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Open Access Publications from the University of California

Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2023)

-arrestins are a recently discovered family of mammalian proteins that function as trafficking and signaling adapters. Like -arrestins, which are structurally similar to -arrestins, -arrestins are known to regulate trafficking and signaling of G protein-coupled receptors (GPCRs). However, little is known about -arrestin functions in health and disease, as well as the molecular mechanisms that control their diverse functions. Thus far, the -arrestin ARRDC3 has been most closely linked to regulation of GPCR function as well as to breast cancer. ARRDC3 was demonstrated to act as a tumor suppressor in invasive breast cancer in vitro and in vivo, in large part due to regulation of GPCR trafficking and signaling. Yet, the molecular mechanisms which control ARRDC3 function are unknown, and are important in order to better understand -arrestin tumor suppressor function. Other arrestins are known to be regulated by post-translational modifications, including ubiquitination and phosphorylation, which suggests that ARRDC3 may be subject to similar regulatory mechanisms. In this dissertation, I explored the molecular mechanisms that control -arrestin ARRDC3 tumor suppressor function. We found that ARRDC3 has the potential to interact with multiple protein modifying enzymes, including ubiquitin ligases, kinases, phosphatases, and more. I then examined ubiquitination as a key regulator of ARRDC3 function. I found that ARRDC3 ubiquitination is mediated primarily by two proline-rich PPXY motifs in the ARRDC3 C-tail domain. The ARRDC4 PPXY motifs are critical for recruiting WWP2 E3 ubiquitin ligase and mediating ARRDC3 ubiquitination. Further, ubiquitination and the PPXY motifs are essential for ARRDC3 function in regulating GPCR trafficking and signaling. Additionally, ubiquitination and the PPXY motifs mediate ARRDC3 protein degradation and dictate ARRDC3 subcellular localization. My work demonstrates an important role for ubiquitination in regulating ARRDC3 function. Taken together, the studies summarized in this dissertation reveal a key mechanism by which ARRDC3 divergent functions are controlled.

    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2023)
    The α-arrestin ARRDC3 is a recently discovered tumor suppressor in invasive breast cancer that functions as a multifaceted adaptor protein to control protein trafficking and cellular signaling. However, the molecular mechanisms that control ARRDC3 function are unknown. Other arrestins are known to be regulated by posttranslational modifications, suggesting that ARRDC3 may be subject to similar regulatory mechanisms. Here we report that ubiquitination is a key regulator of ARRDC3 function and is mediated primarily by two proline-rich PPXY motifs in the ARRDC3 C-tail domain. Ubiquitination and the PPXY motifs are essential for ARRDC3 function in regulating GPCR trafficking and signaling. Additionally, ubiquitination and the PPXY motifs mediate ARRDC3 protein degradation, dictate ARRDC3 subcellular localization, and are required for interaction with the NEDD4-family E3 ubiquitin ligase WWP2. These studies demonstrate a role for ubiquitination in regulating ARRDC3 function and reveal a mechanism by which ARRDC3 divergent functions are controlled.
      Cover page: Divergent regulation of α-arrestin ARRDC3 function by ubiquitination
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2022)
      Significance: Adaptor proteins control the spatiotemporal dynamics of cellular signaling. Dysregulation of adaptor protein function can cause aberrant cell signaling and promote cancer. The arrestin family of adaptor proteins are known to regulate signaling by the superfamily of G protein-coupled receptors (GPCRs). The GPCRs are highly druggable and implicated in cancer progression. However, the molecular mechanisms responsible for arrestin dysregulation and the impact on GPCR function in cancer have yet to be fully elucidated. Recent Advances: A new family of mammalian arrestins, termed the α-arrestins, was recently discovered. The α-arrestin, arrestin domain-containing protein 3 (ARRDC3), in particular, has been identified as a tumor suppressor and is reported to control cellular signaling of GPCRs in cancer. Critical Issues: Compared with the extensively studied mammalian β-arrestins, there is limited information regarding the regulatory mechanisms that control α-arrestin activation and function. Here, we discuss the molecular mechanisms that regulate ARRDC3, which include post-translational modifications such as phosphorylation and ubiquitination. We also provide evidence that ARRDC3 can interact with a wide array of proteins that control diverse biological functions. Future Directions: ARRDC3 interacts with numerous proteins and is likely to display diverse functions in cancer, metabolic disease, and other syndromes. Thus, understanding the regulatory mechanisms of ARRDC3 activity in various cellular contexts is critically important. Recent studies suggest that α-arrestins may be regulated through post-translational modification, which is known to impact adaptor protein function. However, additional studies are needed to determine how these regulatory mechanisms affect ARRDC3 tumor suppressor function. Antioxid. Redox Signal. 36, 1066-1079.
        Cover page: The α-Arrestin ARRDC3 Is an Emerging Multifunctional Adaptor Protein in Cancer
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2023)
        Introduction: Dysfunction of the endothelium impairs its' protective role and promotes inflammation and progression of vascular diseases. Activated Protein C (APC) elicits endothelial cytoprotective responses including barrier stabilization, anti-inflammatory and anti-apoptotic responses through the activation of the G protein-coupled receptor (GPCR) protease-activated receptor-1 (PAR1) and is a promising therapeutic. Despite recent advancements in developing new Activated protein C variants with clinical potential, the mechanism by which APC/PAR1 promotes different cytoprotective responses remains unclear and is important to understand to advance Activated protein C and new targets as future therapeutics. Here we examined the mechanisms by which APC/PAR1 attenuates cytokine-induced pro-inflammatory vascular cell adhesion molecule (VCAM-1) expression, a key mediator of endothelial inflammatory responses. Methods: Quantitative multiplexed mass spectrometry analysis of Activated protein C treated endothelial cells, endothelial cell transcriptomics database (EndoDB) online repository queries, biochemical measurements of protein expression, quantitative real-time polymerase chain reaction (RT-qPCR) measurement of mRNA transcript abundance, pharmacological inhibitors and siRNA transfections of human cultured endothelial cells. Results: Here we report that Activated Protein C modulates phosphorylation of tumor necrosis factor (TNF)-α signaling pathway components and attenuates of TNF-α induced VCAM-1 expression independent of mRNA stability. Unexpectedly, we found a critical role for the G protein-coupled receptor co-receptor sphingosine-1 phosphate receptor-1 (S1PR1) and the G protein receptor kinase-2 (GRK2) in mediating APC/PAR1 anti-inflammatory responses in endothelial cells. Discussion: This study provides new knowledge of the mechanisms by which different APC/PAR1 cytoprotective responses are mediated through discrete β-arrestin-2-driven signaling pathways modulated by specific G protein-coupled receptor co-receptors and GRKs.
          Cover page: Endothelial APC/PAR1 distinctly regulates cytokine-induced pro-inflammatory VCAM-1 expression
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2021)
          The α-arrestin domain containing protein 3 (ARRDC3) is a tumor suppressor in triple-negative breast carcinoma (TNBC), a highly metastatic subtype of breast cancer that lacks targeted therapies. Thus, understanding the mechanisms and targets of ARRDC3 in TNBC is important. ARRDC3 regulates trafficking of protease-activated receptor 1 (PAR1, also known as F2R), a G-protein-coupled receptor (GPCR) implicated in breast cancer metastasis. Loss of ARRDC3 causes overexpression of PAR1 and aberrant signaling. Moreover, dysregulation of GPCR-induced Hippo signaling is associated with breast cancer progression. However, the mechanisms responsible for Hippo dysregulation remain unknown. Here, we report that the Hippo pathway transcriptional co-activator TAZ (also known as WWTR1) is the major effector of GPCR signaling and is required for TNBC migration and invasion. Additionally, ARRDC3 suppresses PAR1-induced Hippo signaling via sequestration of TAZ, which occurs independently of ARRDC3-regulated PAR1 trafficking. The ARRDC3 C-terminal PPXY motifs and TAZ WW domain are crucial for this interaction and are required for suppression of TNBC migration and lung metastasis in vivo. These studies are the first to demonstrate a role for ARRDC3 in regulating GPCR-induced TAZ activity in TNBC and reveal multi-faceted tumor suppressor functions of ARRDC3. This article has an associated First Person interview with the first author of the paper.
            Cover page: α-arrestin ARRDC3 tumor suppressor function is linked to GPCR-induced TAZ activation and breast cancer metastasis
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