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 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 Cyto-friendly polymerization at cell surfaces modulates cell fate by clustering cell-surface receptors

2020 ◽  
Vol 11 (16) ◽  
pp. 4221-4225 ◽  
Author(s):  
Jing Qi ◽  
Weishuo Li ◽  
Xiaoling Xu ◽  
Feiyang Jin ◽  
Di Liu ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Fate ◽  
Cell Surface Receptors ◽  
Cell Surfaces ◽  
Receptor Clustering ◽  
Surface Polymerization ◽  
Surface Receptors ◽  
Anti Cd20 ◽  
In Cells

Cell-surface polymerization of anti-CD20 aptamer modified macromer to induce CD20 receptor clustering, and effectively initiate the apoptotic signals in cells.

Expression and localization of androgen receptor-interacting protein-4 in the testis

2007 ◽  
Vol 292 (2) ◽  
pp. E513-E522 ◽  
Author(s):  
Andrii Domanskyi ◽  
Fu-Ping Zhang ◽  
Mirja Nurmio ◽  
Jorma J. Palvimo ◽  
Jorma Toppari ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Sertoli Cell ◽  
Germ Cells ◽  
Sertoli Cells ◽  
Hormone Receptor ◽  
Luteinizing Hormone Receptor ◽  
Dependent Manner ◽  
Cell Nuclei ◽  
Interacting Protein ◽  
Independent Functions

Androgen receptor-interacting protein 4 (ARIP4) belongs to the SNF2 family of proteins involved in chromatin remodeling, DNA excision repair, and homologous recombination. It is a DNA-dependent ATPase, binds to DNA and mononucleosomes, and interacts with androgen receptor (AR) and modulates AR-dependent transactivation. We have examined in this study the expression and cellular localization of ARIP4 during postnatal development of mouse testis. ARIP4 was detected by immunohistochemistry in Sertoli cell nuclei at all ages studied, starting on day 5, and exhibited the highest expression level in adult mice. At the onset of spermatogenesis, ARIP4 expression became evident in spermatogonia, pachytene, and diplotene spermatocytes. Immunoreactive ARIP4 antigen was present in Leydig cell nuclei. In Sertoli cells ARIP4 was expressed in a stage-dependent manner, with high expression levels at stages II–VI and VII–VIII. ARIP4 expression patterns did not differ significantly in testes of wild-type, follicle-stimulating hormone receptor knockout, and luteinizing hormone receptor knockout mice. In testes of hypogonadal mice, ARIP4 was found mainly in interstitial cells and exhibited lower expression in Sertoli and germ cells. In vitro stimulation of rat seminiferous tubule segments with testosterone, FSH, or forskolin did not significantly change stage-specific levels of ARIP4 mRNA. Heterozygous ARIP4+/− mice were haploinsufficient and had reduced levels of Sertoli-cell specific androgen-regulated Rhox5 (also called Pem) mRNA. Collectively, ARIP4 is an AR coregulator in Sertoli cells in vivo, but the expression in the germ cells implies that it has also AR-independent functions in spermatogenesis.

scholarly journals The Huntingtin-interacting protein SETD2/HYPB is an actin lysine methyltransferase

2020 ◽  
Vol 6 (40) ◽  
pp. eabb7854 ◽  
Author(s):  
Riyad N. H. Seervai ◽  
Rahul K. Jangid ◽  
Menuka Karki ◽  
Durga Nand Tripathi ◽  
Sung Yun Jung ◽  
...  
Keyword(s):  
Cell Migration ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Scaffolding Protein ◽  
Actin Binding ◽  
Set Domain ◽  
Interacting Protein ◽  
Lysine Methyltransferase ◽  
Huntingtin Interacting Protein ◽  
In Cells

The methyltransferase SET domain–containing 2 (SETD2) was originally identified as Huntingtin (HTT) yeast partner B. However, a SETD2 function associated with the HTT scaffolding protein has not been elucidated, and no linkage between HTT and methylation has yet been uncovered. Here, we show that SETD2 is an actin methyltransferase that trimethylates lysine-68 (ActK68me3) in cells via its interaction with HTT and the actin-binding adapter HIP1R. ActK68me3 localizes primarily to the insoluble F-actin cytoskeleton in cells and regulates actin polymerization/depolymerization dynamics. Disruption of the SETD2-HTT-HIP1R axis inhibits actin methylation, causes defects in actin polymerization, and impairs cell migration. Together, these data identify SETD2 as a previously unknown HTT effector regulating methylation and polymerization of actin filaments and provide new avenues for understanding how defects in SETD2 and HTT drive disease via aberrant cytoskeletal methylation.

scholarly journals Structure and function of human Vps20 and Snf7 proteins

2004 ◽  
Vol 377 (3) ◽  
pp. 693-700 ◽  
Author(s):  
Jeremy W. PECK ◽  
Emma T. BOWDEN ◽  
Peter D. BURBELO
Keyword(s):  
Protein Interactions ◽  
Osmotic Shock ◽  
Multivesicular Body ◽  
Protein Protein Interactions ◽  
Interacting Protein ◽  
Endosomal Membrane ◽  
Genomic Studies ◽  
Vacuolar Protein ◽  
And Function

Snf7p (sucrose non-fermenting) and Vps20p (vacuolar protein-sorting) are small coil-coiled proteins involved in yeast MVB (multivesicular body) structure, formation and function. In the present study, we report the identification of three human homologues of yeast Snf7p, designated hSnf7-1, hSnf7-2 and hSnf7-3, and a single human Vps20p homologue, designated hVps20, that may have similar roles in humans. Immunofluorescence studies showed that hSnf7-1 and hSnf7-3 localized in large vesicular structures that also co-localized with late endosomal/lysosomal structures induced by overexpressing an ATPase-defective Vps4-A mutant. In contrast, overexpressed hVps20 showed a typical endosomal membrane-staining pattern, and co-expression of hVps20 with Snf7-1 dispersed the large Snf7-staining vesicles. Interestingly, overexpression of both hSnf7 and hVps20 proteins induced a post-endosomal defect in cholesterol sorting. To explore possible protein–protein interactions involving hSnf7 proteins, we used information from yeast genomic studies showing that yeast Snf7p can interact with proteins involved in MVB function. Using a glutathione S-transferase-capture approach with several mammalian homologues of such yeast Snf7p-interacting proteins, we found that all three hSnf7s interacted with mouse AIP1 [ALG-2 (apoptosis-linked gene 2) interacting protein 1], a mammalian Bro1p [BCK1 (bypass of C kinase)-like resistance to osmotic shock]-containing protein involved in cellular vacuolization and apoptosis. Whereas mapping experiments showed that the N-terminus of AIP1 containing both a Bro1 and an α-helical domain were required for interaction with hSnf7-1, Snf7-1 did not interact with another human Bro1-containing molecule, rhophilin-2. Co-immunoprecipitation experiments confirmed the in vivo interaction of hSnf7-1 and AIP1. Additional immunofluorescence experiments showed that hSnf7-1 recruited cytosolic AIP1 to the Snf7-induced vacuolar-like structures. Together these results suggest that mammalian Vps20, AIP1 and Snf7 proteins, like their yeast counterparts, play roles in MVB function.

scholarly journals Filamin A Links Sphingosine Kinase 1 and Sphingosine-1-Phosphate Receptor 1 at Lamellipodia To Orchestrate Cell Migration

2008 ◽  
Vol 28 (18) ◽  
pp. 5687-5697 ◽  
Author(s):  
Michael Maceyka ◽  
Sergio E. Alvarez ◽  
Sheldon Milstien ◽  
Sarah Spiegel
Keyword(s):  
Critical Role ◽  
Cell Movement ◽  
Sphingosine Kinase ◽  
Lipid Mediator ◽  
Sphingosine Kinase 1 ◽  
Filamin A ◽  
Interacting Protein ◽  
Surface Receptors ◽  
Sphingosine 1 Phosphate ◽  
Lamellipodia Formation

ABSTRACT Sphingosine kinase 1 (SphK1) catalyzes the phosphorylation of sphingosine to produce the potent lipid mediator sphingosine-1-phosphate (S1P), which plays a critical role in cell motility via its cell surface receptors. Here, we have identified filamin A (FLNa), an actin-cross-linking protein involved in cell movement, as a bona fide SphK1-interacting protein. Heregulin stimulated SphK1 activity only in FLNa-expressing A7 melanoma cells but not in FLNa-deficient cells and induced its translocation and colocalization with FLNa at lamellipodia. SphK1 was required for heregulin-induced migration, lamellipodia formation, activation of PAK1, and subsequent FLNa phosphorylation. S1P directly stimulated PAK1 kinase, suggesting that it may be a target of intracellularly generated S1P. Heregulin also induced colocalization of S1P1 (promotility S1P receptor) but not S1P2, with SphK1 and FLNa at membrane ruffles. Moreover, an S1P1 antagonist inhibited the lamellipodia formation induced by heregulin. Hence, FLNa links SphK1 and S1P1 to locally influence the dynamics of actin cytoskeletal structures by orchestrating the concerted actions of the triumvirate of SphK1, FLNa, and PAK1, each of which requires and/or regulates the actions of the others, at lamellipodia to promote cell movement.

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