Adaptor Protein Complex Research Articles

A mouse model for SPG48 reveals a block of autophagic flux upon disruption of adaptor protein complex five.

Hereditary spastic paraplegia is a spastic gait disorder that arises from degeneration of corticospinal axons. The subtype SPG48 is associated with mutations in the zeta subunit of the adaptor protein complex five (AP5). AP5 function and the pathophysiology of SPG48 are only poorly understood. Here, we report an AP5 zeta knockout mouse, which shows an age-dependent degeneration of corticospinal axons. Our analysis of knockout fibroblasts supports a trafficking defect from late endosomes to the transGolgi network and reveals a structural defect of the Golgi. We further show that both autophagic flux and the recycling of lysosomes from autolysosomes were impaired in knockout cells. In vivo, we observe an increase of autophagosomes and autolysosomes and, at later stages, the accumulation of intracellular waste in neurons. Taken together, we propose that loss of AP5 function blocks autophagy and thus leads to the aberrant accumulation of autophagic cargo, which finally results in axon degeneration.

Abi1 loss drives prostate tumorigenesis through activation of EMT and noncanonical WNT signaling.

280 Background: Prostate cancer is characterized by heterogeneity of mechanisms which are poorly understood but pointing to epithelial plasticity as the key mechanism in progression to metastatic disease. ABI1, a member of WAVE complex and actin cytoskeleton regulator and adaptor protein, is proposed to act as tumor suppressor in prostate cancer, but the mechanism of tumor progression due to Abi1 loss is not clear. Methods: To address Abi1’s role in prostate cancer we used CRISPR-based gene editing and retroviral expression to manipulate Abi1 levels in prostate cancer cell lines. Levels of Abi1 expression in prostate organoid tumor cell lines were evaluated by Western blotting and/or RNA sequencing. Association of Abi1 loss with tumor grade was evaluated by immunohistochemistry. Results: Abi1 expression is downregulated in tumor organoid cell lines from metastatic bone and lymph node biopsies. Moreover, low Abi1 expression is associated with high-grade prostate tumors (GG3 or higher, p < 0.001). Disruption of Abi1 gene in a benign prostate epithelial cell line RWPE-1 resulted in a gain of invasive phenotype, which is characterized by loss of cell-cell adhesion markers and increased migratory ability of RWPE-1 Abi1 KO spheroids. Through RNA sequencing and protein expression analysis we discovered that Abi1 loss leads to activation of non-canonical WNT signaling and EMT pathways, which are rescued by re-expression of Abi1. Furthermore, increase in STAT3 phosphorylation upon Abi1 inactivation and evidence for high affinity interaction of FYN-SH2 domain with Abi1 pY421 support the model that Abi1 acts as a gatekeeper of the non-canonical WNT-EMT pathway activation downstream from FZD2 receptor. The gene expression profile of Abi1-EMT-WNT pathway overlaps with the reported gene signature of high-risk prostate tumors. Conclusions: Abi1 contributes to prostate cancer progression and epithelial plasticity through regulation of EMT-WNT pathways. Understanding of Abi1’s role may provide more mechanistic understanding of prostate cancer tumor progression.

Polar Localization of the Borate Exporter BOR1 Requires AP2-Dependent Endocytosis.

Boron (B) is an essential element in plants but is toxic when it accumulates to high levels. In root cells of Arabidopsis (Arabidopsis thaliana), the borate exporter BOR1 is polarly localized in the plasma membrane toward the stele side for directional transport of B. Upon high-B supply, BOR1 is rapidly internalized and degraded in the vacuole. The polar localization and B-induced vacuolar sorting of BOR1 are mediated by endocytosis from the plasma membrane. To dissect the endocytic pathways mediating the polar localization and vacuolar sorting, we investigated the contribution of the clathrin adaptor protein, ADAPTOR PROTEIN2 (AP2) complex, to BOR1 trafficking. In the mutants lacking µ- or σ-subunits of the AP2 complex, the polar localization and constitutive endocytosis of BOR1 under low-B conditions were dramatically disturbed. A coimmunoprecipitation assay showed association of the AP2 complex with BOR1, while it was independent of YxxΦ sorting motifs, which are in a cytosolic loop of BOR1. A yeast two-hybrid assay supported the interaction of the AP2 complex µ-subunit with the C-terminal tail but not with the YxxΦ motifs in the cytosolic loop of BOR1. Intriguingly, lack of the AP2 subunit did not affect the B-induced rapid internalization/vacuolar sorting of BOR1. Consistent with defects in the polar localization, the AP2 complex mutants showed hypersensitivity to B deficiency. Our results indicate that AP2-dependent endocytosis maintains the polar localization of BOR1 to support plant growth under low-B conditions, whereas the B-induced vacuolar sorting of BOR1 is mediated through an AP2-independent endocytic pathway.

The trans-Golgi network is a major site for α-secretase processing of amyloid precursor protein in primary neurons

Amyloid precursor protein (APP) is processed along the amyloidogenic pathway by the β-secretase, BACE1, generating β-amyloid (Aβ), or along the nonamyloidogenic pathway by α-secretase, precluding Aβ production. The plasma membrane is considered the major site for α-secretase-mediated APP cleavage, but other cellular locations have not been rigorously investigated. Here, we report that APP is processed by endogenous α-secretase at the trans-Golgi network (TGN) of both transfected HeLa cells and mouse primary neurons. We have previously shown the adaptor protein complex, AP-4, and small G protein ADP-ribosylation factor-like GTPase 5b (Arl5b) are required for efficient post-Golgi transport of APP to endosomes. We found here that AP-4 or Arl5b depletion results in Golgi accumulation of APP and increased secretion of the soluble α-secretase cleavage product sAPPα. Moreover, inhibition of γ-secretase following APP accumulation in the TGN increases the levels of the membrane-bound C-terminal fragments of APP from both α-secretase cleavage (α-CTF, named C83 according to its band size) and BACE1 cleavage (β-CTF/C99). The level of C83 was ∼4 times higher than that of C99, indicating that α-secretase processing is the major pathway and that BACE1 processing is the minor pathway in the TGN. AP-4 silencing in mouse primary neurons also resulted in the accumulation of endogenous APP in the TGN and enhanced α-secretase processing. These findings identify the TGN as a major site for α-secretase processing in HeLa cells and primary neurons and indicate that both APP processing pathways can occur within the TGN compartment along the secretory pathway.

Defective AP-3-dependent VAMP8 trafficking impairs Weibel-Palade body exocytosis in Hermansky-Pudlak Syndrome type 2 blood outgrowth endothelial cells.

Weibel-Palade bodies are endothelial secretory organelles that contain von Willebrand factor, P-selectin and CD63. Release of von Willebrand factor from Weibel-Palade bodies is crucial for platelet adhesion during primary hemostasis. Endosomal trafficking of proteins like CD63 to Weibel-Palade bodies during maturation is dependent on the adaptor protein complex 3 complex. Mutations in the AP3B1 gene, which encodes the adaptor protein complex 3 β1 subunit, result in Hermansky-Pudlak syndrome 2, a rare genetic disorder that leads to neutropenia and a mild bleeding diathesis. This is caused by abnormal granule formation in neutrophils and platelets due to defects in trafficking of cargo to secretory organelles. The impact of these defects on the secretory pathway of the endothelium is largely unknown. In this study, we investigated the role of adaptor protein complex 3-dependent mechanisms in trafficking of proteins during Weibel-Palade body maturation in endothelial cells. An ex vivo patient-derived endothelial model of Hermansky-Pudlak syndrome type 2 was established using blood outgrowth endothelial cells that were isolated from a patient with compound heterozygous mutations in AP3B1. Hermansky-Pudlak syndrome type 2 endothelial cells and CRISPR-Cas9-engineered AP3B1−/− endothelial cells contain Weibel-Palade bodies that are entirely devoid of CD63, indicative of disrupted endosomal trafficking. Hermansky-Pudlak syndrome type 2 endothelial cells have impaired Ca2+-mediated and cAMP-mediated exocytosis. Whole proteome analysis revealed that, apart from adaptor protein complex 3 β1, also the μ1 subunit and the v-SNARE VAMP8 were depleted. Stimulus-induced von Willebrand factor secretion was impaired in CRISPR-Cas9-engineered VAMP8−/−endothelial cells. Our data show that defects in adaptor protein complex 3-dependent maturation of Weibel-Palade bodies impairs exocytosis by affecting the recruitment of VAMP8.

Preparation of Clathrin-Coated Vesicles From Arabidopsis thaliana Seedlings.

Clathrin coated vesicles (CCVs) mediate endocytosis of plasma membrane proteins and deliver their content to the endosomes for either subsequent recycling to the plasma membrane or transport to the vacuole for degradation. CCVs assemble also at the trans-Golgi network (TGN) and is responsible for the transport of proteins to other membranes. Oligomerization of clathrin and recruitment of adaptor protein complexes promote the budding and the release of CCVs. However, many of the details during plant CCV formation are not completely elucidated. The analysis of isolated CCVs is therefore important to better understand the formation of plant CCVs, their cargos and the regulation of clathrin-mediated transport processes. In this article, we describe an optimized method to isolate CCVs from Arabidopsis thaliana seedlings.

Adaptor protein complex-1 (AP-1) is recruited by the HEATR5 protein Laa1 and its co-factor Laa2 in yeast

Cellular membrane trafficking mediated by the clathrin adaptor protein complex-1 (AP-1) is important for the proper composition and function of organelles of the endolysosomal system. Normal AP-1 function requires proteins of the HEAT repeat-containing 5 (HEATR5) family. Although HEATR5 proteins were first identified based on their ability to interact with AP-1, the functional significance of this interaction was unknown. We used bioinformatics-based phenotypic profiling and information from genome-wide fluorescence microscopy studies in the budding yeast Saccharomyces cerevisiae to identify a protein, Laa2, that mediates the interaction between AP-1 and the yeast HEATR5 protein Laa1. Further characterization of Laa2 revealed that it binds to both Laa1 and AP-1. Laa2 contains a motif similar to the characterized γ-ear-binding sites found in other AP-1-binding proteins. This motif in Laa2 is essential for the Laa1-AP-1 interaction. Moreover, mutation of this motif disrupted AP-1 localization and function and caused effects similar to mutations that remove the γ-ear of AP-1. These results indicate that Laa2 mediates the interaction between Laa1 and AP-1 and reveal that this interaction promotes the stable association of AP-1 with membranes in yeast.

Targeting of Wild-Type and Mutated Forms of Lysosome-Associated Membrane Protein-1 (LAMP-1) to Late Endosomes/Lysosomes Depends on Affinities of Their Cytoplasmic Tail Peptides with a Medium Subunit of Adaptor Protein Complex-3 (AP-3)

Targeting of Wild-Type and Mutated Forms of Lysosome-Associated Membrane Protein-1 (LAMP-1) to Late Endosomes/Lysosomes Depends on Affinities of Their Cytoplasmic Tail Peptides with a Medium Subunit of Adaptor Protein Complex-3 (AP-3)

Pharmacologically diverse antidepressant drugs disrupt the interaction of neurotrophic factor receptor TRKB and endocytic adaptor protein complex AP-2

Pharmacologically diverse antidepressant drugs disrupt the interaction of neurotrophic factor receptor TRKB and endocytic adaptor protein complex AP-2

Investigation of Ldb19/Art1 localization and function at the late Golgi.

The arrestin-related family of proteins (ARTs) are potent regulators of membrane traffic at multiple cellular locations in the yeast Saccharomyces cerevisiae. Several ARTs act at multiple locations, suggesting that ARTs with well-established functions at one location may have additional, as of yet, uncharacterized roles at other locations in the cell. To more fully understand the spectrum of cellular functions regulated by ART proteins, we explored the localization and function of Ldb19/Art1, which has previously been shown to function at the plasma membrane, yet is reported to localize to the trans-Golgi network (TGN). We report that the C-terminal fusion of Ldb19 with GFP is functional and, as previously reported, localizes to the TGN. We further establish that Ldb19 associates with late stages of TGN maturation that are enriched in the clathrin adaptor protein complex-1 (AP-1). Additionally, we present genetic interaction assays that suggest Ldb19 acts at the late TGN in a mechanism related to that of AP-1. However, Ldb19 and AP-1 have dissimilar phenotypes in a subset of assays of membrane traffic, suggesting Ldb19 functions at the TGN are distinct from those of AP-1. Together these results indicate Ldb19 functions at the TGN, in addition to its well-established role in endocytosis.

Phosphoserine acidic cluster motifs bind distinct basic regions on the μ subunits of clathrin adaptor protein complexes

Protein trafficking in the endosomal system involves the recognition of specific signals within the cytoplasmic domains (CDs) of transmembrane proteins by clathrin adaptors. One such signal is the phosphoserine acidic cluster (PSAC), the prototype of which is in the endoprotease furin. How PSACs are recognized by clathrin adaptors has been controversial. We reported previously that HIV-1 Vpu, which modulates cellular immunoreceptors, contains a PSAC that binds to the μ subunits of clathrin adaptor protein (AP) complexes. Here, we show that the CD of furin binds the μ subunits of AP-1 and AP-2 in a phosphorylation-dependent manner. Moreover, we identify a potential PSAC in a cytoplasmic loop of the cellular transmembrane Serinc3, an inhibitor of the infectivity of retroviruses. The two serines within the PSAC of Serinc3 are phosphorylated by casein kinase II and mediate interaction with the μ subunits in vitro The sites of these serines vary among mammals in a manner suggesting host-pathogen conflict, yet the Serinc3 PSAC seems dispensable for anti-HIV activity and for counteraction by HIV-1 Nef. The CDs of Vpu and furin and the PSAC-containing loop of Serinc3 each bind the μ subunit of AP-2 (μ2) with similar affinities, but they appear to utilize different basic regions on μ2. The Serinc3 loop requires a region previously reported to bind the acidic plasma membrane lipid phosphatidylinositol 4,5-bisphosphate. These data suggest that the PSACs within different proteins recognize different basic regions on the μ surface, providing the potential to inhibit the activity of viral proteins without necessarily affecting cellular protein trafficking.

The AP-1 Complex is Required for Proper Mucilage Formation in Arabidopsis Seeds.

The adaptor protein (AP) complexes play crucial roles in vesicle formation in post-Golgi trafficking. Land plants have five types of AP complexes (AP-1 to AP-5), each of which consists of two large subunits, one medium subunit and one small subunit. Here, we show that the Arabidopsis AP-1 complex mediates the polarized secretion and accumulation of a pectic polysaccharide called mucilage in seed coat cells. Previously, a loss-of-function mutant of AP1M2, the medium subunit of AP-1, has been shown to display deleterious growth defects because of defective cytokinesis. To investigate the function of AP-1 in interphase, we generated transgenic Arabidopsis plants expressing AP1M2-GFP (green fluorescent protein) under the control of the cytokinesis-specific KNOLLE (KN) promoter in the ap1m2 background. These transgenic plants, designated pKN lines, successfully rescued the cytokinesis defect and dwarf phenotype of ap1m2. pKN lines showed reduced mucilage extrusion from the seed coat. Furthermore, abnormal accumulation of mucilage was found in the vacuoles of the outermost integument cells of pKN lines. During seed development, the accumulation of AP1M2-GFP was greatly reduced in the integument cells of pKN lines. These results suggest that trans-Golgi network (TGN)-localized AP-1 is involved in the trafficking of mucilage components to the outer surface of seed coat cells. Our study highlights an evolutionarily conserved function of AP-1 in polarized sorting in eukaryotic cells.

A genetic variant near adaptor-related protein complex 2 alpha 2 subunit gene is associated with coronary artery disease in a Chinese population

BackgroundAdaptor-related protein complex 2 alpha 2 subunit (AP2A2) gene encodes a protein-a subunit of the AP-2 adaptor protein complex. Evidence has revealed that benzodiazepine receptor-associated protein 1 (BZRAP1) is abundant in the hippocampus with potential effects on brain diseases. Recently, an epidemiological study reported that two variants (rs7396366 and rs2526378) closest to the AP2A2 and BZRAP1 genes are associated with higher plasma lipids and Alzheimer’s disease. Whether the two single nucleotide polymorphisms (SNPs) are actually relevant to coronary artery disease (CAD) and CAD severity remains elusive. Our aim was to assess whether these two SNPs are relevant to CAD and its severity in a Chinese population.MethodsThree hundred and thirty-five patients with documented CAD (282 stable CAD, 28 non-ST-segment elevation myocardial infarction, 25 ST-segment elevation myocardial infarction), and 372 non-CAD controls were included in the study. The participants were divided into two groups according to coronary angiography results. CAD patients were further demarcated into subgroups with one-, two-, or three-vessel stenosis. Genotypes at rs7396366 and rs2526378 were examined using polymerase chain reaction-ligase detection reaction. The association between these two SNPs with CAD and its severity were analyzed.ResultsThe frequency of the rs7396366 TT genotype was significantly higher in CAD patients than in controls (13.7% vs. 7.8%, 95% CI: 1.15–3.07, P = 0.014). Subjects with a variant genotype T allele had an increased risk of CAD compared with G allele carriers (additive model: 95% CI: 1.21–3.35, P = 0.008). After adjustment for traditional cardiovascular risk factors, analysis of the dominant models involving rs7396366 also showed that T allele carriers had a significantly higher risk for CAD than G allele carriers had (dominant model: OR 1.48, 95% CI: 1.03–2.14, P = 0.035). Age, sex, type 2 diabetes mellitus, fasting plasma glucose, and the TT genotype in rs7396366 were significantly associated with three-vessel lesions. Despite these significant outcomes of rs7396366, information on rs2526378 showed no significant difference between CAD patients and non-CAD controls.ConclusionOur results show that the T allele and TT genotype in rs7396366, closest to the AP2A2 gene, are linked to an increased risk of CAD and its severity in a Chinese population.

Intracellular localization of GGA accessory protein p56 in cell lines and central nervous system neurons.

Adaptor protein complex-1 (AP-1) and Golgi associated, γ-adaptin ear containing, Arf binding proteins (GGAs) are clathrin adaptors that regulate membrane trafficking between the trans-Golgi network (TGN) and endosomes. p56 is a clathrin adaptor accessory protein that may modulate the function of GGAs in mammalian cell lines. However, the precise relationship between p56 and the three GGAs (GGA1-3), as well as the physiological role of p56 in tissue cells, remain unknown. To this end, we generated an antibody against p56 and determined its cellular localization. In ARPE-19 cells and mouse embryonic fibroblasts, p56 was found to be localized as fine puncta in the TGN. Interestingly, the depletion of each clathrin adaptor by RNAi revealed that this localization was dependent on the expression of GGA1, but not that of GGA2, GGA3, or AP-1. Using immunohistofluorescence microscopy in the mouse central nervous system (CNS), p56 was clearly detected as scattered cytoplasmic puncta in spinal motor neurons, cerebellar Purkinje cells, and pyramidal neurons of the hippocampus and cerebral cortex. Moreover, double labeling with organelle markers revealed that the majority of these puncta were closely associated with the TGN; however, a small fraction was associated with endosomes or lysosomes in spinal motor neurons. Collectively, these results indicate a functional association of p56 with GGA1, suggesting an important role of p56 in larger CNS neurons.

Varicella-Zoster Virus ORF9p Binding to Cellular Adaptor Protein Complex 1 Is Important for Viral Infectivity.

ORF9p (homologous to herpes simplex virus 1 [HSV-1] VP22) is a varicella-zoster virus (VZV) tegument protein essential for viral replication. Even though its precise functions are far from being fully described, a role in the secondary envelopment of the virus has long been suggested. We performed a yeast two-hybrid screen to identify cellular proteins interacting with ORF9p that might be important for this function. We found 31 ORF9p interaction partners, among which was AP1M1, the μ subunit of the adaptor protein complex 1 (AP-1). AP-1 is a heterotetramer involved in intracellular vesicle-mediated transport and regulates the shuttling of cargo proteins between endosomes and the trans-Golgi network via clathrin-coated vesicles. We confirmed that AP-1 interacts with ORF9p in infected cells and mapped potential interaction motifs within ORF9p. We generated VZV mutants in which each of these motifs was individually impaired and identified leucine 231 in ORF9p to be critical for the interaction with AP-1. Disrupting ORF9p binding to AP-1 by mutating leucine 231 to alanine in ORF9p strongly impaired viral growth, most likely by preventing efficient secondary envelopment of the virus. Leucine 231 is part of a dileucine motif conserved among alphaherpesviruses, and we showed that VP22 of Marek's disease virus and HSV-2 also interacts with AP-1. This indicates that the function of this interaction in secondary envelopment might be conserved as well.IMPORTANCE Herpesviruses are responsible for infections that, especially in immunocompromised patients, can lead to severe complications, including neurological symptoms and strokes. The constant emergence of viral strains resistant to classical antivirals (mainly acyclovir and its derivatives) pleads for the identification of new targets for future antiviral treatments. Cellular adaptor protein (AP) complexes have been implicated in the correct addressing of herpesvirus glycoproteins in infected cells, and the discovery that a major constituent of the varicella-zoster virus tegument interacts with AP-1 reveals a previously unsuspected role of this tegument protein. Unraveling the complex mechanisms leading to virion production will certainly be an important step in the discovery of future therapeutic targets.

Activation of Self-Incompatibility Signaling in Transgenic Arabidopsis thaliana Is Independent of AP2-Based Clathrin-Mediated Endocytosis.

Internalization of plasma membrane (PM)-localized ligand-activated receptor kinases and their trafficking to sorting endosomes have traditionally been viewed as functioning primarily in the down-regulation of receptor signaling, but are now considered to be also essential for signaling by some receptors. A major mechanism for internalization of PM proteins is clathrin-mediated endocytosis (CME). CME is mediated by the Adaptor Protein Complex 2 (AP2), which is involved in interaction of the AP2 μ-adaptin subunit with a tyrosine-based Yxxϕ motif located in the cytoplasmic domain of the cargo protein. In this study, we investigated the role of AP2-mediated CME for signaling by the S-locus receptor kinase (SRK), a protein localized in the PM of stigma epidermal cells, which, together with its pollen coat-localized S-locus cysteine-rich (SCR) ligand, functions in the self-incompatibility (SI) response of the Brassicaceae. Using Arabidopsis thaliana plants that were made self-incompatible by transformation with an A. lyrata-derived SRK/SCR gene pair, we tested the effect on SI of site-directed mutations in each of the two Yxxϕ motifs in SRK and of a CRISPR/Cas9-induced null mutation in the AP2 μ-adaptin gene AP2M. Both in vitro SRK kinase activity and the in planta SI response were abolished by substitution of tyrosine in one of the two Yxxϕ motifs, but were unaffected by elimination of either the second Yxxϕ motif or AP2M function. Thus, AP2-mediated CME is considered to be unnecessary for SRK signaling in the SI response.

NLRX1 resides in mitochondrial RNA granules and regulates mitochondrial RNA processing and bioenergetic adaptation

The role of mitochondria is emerging in regulation of innate immunity, inflammation and cell death beyond its primary role in energy metabolism. Mitochondria act as molecular platform for immune adaptor protein complexes, which participate in innate immune signaling. The mitochondrial localized immune adaptors are widely expressed in non-immune cells, however their role in regulation of mitochondrial function and metabolic adaption is not well understood. NLRX1, a member of NOD family receptor proteins, localizes to mitochondria and is a negative regulator of anti-viral signaling. However, the submitochondrial localization of NLRX1 and its implication in regulation of mitochondrial functions remains elusive. Here, we confirm that NLRX1 translocates to mitochondrial matrix and associates with mitochondrial FASTKD5 (Fas-activated serine-threonine kinase family protein-5), a bonafide component of mitochondrial RNA granules (MRGs). The association of NLRX1 with FASTKD5 negatively regulates the processing of mitochondrial genome encoded transcripts for key components of complex-I and complex-IV, to modulate its activity and supercomplexes formation. The evidences, here, suggest an important role of NLRX1 in regulating the post-transcriptional processing of mitochondrial RNA, which may have an important implication in bioenergetic adaptation during metabolic stress, oncogenic transformation and innate immunity.

Spatial regulation of signaling by the coordinated action of the protein tyrosine kinases MET and FER

A critical aspect of understanding the regulation of signal transduction is not only to identify the protein-protein interactions that govern assembly of signaling pathways, but also to understand how those pathways are regulated in time and space. In this report, we have applied both gain-of-function and loss-of-function analyses to assess the role of the non-receptor protein tyrosine kinase FER in activation of the HGF Receptor protein tyrosine kinase MET. Overexpression of FER led to direct phosphorylation of several signaling sites in MET, including Tyr1349, but not the activation loop residues Tyr1234/5; in contrast, suppression of FER by RNAi revealed that phosphorylation of both a C-terminal signaling site (Tyr1349) and the activation loop (Tyr1234/5) were influenced by the function of this kinase. Adaptin β, a component of the adaptor protein complex 2 (AP-2) that links clathrin to receptors in coated vesicles, was recruited to MET following FER-mediated phosphorylation. Furthermore, we provide evidence to support a role of FER in maintaining plasma membrane distribution of MET and thereby delaying protein-tyrosine phosphatase PTP1B-mediated inactivation of the receptor. Simultaneous up-regulation of FER and down-regulation of PTP1B observed in ovarian carcinoma-derived cell lines would be expected to contribute to persistent activation of HGF-MET signaling, suggesting that targeting of both FER and MET may be an effective strategy for therapeutic intervention in ovarian cancer.

A mechanism for differential sorting of the planar cell polarity proteins Frizzled6 and Vangl2 at the trans-Golgi network

In planar cell polarity (PCP), the epithelial cells are polarized along the plane of the cell surface perpendicular to the classical apical-basal axis, a process mediated by several conserved signaling receptors. Two PCP-signaling proteins, VANGL planar cell polarity protein 2 (Vangl2) and Frizzled6 (Fzd6), are located asymmetrically on opposite boundaries of the cell. Examining sorting of these two proteins at the trans-Golgi network (TGN), we demonstrated previously that the GTP-binding protein ADP-ribosylation factor-related protein 1 (Arfrp1) and the clathrin-associated adaptor protein complex 1 (AP-1) are required for Vangl2 transport from the TGN. In contrast, TGN export of Frizzled6 does not depend on Arfrp1 or AP-1. Here, to further investigate the TGN sorting process in mammalian cells, we reconstituted release of Vangl2 and Frizzled6 from the TGN into vesicles in vitro Immunoblotting of released vesicles indicated that Vangl2 and Frizzled6 exit the TGN in separate compartments. Knockdown analysis revealed that a clathrin adaptor, epsinR, regulates TGN export of Frizzled6 but not of Vangl2. Protein interaction analysis suggested that epsinR forms a stable complex with clathrin and that this complex interacts with a conserved polybasic motif in the Frizzled6 cytosolic domain to package Frizzled6 into transport vesicles. Moreover, we found that Frizzled6-epsinR binding dissociates epsinR from AP-1, which may separate these two cargo adaptors from each other to perform distinct cargo-sorting functions. Our results suggest that Vangl2 and Frizzled6 are packaged into separate vesicles that are regulated by different clathrin adaptors at the TGN, which may contribute to their asymmetric localizations.

Endothelial-derived VWF, But Not Platelet-Derived, Exacerbates Atherosclerosis in Apoe-null Mice by Promoting Platelet and Leukocyte Adhesion

Hermansky-Pudlak syndrome (HPS) is a recessive disorder with bleeding diathesis, which has been linked to platelet granule defects. Both platelet granules and endothelial Weibel-Palade bodies (WPBs) are members of lysosome-related organelles (LROs) whose formation is regulated by HPS protein associated complexes such as BLOC (biogenesis of lysosome-related organelles complex) -1, -2, -3, AP-3 (adaptor protein complex-3) and HOPS (homotypic fusion and protein sorting complex). Von Willebrand factor (VWF) is critical to hemostasis, which is stored in a highly-multimerized form as tubules in the WPBs. In this study, we found the defective, but varying, release of VWF into plasma after desmopressin (DDAVP) stimulation in HPS1 (BLOC-3 subunit), HPS6 (BLOC-2 subunit), and HPS9 (BLOC-1 subunit) deficient mice. In particular, VWF tubulation, a critical step in VWF maturation, was impaired in HPS6 deficient WPBs. This likely reflects a defective endothelium, contributing to the bleeding tendency in HPS mice or patients. The differentially defective regulated release of VWF in these HPS mouse models suggests the need for precise HPS genotyping before DDAVP administration to HPS patients.