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 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 The deubiquitinating enzyme DUB2A enhances CSF3 signalling by attenuating lysosomal routing of the CSF3 receptor

2011 ◽  
Vol 434 (2) ◽  
pp. 343-351 ◽  
Author(s):  
Annemarie Meenhuis ◽  
Carola Verwijmeren ◽  
Onno Roovers ◽  
Ivo P. Touw
Keyword(s):  
E3 Ligase ◽  
Deubiquitinating Enzyme ◽  
Deubiquitinating Enzymes ◽  
Lysosomal Degradation ◽  
Feedback Mechanisms ◽  
Endosomal Sorting ◽  
Adaptor Molecule ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Stimulating Factor

Ubiquitination of the CSF3R [CSF3 (colony-stimulating factor 3) receptor] occurs after activated CSF3Rs are internalized and reside in early endosomes. CSF3R ubiquitination is crucial for lysosomal routing and degradation. The E3 ligase SOCS3 (suppressor of cytokine signalling 3) has been shown to play a major role in this process. Deubiquitinating enzymes remove ubiquitin moieties from target proteins by proteolytic cleavage. Two of these enzymes, AMSH [associated molecule with the SH3 domain of STAM (signal transducing adaptor molecule)] and UBPY (ubiquitin isopeptidase Y), interact with the general endosomal sorting machinery. Whether deubiquitinating enzymes control CSF3R trafficking from early towards late endosomes is unknown. In the present study, we asked whether AMSH, UBPY or a murine family of deubiquitinating enzymes could fulfil such a role. This DUB family (deubiquitin enzyme family) comprises four members (DUB1, DUB1A, DUB2 and DUB2A), which were originally described as being haematopoietic-specific and cytokine-inducible, but their function in cytokine receptor routing and signalling has remained largely unknown. We show that DUB2A expression is induced by CSF3 in myeloid 32D cells and that DUB2 decreases ubiquitination and lysosomal degradation of the CSF3R, leading to prolonged signalling. These results support a model in which CSF3R ubiquitination is dynamically controlled at the early endosome by feedback mechanisms involving CSF3-induced E3 ligase (SOCS3) and deubiquitinase (DUB2A) activities.

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 The ubiquitin ligase deltex-3l regulates endosomal sorting of the G protein–coupled receptor CXCR4

2014 ◽  
Vol 25 (12) ◽  
pp. 1892-1904 ◽  
Author(s):  
Justine Holleman ◽  
Adriano Marchese
Keyword(s):  
G Protein ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Lysosomal Degradation ◽  
G Protein Coupled Receptor ◽  
Interacting Protein ◽  
Endosomal Sorting ◽  
Early Endosomes ◽  
New Gene ◽  
G Protein Coupled

G protein–coupled receptor (GPCR) sorting into the degradative pathway is important for limiting the duration and magnitude of signaling. Agonist activation of the GPCR CXCR4 induces its rapid ubiquitination and sorting to lysosomes via the endosomal sorting complex required for transport (ESCRT) pathway. We recently reported that ESCRT-0 ubiquitination is linked to the efficiency with which CXCR4 is sorted for lysosomal degradation; however mechanistic insight is lacking. Here we define a novel role for the really interesting new gene–domain E3 ubiquitin ligase deltex-3-like (DTX3L) in regulating CXCR4 sorting from endosomes to lysosomes. We show that DTX3L localizes to early endosomes upon CXCR4 activation and interacts directly with and inhibits the activity of the E3 ubiquitin ligase atrophin-1 interacting protein 4. This serves to limit the extent to which ESCRT-0 is ubiquitinated and is able to sort CXCR4 for lysosomal degradation. Therefore we define a novel role for DTX3L in GPCR endosomal sorting and reveal an unprecedented link between two distinct E3 ubiquitin ligases to control the activity of the ESCRT machinery.

scholarly journals ESCRT-dependent targeting of plasma membrane localized KCa3.1 to the lysosomes

2010 ◽  
Vol 299 (5) ◽  
pp. C1015-C1027 ◽  
Author(s):  
Corina M. Balut ◽  
Yajuan Gao ◽  
Sandra A. Murray ◽  
Patrick H. Thibodeau ◽  
Daniel C. Devor
Keyword(s):  
Plasma Membrane ◽  
Molecular Mechanisms ◽  
Close Association ◽  
Significant Inhibition ◽  
Dominant Negative ◽  
Human Embryonic Kidney Cells ◽  
Endosomal Sorting ◽  
Escrt Machinery ◽  
Interfering Rna

The number of intermediate-conductance, Ca2+-activated K+ channels (KCa3.1) present at the plasma membrane is deterministic in any physiological response. However, the mechanisms by which KCa3.1 channels are removed from the plasma membrane and targeted for degradation are poorly understood. Recently, we demonstrated that KCa3.1 is rapidly internalized from the plasma membrane, having a short half-life in both human embryonic kidney cells (HEK293) and human microvascular endothelial cells (HMEC-1). In this study, we investigate the molecular mechanisms controlling the degradation of KCa3.1 heterologously expressed in HEK and HMEC-1 cells. Using immunofluorescence and electron microscopy, as well as quantitative biochemical analysis, we demonstrate that membrane KCa3.1 is targeted to the lysosomes for degradation. Furthermore, we demonstrate that either overexpressing a dominant negative Rab7 or short interfering RNA-mediated knockdown of Rab7 results in a significant inhibition of channel degradation rate. Coimmunoprecipitation confirmed a close association between Rab7 and KCa3.1. On the basis of these findings, we assessed the role of the ESCRT machinery in the degradation of heterologously expressed KCa3.1, including TSG101 [endosomal sorting complex required for transport (ESCRT)-I] and CHMP4 (ESCRT-III) as well as VPS4, a protein involved in the disassembly of the ESCRT machinery. We demonstrate that TSG101 is closely associated with KCa3.1 via coimmunoprecipitation and that a dominant negative TSG101 inhibits KCa3.1 degradation. In addition, both dominant negative CHMP4 and VPS4 significantly decrease the rate of membrane KCa3.1 degradation, compared with wild-type controls. These results are the first to demonstrate that plasma membrane-associated KCa3.1 is targeted for lysosomal degradation via a Rab7 and ESCRT-dependent pathway.

scholarly journals KIF13A mediates the activity-dependent transport of ESCRT-0 proteins in axons

2020 ◽  
Author(s):  
Veronica Birdsall ◽  
Yuuta Imoto ◽  
Shigeki Watanabe ◽  
Clarissa L. Waites
Keyword(s):  
Axonal Transport ◽  
Neuronal Firing ◽  
Dependent Manner ◽  
Endosomal Sorting ◽  
Transport Dynamics ◽  
Transport Vesicles ◽  
Early Endosomes ◽  
Dependent Transport ◽  
Activity Dependent ◽  
Dependent Mechanism

AbstractTurnover of synaptic vesicle (SV) proteins is vital for the maintenance of healthy, functional synapses in neurons. Our previous work showed that the degradation of SV proteins is mediated by the endosomal sorting complex required for transport (ESCRT) pathway in an activity-dependent manner. Here, we characterize the axonal transport dynamics of ESCRT-0 proteins Hrs and STAM1, the first components of the ESCRT pathway critical for initiating SV protein degradation. Hrs- and STAM1-positive transport vesicles exhibit increased anterograde and bidirectional motility in response to neuronal firing, and frequent colocalization with SV pools. These ESCRT-0 vesicles are a subset of Rab5-positive structures in axons, likely representing pro-degradative early endosomes. Further, we identify kinesin motor protein KIF13A as essential for the activity-dependent transport of ESCRT-0 vesicles as well as the degradation of SV membrane proteins. Together, these data demonstrate a novel KIF13A-dependent mechanism for mobilizing axonal transport of ESCRT machinery to facilitate the degradation of SV proteins.

scholarly journals N-glycans are direct determinants of CFTR folding and stability in secretory and endocytic membrane traffic

2009 ◽  
Vol 184 (6) ◽  
pp. 847-862 ◽  
Author(s):  
Rina Glozman ◽  
Tsukasa Okiyoneda ◽  
Cory M. Mulvihill ◽  
James M. Rini ◽  
Herve Barriere ◽  
...  
Keyword(s):  
Cell Surface ◽  
Conformational Stability ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Membrane Expression ◽  
Kinase Substrate ◽  
Endosomal Sorting ◽  
Early Endosomes ◽  
Polytopic Membrane Protein

N-glycosylation, a common cotranslational modification, is thought to be critical for plasma membrane expression of glycoproteins by enhancing protein folding, trafficking, and stability through targeting them to the ER folding cycles via lectin-like chaperones. In this study, we show that N-glycans, specifically core glycans, enhance the productive folding and conformational stability of a polytopic membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), independently of lectin-like chaperones. Defective N-glycosylation reduces cell surface expression by impairing both early secretory and endocytic traffic of CFTR. Conformational destabilization of the glycan-deficient CFTR induces ubiquitination, leading to rapid elimination from the cell surface. Ubiquitinated CFTR is directed to lysosomal degradation instead of endocytic recycling in early endosomes mediated by ubiquitin-binding endosomal sorting complex required for transport (ESCRT) adaptors Hrs (hepatocyte growth factor–regulated tyrosine kinase substrate) and TSG101. These results suggest that cotranslational N-glycosylation can exert a chaperone-independent profolding change in the energetic of CFTR in vivo as well as outline a paradigm for the peripheral trafficking defect of membrane proteins with impaired glycosylation.

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 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.

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