vascular leakage, a hallmark of inflammation. Thrombin activates endothelial protease-activated receptor-1 (PAR1) to disrupt endothelial barrier through the RhoA/myosin light chain (MLC) pathway. However, thrombin/PAR1-stimulated p38 mitogen-activated protein kinase (MAPK) signaling also promotes barrier permeability through a pathway that does not integrate with the RhoA/MLC signaling, suggesting that p38 acts through an uncharacterized pathway to promote inflammatory responses in endothelial cells. Our previous work showed that thrombin-activated PAR1 ubiquitination drives the recruitment of TAB2-TAB1 protein complexes on endosomes to trigger non-canonical p38 activation and endothelial barrier disruption. The mechanisms that regulate PAR1 ubiquitination/de-ubiquitination are not known. This proposal aims to understand the molecular processes that regulate PAR1-ubiquitin-driven p38 signaling and most importantly its impact on endothelial barrier integrity. The central hypothesis is that de-ubiquitination of PAR1 leads to termination of p38-mediated pro-inflammatory signaling. Using advanced biochemical, microscopy and genome-wide RNAi screening methods, we for the first time investigate molecular determinants that regulate the dynamics of PAR1-ubiquitination and the impact on endothelial proinflammatory responses in vitro using human endothelial cells. We reveal new information regarding the function of PAR1 ubiquitination in driving non-canonical endosomal p38 MAPK signaling in endothelial barrier disruption, a hallmark of inflammation, which will provide an opportunity for the development new strategies for the treatment of inflammatory responses associated with CVD.

    • 2 supplemental ZIPs
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2021)
    G protein-coupled receptors (GPCRs) are a large family comprising >800 signaling receptors that regulate numerous cellular and physiologic responses. GPCRs have been implicated in numerous diseases and represent the largest class of drug targets. Although advances in GPCR structure and pharmacology have improved drug discovery, the regulation of GPCR function by diverse post-translational modifications (PTMs) has received minimal attention. Over 200 PTMs are known to exist in mammalian cells, yet only a few have been reported for GPCRs. Early studies revealed phosphorylation as a major regulator of GPCR signaling, whereas later reports implicated a function for ubiquitination, glycosylation, and palmitoylation in GPCR biology. Although our knowledge of GPCR phosphorylation is extensive, our knowledge of the modifying enzymes, regulation, and function of other GPCR PTMs is limited. In this review we provide a comprehensive overview of GPCR post-translational modifications with a greater focus on new discoveries. We discuss the subcellular location and regulatory mechanisms that control post-translational modifications of GPCRs. The functional implications of newly discovered GPCR PTMs on receptor folding, biosynthesis, endocytic trafficking, dimerization, compartmentalized signaling, and biased signaling are also provided. Methods to detect and study GPCR PTMs as well as PTM crosstalk are further highlighted. Finally, we conclude with a discussion of the implications of GPCR PTMs in human disease and their importance for drug discovery. SIGNIFICANCE STATEMENT: Post-translational modification of G protein-coupled receptors (GPCRs) controls all aspects of receptor function; however, the detection and study of diverse types of GPCR modifications are limited. A thorough understanding of the role and mechanisms by which diverse post-translational modifications regulate GPCR signaling and trafficking is essential for understanding dysregulated mechanisms in disease and for improving and refining drug development for GPCRs.
      Cover page: Post-Translational Modifications of G Protein–Coupled Receptors Control Cellular Signaling Dynamics in Space and Time
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2022)
      Endothelial dysfunction is a hallmark of inflammation and is mediated by inflammatory factors that signal through G protein-coupled receptors including protease-activated receptor-1 (PAR1). PAR1, a receptor for thrombin, signals via the small GTPase RhoA and myosin light chain intermediates to facilitate endothelial barrier permeability. PAR1 also induces endothelial barrier disruption through a p38 mitogen-activated protein kinase-dependent pathway, which does not integrate into the RhoA/MLC pathway; however, the PAR1-p38 signaling pathways that promote endothelial dysfunction remain poorly defined. To identify effectors of this pathway, we performed a global phosphoproteome analysis of thrombin signaling regulated by p38 in human cultured endothelial cells using multiplexed quantitative mass spectrometry. We identified 5491 unique phosphopeptides and 2317 phosphoproteins, four distinct dynamic phosphoproteome profiles of thrombin-p38 signaling, and an enrichment of biological functions associated with endothelial dysfunction, including modulators of endothelial barrier disruption and a subset of kinases predicted to regulate p38-dependent thrombin signaling. Using available antibodies to detect identified phosphosites of key p38-regulated proteins, we discovered that inhibition of p38 activity and siRNA-targeted depletion of the p38α isoform increased basal phosphorylation of extracellular signal-regulated protein kinase 1/2, resulting in amplified thrombin-stimulated extracellular signal-regulated protein kinase 1/2 phosphorylation that was dependent on PAR1. We also discovered a role for p38 in the phosphorylation of α-catenin, a component of adherens junctions, suggesting that this phosphorylation may function as an important regulatory process. Taken together, these studies define a rich array of thrombin- and p38-regulated candidate proteins that may serve important roles in endothelial dysfunction.
        Cover page: Phosphoproteomic analysis of thrombin- and p38 MAPK-regulated signaling networks in endothelial cells
        Top