Neil J. Grimsey

Ubiquitin plays an atypical role in GPCR-induced p38 MAP kinase activation on endosomes

K63-linked ubiquitination of GPCRs mediated by the NEDD4-2 E3 ubiquitin ligase regulates recruitment of a TAB1–TAB2 complex on endosomes and stimulates p38 MAPK through a noncanonical pathway, which is critical for endothelial barrier disruption.

The α-arrestin ARRDC3 mediates ALIX ubiquitination and G protein-coupled receptor lysosomal sorting

The novel ALIX-dependent GPCR sorting pathway is regulated by the a-arrestin ARRDC3. A critical role is also shown for the E3 ubiquitin ligase WWP2 in regulation of ALIX ubiquitination and lysosomal sorting of GPCRs.

ALIX Regulates the Ubiquitin-Independent Lysosomal Sorting of the P2Y1 Purinergic Receptor via a YPX3L Motif.

Endocytic sorting and lysosomal degradation are integral to the regulation of G protein-coupled receptor (GPCR) function. Upon ligand binding, classical GPCRs are activated, internalized and recycled or sorted to lysosomes for degradation, a process that requires receptor ubiquitination. However, recent studies have demonstrated that numerous GPCRs are sorted to lysosomes independent of receptor ubiquitination. Here, we describe an ubiquitin-independent lysosomal sorting pathway for the purinergic GPCR P2Y1. After activation, P2Y1 sorts to lysosomes for degradation independent of direct ubiquitination that is mediated by a YPX3L motif within the second intracellular loop that serves as a binding site for the adaptor protein ALIX. Depletion of ALIX or site-directed mutation of the YPX3L motif inhibits P2Y1 sorting into the lumen of multivesicular endosomes/lysosomes and degradation. These findings confirm the function of YPX3L motifs as lysosomal targeting sequences for GPCRs and demonstrate that ALIX mediates the ubiquitin-independent degradation of certain GPCRs.

Regulation of protease‐activated receptor signaling by post‐translational modifications

Protease‐activated receptors (PARs) are a unique family of G‐protein‐coupled receptors (GPCRs) that are irreversibly activated following proteolytic cleavage of their extracellular N‐terminus. PARs play critical functions in hemostasis, thrombosis, inflammation, embryonic development, and cancer progression. Because of the irreversible proteolytic nature of PAR activation, signaling by the receptors is tightly regulated. Three distinct processes including desensitization, internalization, and lysosomal degradation, regulate the temporal and spatial aspects of activated PAR signaling. Post‐translational modifications play a critical role in regulating each of these processes and here we review the nature of PAR post‐translational modifications and their importance in signal regulation. The PARs are activated by numerous proteases, and some can elicit distinct cellular responses, how this biased agonism is determined is unknown. Further study of the function of post‐translational modifications of the PARs will lead to a greater understanding of the physiological regulation of baised agonism and how PAR signaling is precisely controlled in different cellular contexts.

The unfolded protein response regulator ATF6 promotes mesodermal differentiation

The proteostasis stress-response protein ATF6 directs the mesodermal lineage during vertebrate embryogenesis and early development. A primitive role for ATF6 The endoplasmic reticulum (ER) mediates and monitors the folding, packaging, and transport of proteins in cells. The unfolded protein response (UPR) preserves ER homeostasis in the adult organism; however, inactivating mutations in the UPR-associated transcription factor ATF6 cause congenital vision defects, suggesting an embryonic role as well. Kroeger et al. found that ATF6 was critical to the differentiation of stem cells to the mesodermal lineage, at least in part, by promoting the growth and maturation of the ER, which presumably enables cells to stably produce the abundance of proteins necessary for development. Thus, the ER homeostasis protein in adult cells first directs ER development in embryonic cells. Activating ATF6 promoted the development of functional vascular endothelial cells from stem cells in culture dishes, suggesting that manipulating ATF6 may facilitate the production of mesodermal tissues for research or therapy. ATF6 encodes a transcription factor that is anchored in the endoplasmic reticulum (ER) and activated during the unfolded protein response (UPR) to protect cells from ER stress. Deletion of the isoform activating transcription factor 6α (ATF6α) and its paralog ATF6β results in embryonic lethality and notochord dysgenesis in nonhuman vertebrates, and loss-of-function mutations in ATF6α are associated with malformed neuroretina and congenital vision loss in humans. These phenotypes implicate an essential role for ATF6 during vertebrate development. We investigated this hypothesis using human stem cells undergoing differentiation into multipotent germ layers, nascent tissues, and organs. We artificially activated ATF6 in stem cells with a small-molecule ATF6 agonist and, conversely, inhibited ATF6 using induced pluripotent stem cells from patients with ATF6 mutations. We found that ATF6 suppressed pluripotency, enhanced differentiation, and unexpectedly directed mesodermal cell fate. Our findings reveal a role for ATF6 during differentiation and identify a new strategy to generate mesodermal tissues through the modulation of the ATF6 arm of the UPR.

Recycling and Endosomal Sorting of Protease-activated Receptor-1 Is Distinctly Regulated by Rab11A and Rab11B Proteins

Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor that undergoes proteolytic irreversible activation by coagulant and anti-coagulant proteases. Given the irreversible activation of PAR1, signaling by the receptor is tightly regulated through desensitization and intracellular trafficking. PAR1 displays both constitutive and agonist-induced internalization. Constitutive internalization of PAR1 is important for generating an internal pool of naïve receptors that replenish the cell surface and facilitate resensitization, whereas agonist-induced internalization of PAR1 is critical for terminating G protein signaling. We showed that PAR1 constitutive internalization is mediated by the adaptor protein complex-2 (AP-2), whereas AP-2 and epsin control agonist-induced PAR1 internalization. However, the mechanisms that regulate PAR1 recycling are not known. In the present study we screened a siRNA library of 140 different membrane trafficking proteins to identify key regulators of PAR1 intracellular trafficking. In addition to known mediators of PAR1 endocytosis, we identified Rab11B as a critical regulator of PAR1 trafficking. We found that siRNA-mediated depletion of Rab11B and not Rab11A blocks PAR1 recycling, which enhanced receptor lysosomal degradation. Although Rab11A is not required for PAR1 recycling, depletion of Rab11A resulted in intracellular accumulation of PAR1 through disruption of basal lysosomal degradation of the receptor. Moreover, enhanced degradation of PAR1 observed in Rab11B-deficient cells is blocked by depletion of Rab11A and the autophagy related-5 protein, suggesting that PAR1 is shuttled to an autophagic degradation pathway in the absence of Rab11B recycling. Together these findings suggest that Rab11A and Rab11B differentially regulate intracellular trafficking of PAR1 through distinct endosomal sorting mechanisms.

Integration of endothelial protease-activated receptor-1 inflammatory signaling by ubiquitin

Purpose of reviewThe maintenance and integrity of the endothelial barrier is essential for vascular homeostasis. Endothelial barrier dysfunction is mediated by various inflammatory factors, many of which act through G protein-coupled receptors including protease-activated receptors (PARs). PARs are expressed in multiple cell types in the vasculature and mediate cellular responses to thrombin, the key effector protease of the coagulation cascade. Thrombin activation of PAR1 induces endothelial barrier permeability through multiple pathways. Here, we discuss the mechanism by which thrombin activation of PAR1 promotes endothelial barrier breakdown and highlight recent advances that have provided new insight into molecular mechanisms that control endothelial barrier integrity. Recent findingsAlthough the signal transduction pathways induced by thrombin activation of PAR1 in endothelial cells have been extensively studied, the key regulatory mechanisms remain poorly understood. Posttranslational modifications are integral to the regulation of PAR1 signaling and recent studies suggest a novel function for ubiquitination of PAR1 in regulation of endothelial barrier permeability. SummaryAn understanding of how endothelial barrier permeability is regulated by thrombin activation of PAR1 is important for the discovery of new drug targets that can be manipulated to control endothelial barrier permeability and prevent progression of vascular inflammation.

A Tyrosine Switch on NEDD4-2 E3 Ligase Transmits GPCR Inflammatory Signaling

SUMMARY Ubiquitination is essential for protein degradation and signaling and pivotal to many physiological processes. Ubiquitination of a subset of G-protein-coupled receptors (GPCRs) by the E3 ligase NEDD4–2 is required for p38 activation, but how GPCRs activate NEDD4–2 to promote ubiquitinmediated signaling is not known. Here, we report that the GPCR protease-activated receptor-1 (PAR1) stimulates c-Src-mediated tyrosine phosphorylation and activation of NEDD4–2 to promote p38 signaling and endothelial barrier disruption. Using mass spectrometry, we identified a unique phosphorylated tyrosine (Y)-485 within the 2,3-linker peptide between WW domain 2 and 3 of NEDD4–2 in agonist-stimulated cells. Mutation of NEDD4–2 Y485 impaired E3 ligase activity and failed to rescue PAR1-stimulated p38 activation and endothelial barrier permeability. The purinergic P2Y1 receptor also required c-Src and NEDD4–2 tyrosine phosphorylation for p38 activation. These studies reveal a novel role for c-Src in GPCR-induced NEDD4–2 activation, which is critical for driving ubiquitin-mediated p38 inflammatory signaling.