scholarly journals ALIX binds a YPX3L motif of the GPCR PAR1 and mediates ubiquitin-independent ESCRT-III/MVB sorting

2012 ◽  
Vol 197 (3) ◽  
pp. 407-419 ◽  
Author(s):  
Michael R. Dores ◽  
Buxin Chen ◽  
Huilan Lin ◽  
Unice J.K. Soh ◽  
May M. Paing ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Interacting Protein ◽  
Kinase Substrate ◽  
Endosomal Sorting ◽  
Ubiquitin Binding ◽  
Lysosomal Sorting ◽  
Protease Activated Receptor 1 ◽  
Signaling Receptors ◽  
G Protein Coupled ◽  
Hepatocyte Growth

The sorting of signaling receptors to lysosomes is an essential regulatory process in mammalian cells. During degradation, receptors are modified with ubiquitin and sorted by endosomal sorting complex required for transport (ESCRT)–0, –I, –II, and –III complexes into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). However, it remains unclear whether a single universal mechanism mediates MVB sorting of all receptors. We previously showed that protease-activated receptor 1 (PAR1), a G protein–coupled receptor (GPCR) for thrombin, is internalized after activation and sorted to lysosomes independent of ubiquitination and the ubiquitin-binding ESCRT components hepatocyte growth factor–regulated tyrosine kinase substrate and Tsg101. In this paper, we report that PAR1 sorted to ILVs of MVBs through an ESCRT-III–dependent pathway independent of ubiquitination. We further demonstrate that ALIX, a charged MVB protein 4–ESCRT-III interacting protein, bound to a YPX3L motif of PAR1 via its central V domain to mediate lysosomal degradation. This study reveals a novel MVB/lysosomal sorting pathway for signaling receptors that bypasses the requirement for ubiquitination and ubiquitin-binding ESCRTs and may be applicable to a subset of GPCRs containing YPXnL motifs.

scholarly journals AP-3 regulates PAR1 ubiquitin-independent MVB/lysosomal sorting via an ALIX-mediated pathway

2012 ◽  
Vol 23 (18) ◽  
pp. 3612-3623 ◽  
Author(s):  
Michael R. Dores ◽  
May M. Paing ◽  
Huilan Lin ◽  
William A. Montagne ◽  
Adriano Marchese ◽  
...  
Keyword(s):  
Adaptor Protein ◽  
Kinase Substrate ◽  
Endosomal Sorting ◽  
Escrt Machinery ◽  
Early Endosomes ◽  
Lysosomal Sorting ◽  
Protease Activated Receptor 1 ◽  
Signaling Receptors ◽  
G Protein Coupled ◽  
Hepatocyte Growth

The sorting of signaling receptors within the endocytic system is important for appropriate cellular responses. After activation, receptors are trafficked to early endosomes and either recycled or sorted to lysosomes and degraded. Most receptors trafficked to lysosomes are modified with ubiquitin and recruited into an endosomal subdomain enriched in hepatocyte growth factor–regulated tyrosine kinase substrate (HRS), a ubiquitin-binding component of the endosomal-sorting complex required for transport (ESCRT) machinery, and then sorted into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs)/lysosomes. However, not all receptors use ubiquitin or the canonical ESCRT machinery to sort to MVBs/lysosomes. This is exemplified by protease-activated receptor-1 (PAR1), a G protein–coupled receptor for thrombin, which sorts to lysosomes independent of ubiquitination and HRS. We recently showed that the adaptor protein ALIX binds to PAR1, recruits ESCRT-III, and mediates receptor sorting to ILVs of MVBs. However, the mechanism that initiates PAR1 sorting at the early endosome is not known. We now report that the adaptor protein complex-3 (AP-3) regulates PAR1 ubiquitin-independent sorting to MVBs through an ALIX-dependent pathway. AP-3 binds to a PAR1 cytoplasmic tail–localized tyrosine-based motif and mediates PAR1 lysosomal degradation independent of ubiquitination. Moreover, AP-3 facilitates PAR1 interaction with ALIX, suggesting that AP-3 functions before PAR1 engagement of ALIX and MVB/lysosomal sorting.

scholarly journals Arrestin-2 Interacts with the Endosomal Sorting Complex Required for Transport Machinery to Modulate Endosomal Sorting of CXCR4

2010 ◽  
Vol 21 (14) ◽  
pp. 2529-2541 ◽  
Author(s):  
Rohit Malik ◽  
Adriano Marchese
Keyword(s):  
Molecular Mechanisms ◽  
Lysosomal Degradation ◽  
Kinase Substrate ◽  
Endosomal Sorting ◽  
Adaptor Molecule ◽  
Escrt Machinery ◽  
Early Endosomes ◽  
G Protein Coupled ◽  
Hepatocyte Growth ◽  
Dependent Mechanism

The chemokine receptor CXCR4, a G protein-coupled receptor, is targeted for lysosomal degradation via a ubiquitin-dependent mechanism that involves the endosomal sorting complex required for transport (ESCRT) machinery. We have reported recently that arrestin-2 also targets CXCR4 for lysosomal degradation; however, the molecular mechanisms by which this occurs remain poorly understood. Here, we show that arrestin-2 interacts with ESCRT-0, a protein complex that recognizes and sorts ubiquitinated cargo into the degradative pathway. Signal-transducing adaptor molecule (STAM)-1, but not related STAM-2, interacts directly with arrestin-2 and colocalizes with CXCR4 on early endosomal antigen 1-positive early endosomes. Depletion of STAM-1 by RNA interference and disruption of the arrestin-2/STAM-1 interaction accelerates agonist promoted degradation of CXCR4, suggesting that STAM-1 via its interaction with arrestin-2 negatively regulates CXCR4 endosomal sorting. Interestingly, disruption of this interaction blocks agonist promoted ubiquitination of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) but not CXCR4 and STAM-1 ubiquitination. Our data suggest a mechanism whereby arrestin-2 via its interaction with STAM-1 modulates CXCR4 sorting by regulating the ubiquitination status of HRS.

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

2015 ◽  
Vol 26 (25) ◽  
pp. 4660-4673 ◽  
Author(s):  
Michael R. Dores ◽  
Huilan Lin ◽  
Neil J. Grimsey ◽  
Francisco Mendez ◽  
JoAnn Trejo
Keyword(s):  
G Protein ◽  
Critical Role ◽  
Adaptor Protein ◽  
G Protein Coupled Receptors ◽  
Late Endosomes ◽  
Ubiquitin Binding ◽  
Lysosomal Sorting ◽  
Protease Activated Receptor 1 ◽  
Interfering Rna ◽  
G Protein Coupled

The sorting of G protein–coupled receptors (GPCRs) to lysosomes is critical for proper signaling and cellular responses. We previously showed that the adaptor protein ALIX regulates lysosomal degradation of protease-activated receptor-1 (PAR1), a GPCR for thrombin, independent of ubiquitin-binding ESCRTs and receptor ubiquitination. However, the mechanisms that regulate ALIX function during PAR1 lysosomal sorting are not known. Here we show that the mammalian α-arrestin arrestin domain–containing protein-3 (ARRDC3) regulates ALIX function in GPCR sorting via ubiquitination. ARRDC3 colocalizes with ALIX and is required for PAR1 sorting at late endosomes and degradation. Depletion of ARRDC3 by small interfering RNA disrupts ALIX interaction with activated PAR1 and the CHMP4B ESCRT-III subunit, suggesting that ARRDC3 regulates ALIX activity. We found that ARRDC3 is required for ALIX ubiquitination induced by activation of PAR1. A screen of nine mammalian NEDD4-family E3 ubiquitin ligases revealed a critical role for WWP2. WWP2 interacts with ARRDC3 and not ALIX. Depletion of WWP2 inhibited ALIX ubiquitination and blocked ALIX interaction with activated PAR1 and CHMP4B. These findings demonstrate a new role for the α-arrestin ARRDC3 and the E3 ubiquitin ligase WWP2 in regulation of ALIX ubiquitination and lysosomal sorting of GPCRs.

scholarly journals An Essential Role for SNX1 in Lysosomal Sorting of Protease-activated Receptor-1: Evidence for Retromer-, Hrs-, and Tsg101-independent Functions of Sorting Nexins

2006 ◽  
Vol 17 (3) ◽  
pp. 1228-1238 ◽  
Author(s):  
Anuradha Gullapalli ◽  
Breann L. Wolfe ◽  
Courtney T. Griffin ◽  
Terry Magnuson ◽  
JoAnn Trejo
Keyword(s):  
Essential Role ◽  
Resistant Mutant ◽  
Interacting Protein ◽  
Sorting Nexins ◽  
Surface Receptors ◽  
Endosomal Membrane ◽  
Independent Functions ◽  
Lysosomal Sorting ◽  
Protease Activated Receptor 1 ◽  
In Cells

Sorting nexin 1 (SNX1) and SNX2 are the mammalian homologues of the yeast Vps5p retromer component that functions in endosome-to-Golgi trafficking. SNX1 is also implicated in endosome-to-lysosome sorting of cell surface receptors, although its requirement in this process remains to be determined. To assess SNX1 function in endocytic sorting of protease-activated receptor-1 (PAR1), we used siRNA to deplete HeLa cells of endogenous SNX1 protein. PAR1, a G-protein-coupled receptor, is proteolytically activated by thrombin, internalized, sorted predominantly to lysosomes, and efficiently degraded. Strikingly, depletion of endogenous SNX1 by siRNA markedly inhibited agonist-induced PAR1 degradation, whereas expression of a SNX1 siRNA-resistant mutant protein restored agonist-promoted PAR1 degradation in cells lacking endogenous SNX1, indicating that SNX1 is necessary for lysosomal degradation of PAR1. SNX1 is known to interact with components of the mammalian retromer complex and Hrs, an early endosomal membrane-associated protein. However, activated PAR1 degradation was not affected in cells depleted of retromer Vps26/Vps35 subunits, Hrs or Tsg101, an Hrs-interacting protein. We further show that SNX2, which dimerizes with SNX1, is not essential for lysosomal sorting of PAR1, but rather can regulate PAR1 degradation by disrupting endosomal localization of endogenous SNX1 when ectopically expressed. Together, our findings establish an essential role for endogenous SNX1 in sorting activated PAR1 to a distinct lysosomal degradative pathway that is independent of retromer, Hrs, and Tsg101.

scholarly journals Host ESCRT factors are recruited during chikungunya virus infection and are required for the intracellular viral replication cycle

2020 ◽  
Vol 295 (23) ◽  
pp. 7941-7957 ◽  
Author(s):  
Shiho Torii ◽  
Yasuko Orba ◽  
Michihito Sasaki ◽  
Koshiro Tabata ◽  
Yuji Wada ◽  
...  
Keyword(s):  
Chikungunya Virus ◽  
Host Factors ◽  
Genome Replication ◽  
Sirna Screen ◽  
Chikv Infection ◽  
Kinase Substrate ◽  
Endosomal Sorting ◽  
Viral Genome Replication ◽  
Human Host Factors ◽  
Hepatocyte Growth

Chikungunya fever is a re-emerging zoonotic disease caused by chikungunya virus (CHIKV), a member of the Alphavirus genus in the Togaviridae family. Only a few studies have reported on the host factors required for intracellular CHIKV trafficking. Here, we conducted an imaging-based siRNA screen to identify human host factors for intracellular trafficking that are involved in CHIKV infection, examined their interactions with CHIKV proteins, and investigated the contributions of these proteins to CHIKV infection. The results of the siRNA screen revealed that host endosomal sorting complexes required for transport (ESCRT) proteins are recruited during CHIKV infection. Co-immunoprecipitation analyses revealed that both structural and nonstructural CHIKV proteins interact with hepatocyte growth factor–regulated tyrosine kinase substrate (HGS), a component of the ESCRT-0 complex. We also observed that HGS co-localizes with the E2 protein of CHIKV and with dsRNA, a marker of the replicated CHIKV genome. Results from gene knockdown analyses indicated that, along with other ESCRT factors, HGS facilitates both genome replication and post-translational steps during CHIKV infection. Moreover, we show that ESCRT factors are also required for infections with other alphaviruses. We conclude that during CHIKV infection, several ESCRT factors are recruited via HGS and are involved in viral genome replication and post-translational processing of viral proteins.

scholarly journals Down-Regulation of Protease-activated Receptor-1 Is Regulated by Sorting Nexin 1

2002 ◽  
Vol 13 (6) ◽  
pp. 1965-1976 ◽  
Author(s):  
Yingjie Wang ◽  
Yixing Zhou ◽  
Katalin Szabo ◽  
Carol Renfrew Haft ◽  
JoAnn Trejo
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptor ◽  
Down Regulation ◽  
Sorting Nexin ◽  
Early Endosomes ◽  
Lysosomal Sorting ◽  
Protease Activated Receptor 1 ◽  
Phox Homology ◽  
G Protein Coupled ◽  
Sorting Nexin 1

Degradation or “down-regulation” of protease-activated receptor-1 (PAR1), a G protein-coupled receptor for thrombin, is critical for termination of receptor signaling. Toward understanding the molecular mechanisms by which activated PAR1 is internalized, sorted to lysosomes, and degraded, we investigated whether PAR1 interacted with sorting nexin 1 (SNX1). SNX1 is a membrane-associated protein that functions in lysosomal sorting of the epidermal growth factor receptor. In vitro biochemical binding assays revealed a specific interaction between a glutathione S-transferase fusion of SNX1 and PAR1. In HeLa cells, activated PAR1 colocalized with endogenous SNX1 and coimmunoprecipitated SNX1. SNX1 contains a phox homology domain predicted to bind phosphatidylinositol-3-phosphate and a C-terminal coiled-coil region. To assess SNX1 function, we examined the effects of SNX1 deletion mutants on PAR1 trafficking. Neither the N terminus nor phox homology domain of SNX1 affected PAR1 trafficking. By contrast, overexpression of SNX1 C-terminal domain markedly inhibited agonist-induced degradation of PAR1, whereas internalization remained virtually intact. Immunofluorescence microscopy studies revealed substantial PAR1 accumulation in an early endosome antigen-1-positive compartment in agonist-treated cells expressing SNX1 C terminus. By contrast, lysosome-associated membrane protein-1 distribution was unperturbed. Together, these findings strongly suggest a role for SNX1 in sorting of PAR1 from early endosomes to lysosomes. Moreover, this study provides the first example of a protein involved in lysosomal sorting of a G protein-coupled receptor in mammalian cells.

scholarly journals Plasticity of Polyubiquitin Recognition as Lysosomal Targeting Signals by the Endosomal Sorting Machinery

2007 ◽  
Vol 18 (10) ◽  
pp. 3952-3965 ◽  
Author(s):  
Herve Barriere ◽  
Csilla Nemes ◽  
Kai Du ◽  
Gergely L. Lukacs
Keyword(s):  
Multivesicular Body ◽  
Invariant Chain ◽  
Endosomal Sorting ◽  
Cytoplasmic Tails ◽  
Lysosomal Sorting ◽  
Targeting Signals ◽  
Lysosomal Targeting ◽  
Necessary And Sufficient ◽  
Kinetics Of ◽  
Hepatocyte Growth

Lysosomal targeting is fundamental for the regulated disposal of ubiquitinated membrane proteins from the cell surface. To elucidate ubiquitin (Ub) configurations that are necessary and sufficient as multivesicular body (MVB)/lysosomal-sorting motifs, the intraendosomal destination and transport kinetics of model transmembrane cargo molecules bearing monoubiquitinated, multi-monoubiquitinated, or polyubiquitinated cytoplasmic tails were determined. Monomeric CD4 chimeras with K63-linked poly-Ub chains and tetrameric CD4-mono-Ub chimeras were rapidly targeted to the lysosome. In contrast, lysosomal delivery of CD4 chimeras exposing K48-linked Ub chains was delayed, whereas delivery of monoubiquitinated CD4 chimeras was undetectable. Similar difference was observed in the lysosomal targeting of mono- versus polyubiquitinated invariant chain and CD4 ubiquitinated by the MARCH (membrane-associated RING-CH) IV Ub ligase. Consistent with this, Hrs (hepatocyte growth factor regulated tyrosine kinase phosphorylated substrate), an endosomal sorting adaptor, binds preferentially to K63-Ub chain and negligibly to mono-Ub. These results highlight the plasticity of Ub as a sorting signal and its recognition by the endosomal sorting machinery, and together with previous data, suggest a regulatory role for assembly and disassembly of Ub chains of specific topology in lysosomal cargo sorting.

scholarly journals The ESCRT-0 subcomplex component Hrs/Hgs is a master regulator of myogenesis via modulation of signaling and degradation pathways

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
L. Coudert ◽  
A. Osseni ◽  
Y. G. Gangloff ◽  
L. Schaeffer ◽  
P. Leblanc
Keyword(s):  
Muscle Development ◽  
Signaling Cascades ◽  
Degradation Pathways ◽  
Master Regulator ◽  
Kinase Substrate ◽  
Endosomal Sorting ◽  
Spatial Regulation ◽  
First Time ◽  
Inhibition Of Differentiation ◽  
Hepatocyte Growth

Abstract Background Myogenesis is a highly regulated process ending with the formation of myotubes, the precursors of skeletal muscle fibers. Differentiation of myoblasts into myotubes is controlled by myogenic regulatory factors (MRFs) that act as terminal effectors of signaling cascades involved in the temporal and spatial regulation of muscle development. Such signaling cascades converge and are controlled at the level of intracellular trafficking, but the mechanisms by which myogenesis is regulated by the endosomal machinery and trafficking is largely unexplored. The Endosomal Sorting Complex Required for Transport (ESCRT) machinery composed of four complexes ESCRT-0 to ESCRT-III regulates the biogenesis and trafficking of endosomes as well as the associated signaling and degradation pathways. Here, we investigate its role in regulating myogenesis. Results We uncovered a new function of the ESCRT-0 hepatocyte growth factor-regulated tyrosine kinase substrate Hrs/Hgs component in the regulation of myogenesis. Hrs depletion strongly impairs the differentiation of murine and human myoblasts. In the C2C12 murine myogenic cell line, inhibition of differentiation was attributed to impaired MRF in the early steps of differentiation. This alteration is associated with an upregulation of the MEK/ERK signaling pathway and a downregulation of the Akt2 signaling both leading to the inhibition of differentiation. The myogenic repressors FOXO1 as well as GSK3β were also found to be both activated when Hrs was absent. Inhibition of the MEK/ERK pathway or of GSK3β by the U0126 or azakenpaullone compounds respectively significantly restores the impaired differentiation observed in Hrs-depleted cells. In addition, functional autophagy that is required for myogenesis was also found to be strongly inhibited. Conclusions We show for the first time that Hrs/Hgs is a master regulator that modulates myogenesis at different levels through the control of trafficking, signaling, and degradation pathways.

Ubiquitin and endocytic protein sorting

2005 ◽  
Vol 41 ◽  
pp. 81-98 ◽  
Author(s):  
Sylvie Urbé
Keyword(s):  
Membrane Proteins ◽  
Tyrosine Kinases ◽  
Mammalian Cells ◽  
Degradation Process ◽  
Deubiquitinating Enzymes ◽  
Endosomal Sorting ◽  
Binding Domains ◽  
C Terminus ◽  
Lysosomal Sorting ◽  
Ubiquitin Ligase E3

Ubiquitin plays a fundamental role not only in proteasome-mediated protein degradation but also in the targeting of membrane proteins for degradation inside the lysosome. Ubiquitination provides a key signal for endosomal sorting of membrane proteins into the MVB (multi-vesicular body), which delivers its cargo to the proteolytic interior of the lysosome. Attachment of single ubiquitin molecules, rather than ubiquitin chains, to one or multiple lysines of the cytoplasmic domains of many growth factor receptors, ion channels and other membrane transporters is sufficient to target these proteins to a complex sorting apparatus on the endosome. This machinery selects ubiquitinated proteins for lysosomal sorting through consecutive interactions with a variety of ubiquitin-binding domains. The major ubiquitin ligase (E3) responsible for ubiquitination in this pathway in yeast is the HECT [homologous to E6-AP (E6-associated protein) C-terminus]-ligase, Rsp5, whereas in mammalian cells the RING (really interesting new gene)-ligase Cbl has been implicated in the down-regulation of several RTKs (receptor tyrosine kinases). Ubiquitinated receptors can be rescued from degradation by the activity of DUBs (deubiquitinating enzymes), which may provide a proofreading mechanism that enhances the fidelity of this sorting and degradation process. DUBs also allow for recycling of the ubiquitin moieties from proteins prior to their final commitment to the MVB and lysosome interior.

scholarly journals SNX-1 and RME-8 oppose the assembly of HGRS-1/ESCRT-0 degradative microdomains on endosomes

2017 ◽  
Vol 114 (3) ◽  
pp. E307-E316 ◽  
Author(s):  
Anne Norris ◽  
Prasad Tammineni ◽  
Simon Wang ◽  
Julianne Gerdes ◽  
Alexandra Murr ◽  
...  
Keyword(s):  
Kinase Substrate ◽  
Endosomal Sorting ◽  
Sorting Nexin ◽  
Binding Partner ◽  
Regulatory Interactions ◽  
C Elegans ◽  
J Domain ◽  
Vacuolar Protein ◽  
Hepatocyte Growth

After endocytosis, transmembrane cargo reaches endosomes, where it encounters complexes dedicated to opposing functions: recycling and degradation. Microdomains containing endosomal sorting complexes required for transport (ESCRT)-0 component Hrs [hepatocyte growth factor-regulated tyrosine kinase substrate (HGRS-1) in Caenorhabditis elegans] mediate cargo degradation, concentrating ubiquitinated cargo and organizing the activities of ESCRT. At the same time, retromer associated sorting nexin one (SNX-1) and its binding partner, J-domain protein RME-8, sort cargo away from degradation, promoting cargo recycling to the Golgi. Thus, we hypothesized that there could be important regulatory interactions between retromer and ESCRT that balance degradative and recycling functions. Taking advantage of the naturally large endosomes of the C. elegans coelomocyte, we visualized complementary ESCRT-0 and RME-8/SNX-1 microdomains in vivo and assayed the ability of retromer and ESCRT microdomains to regulate one another. We found in snx-1(0) and rme-8(ts) mutants increased endosomal coverage and intensity of HGRS-1–labeled microdomains, as well as increased total levels of HGRS-1 bound to membranes. These effects are specific to SNX-1 and RME-8, as loss of other retromer components SNX-3 and vacuolar protein sorting-associated protein 35 (VPS-35) did not affect HGRS-1 microdomains. Additionally, knockdown of hgrs-1 had little to no effect on SNX-1 and RME-8 microdomains, suggesting directionality to the interaction. Separation of the functionally distinct ESCRT-0 and SNX-1/RME-8 microdomains was also compromised in the absence of RME-8 and SNX-1, a phenomenon we observed to be conserved, as depletion of Snx1 and Snx2 in HeLa cells also led to greater overlap of Rme-8 and Hrs on endosomes.

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