Sorting Receptor Research Articles

Signalling, sorting and scaffolding adaptors for Toll-like receptors.

Toll-like receptors (TLRs) are danger-sensing receptors that typically propagate self-limiting inflammatory responses, but can unleash uncontrolled inflammation in non-homeostatic or disease settings. Activation of TLRs by pathogen- and/or host-derived stimuli triggers a range of signalling and transcriptional pathways to programme inflammatory and anti-microbial responses, including the production of a suite of inflammatory cytokines and other mediators. Multiple sorting and signalling adaptors are recruited to receptor complexes on the plasma membrane or endosomes where they act as scaffolds for downstream signalling kinases and effectors at these sites. So far, seven proximal TLR adaptors have been identified: MyD88, MAL, TRIF (also known as TICAM1), TRAM (TICAM2), SARM (SARM1), BCAP (PIK3AP1) and SCIMP. Most adaptors tether directly to TLRs through homotypic Toll/interleukin-1 receptor domain (TIR)-TIR interactions, whereas SCIMP binds to TLRs through an atypical TIR-non-TIR interaction. In this Review, we highlight the key roles for these adaptors in TLR signalling, scaffolding and receptor sorting and discuss how the adaptors thereby direct the differential outcomes of TLR-mediated responses. We further summarise TLR adaptor regulation and function, and make note of human diseases that might be associated with mutations in these adaptors.

Control of Rab7a activity and localization through endosomal type Igamma PIP 5‐kinase is required for endosome maturation and lysosome function

The small GTPase Ras-related protein Rab-7a (Rab7a) serves as a key organizer of the endosomal-lysosomal system. However, molecular mechanisms controlling Rab7a activation levels and subcellular translocation are still poorly defined. Here, we demonstrate that type Igamma phosphatidylinositol phosphate 5-kinase i5 (PIPKIγi5), an endosome-localized enzyme that produces phosphatidylinositol 4,5-bisphosphate, directly interacts with Rab7a and plays critical roles in the control of the endosomal-lysosomal system. The loss of PIPKIγi5 blocks Rab7a recruitment to early endosomes, which prevents the maturation of early to late endosomes. PIPKIγi5 loss disturbs retromer complex connection with Rab7a, which blocks the retrograde sorting of Cation-independent Mannose 6-Phosphate Receptor from late endosomes. This leads to the decreased sorting of hydrolases to lysosomes and reduces the autophagic degradation. By modulating the retromer-Rab7a connection, PIPKIγi5 is also required for the recruitment of the GTPase-activating protein TBC1 domain family member 5 to late endosomes, which controls the conversion of Rab7a from the active state to the inactive state. Thus, PIPKIγi5 is critical for the modulation of Rab7a activity, localization, and function in the endosomal-lysosomal system.

LC3C-Mediated Autophagy Selectively Regulates the Met RTK and HGF-Stimulated Migration and Invasion

The Met/hepatocyte growth factor (HGF) receptor tyrosine kinase (RTK) is deregulated in many cancers and is a recognized target for cancer therapies. Following HGF stimulation, the signaling output of Met is tightly controlled by receptor internalization and sorting for degradation or recycling. Here, we uncover a role for autophagy in selective degradation of Met and regulation of Met-dependent cell migration and invasion. Met engagement with the autophagic pathway is dependent on complex formation with the mammalian ATG8 family member MAP1LC3C. LC3C deletion abrogates Met entry into the autophagy-dependent degradative pathway, allowing identification of LC3C domains required for rescue. Cancer cells with low LC3C levels show enhanced Met stability, signaling, and cell invasion. These findings provide mechanistic insight into RTK recruitment to autophagosomes and establish distinct roles for ATG8 proteins in this process, supporting that differential expression of ATG8 proteins can shape the functional consequences of autophagy in cancer development and progression.

Asymmetric distribution of TLR3 leads to a polarized immune response in human intestinal epithelial cells.

Intestinal epithelial cells (IECs) act as a physical barrier separating the commensal-containing intestinal tract from the sterile interior. These cells have found a complex balance allowing them to be prepared for pathogen attacks while still tolerating the presence of bacterial or viral stimuli present in the lumen of the gut. Using primary human IECs, we probed the mechanisms that allow for such a tolerance. We discovered that viral infections emanating from the basolateral side of IECs elicit a stronger intrinsic immune response in comparison to lumenal apical infections. We determined that this asymmetric immune response is driven by the clathrin-sorting adaptor AP-1B, which mediates the polarized sorting of Toll-like receptor 3 (TLR3) towards the basolateral side of IECs. Mice and human IECs lacking AP-1B showed an exacerbated immune response following apical stimulation. Together, these results suggest a model where the cellular polarity program plays an integral role in the ability of IECs to partially tolerate apical commensals while remaining fully responsive to invasive basolateral pathogens.

SorCS1-mediated sorting in dendrites maintains neurexin axonal surface polarization required for synaptic function.

The pre- and postsynaptic membranes comprising the synaptic junction differ in protein composition. The membrane trafficking mechanisms by which neurons control surface polarization of synaptic receptors remain poorly understood. The sorting receptor Sortilin-related CNS expressed 1 (SorCS1) is a critical regulator of trafficking of neuronal receptors, including the presynaptic adhesion molecule neurexin (Nrxn), an essential synaptic organizer. Here, we show that SorCS1 maintains a balance between axonal and dendritic Nrxn surface levels in the same neuron. Newly synthesized Nrxn1α traffics to the dendritic surface, where it is endocytosed. Endosomal SorCS1 interacts with the Rab11 GTPase effector Rab11 family-interacting protein 5 (Rab11FIP5)/Rab11 interacting protein (Rip11) to facilitate the transition of internalized Nrxn1α from early to recycling endosomes and bias Nrxn1α surface polarization towards the axon. In the absence of SorCS1, Nrxn1α accumulates in early endosomes and mispolarizes to the dendritic surface, impairing presynaptic differentiation and function. Thus, SorCS1-mediated sorting in dendritic endosomes controls Nrxn axonal surface polarization required for proper synapse development and function.

Endocytic sorting and downregulation of the M2 acetylcholine receptor is regulated by ubiquitin and the ESCRT complex

Cholinergic dysfunction plays a critical role in a number of disease states, and the loss of functional muscarinic acetylcholine receptors plays a key role in disease pathogenesis. Therefore, preventing receptor downregulation would maintain functional receptor number, and be predicted to alleviate symptoms. However, the molecular mechanism(s) underlying muscarinic receptor downregulation are currently unknown. Here we demonstrate that the M2 muscarinic receptor undergoes rapid lysosomal proteolysis, and this lysosomal trafficking is facilitated by ubiquitination of the receptor. Importantly, we show that this trafficking is driven specifically by ESCRT mediated involution. Critically, we provide evidence that disruption of this process leads to a re-routing of the trafficking of the M2 receptor away from the lysosome and into recycling pathway, and eventually back to the plasma membrane. This study is the first to identify the process by which the M2 muscarinic acetylcholine receptor undergoes endocytic sorting, and critically reveals a regulatory checkpoint that represents a target to pharmacologically increase the number of functional muscarinic receptors within the central nervous system.

Up-Regulation of SorCS1, an Important Sorting Receptor, in the Retina of a Form-Deprivation Rat Model.

Visually guided regulation is a sophisticated and active process, whereby sensory input helps to shape ocular development. Here, we sought to investigate the potential involvement of SorCS1, an important protein in synaptic transmission in neuron, in retinal development. A form-deprivation (FD) rat model was established. Ocular variations induced by FD were examined, including changes to eye axial length and retinal thickness. Scotopic electroretinogram (ERG) was used to examine retinal function. RD-PCR assays were screened for differentially expressed genes in FD rat eyes. Immunofluorescence staining identified the expression pattern and localization of SorCS1 in rat retina, with or without FD treatment. Additionally, primary retinal neural cells were cultured and incubated with or without a light-dark cycle, and western blot and real-time PCR assays were used to examine the expression of SorCS1. Retinal neural cells were treated with recombinant SorCS1 (h-SorCS1) coated with beads in serum-free conditions to test for effects on cellular physiology and expression of neurotransmitters involved in visual development. To monitor cell viability, a CCK8 assay was employed. Our data demonstratedthat FD led to ocular axial elongation and retinal thinning. ERG tests showed FD impaired electrophysiological function in rat. An age-related expression pattern of SorCS1 was observed in the rat retina, and SorCS1 was significantly up-regulated in the FD rat retina. In addition, in vitro evidence suggested a strong correlation between light exposure and SorCS1 expression. Furthermore, treatment of retinal neural cells with h-SorCS1-beads promoted cell viability, neurite outgrowth, and up-regulation of inhibitory neurotransmitter expression, which implies that over-expression of SorCS1 may cause abnormal retinal development. Our findings suggest that SorCS1 is involved in the physiological processes of light/visually guided ocular growth.

Flotillins promote T cell receptor sorting through a fast Rab5\u2013Rab11 endocytic recycling axis

The targeted endocytic recycling of the T cell receptor (TCR) to the immunological synapse is essential for T cell activation. Despite this, the mechanisms that underlie the sorting of internalised receptors into recycling endosomes remain poorly understood. To build a comprehensive picture of TCR recycling during T cell activation, we developed a suite of new imaging and quantification tools centred on photoactivation of fluorescent proteins. We show that the membrane-organising proteins, flotillin-1 and -2, are required for TCR to reach Rab5-positive endosomes immediately after endocytosis and for transfer from Rab5- to Rab11a-positive compartments. We further observe that after sorting into in Rab11a-positive vesicles, TCR recycles to the plasma membrane independent of flotillin expression. Our data suggest a mechanism whereby flotillins delineate a fast Rab5-Rab11a endocytic recycling axis and functionally contribute to regulate the spatial organisation of these endosomes.

Syndecan-1 Mediates Sorting of Soluble Lipoprotein Lipase with Sphingomyelin-Rich Membrane in the Golgi Apparatus.

In the secretory pathway, budding of vesicular transport carriers from the trans-Golgi network (TGN) must coordinate specification of lipid composition with selection of secreted proteins. We elucidate a mechanism of soluble protein cargo sorting into secretory vesicles with a sphingomyelin-rich membrane; the integral membrane proteoglycan Syndecan-1 (SDC1) acts as a sorting receptor, capturing the soluble enzyme lipoprotein lipase (LPL) during export from the TGN. Sorting of LPL requires bivalent interactions between LPL and SDC1-linked heparan sulfate chains and between LPL and the Golgi membrane. Physical features of the SDC1 transmembrane domain, rather than a specific sequence, confer targeting of SDC1 and bound LPL into the sphingomyelin secretion pathway. This study establishes that physicochemical properties of a protein transmembrane domain that drive lateral heterogeneity of the plasma membrane also operate at the TGN to confer sorting of an integral membrane protein and its ligand within the biosynthetic secretory pathway.

INVESTIGATING THE ROLE OF SORTILIN-RELATED RECEPTOR (SORL1/SORLA) IN HUMAN INDUCED PLURIPOTENT STEM CELL DERIVED NEURAL CELLS AS A THERAPEUTIC TARGET FOR ALZHEIMER’S DISEASE

Recent compelling genetic evidence has identified genes involved in endocytosis and vesicular trafficking including SORL1 as AD risk genes. Decreased expression of SORLA has been repeatedly observed in postmortem human AD brains especially in late onset sporadic AD (SAD) which is the most common form of AD. Eventually, several studies linked SORLA expression with production and deposition of the pathogenic protein β amyloid peptide which is one of the hallmarks of AD pathogenesis. However, most of these studies were done using mouse models and non-human cell lines and role of SORLA in amyloid β production in human neural cells remains unclear. SORL1 was overexpressed in clonal populations of human pluripotent stem cells (hiPSCs) using the piggybac transposon system. Control and SORL1overexpressed hiPSCs were then differentiated into neural cells. Amount of Aβ-40 and Aβ-42 released was measured using ELISA in hiPSC derived neural cells. Additionally, intra-cellular trafficking was examined using immunocytochemistry and cell biology assays. Specifically, to assess transport into lysosomes, pH sensitive fluorescent dye Phrodo conjugated with Aβ was used. Overexpression of SORL1 in hiPSC derived human neural cells resulted in decreased Aβ-40 and Aβ-42 released into media as measured by ELISA. This decrease can be caused by a possible increase in Aβ transport into lysosomes for degradation. In support of this idea, increased Aβ transport into lysosomes was observed in SORL1 overexpressed neural cells as compared to control cells using the Phrodo assay. Decreased Aβ release could also result from increased degradation of Amyloid precursor protein (APP). In support of this hypothesis, we showed increased colocalization of APP with a lysosomal marker, Lysosome associated membrane protein (LAMP1) using immunocytochemistry. Overall, these results suggest that overexpression of SORL1 could lead to decreased amyloid β production in part due to increased transport of Aβ and APP into lysosomes for degradation in human neural cells. Our findings reveal a previously unidentified role of SORL1 in hiPSC derived neural cells, information that may be useful in exploring not just SORLA, but other sorting receptors expressed in human neurons as therapeutic targets for AD.

The BBSome in POMC and AgRP Neurons Is Necessary for Body Weight Regulation and Sorting of Metabolic Receptors.

The BBSome, a complex of eight Bardet-Biedl syndrome (BBS) proteins involved in cilia function, has emerged as an important regulator of energy balance, but the underlying cellular and molecular mechanisms are not fully understood. Here, we show that the control of energy homeostasis by the anorexigenic proopiomelanocortin (POMC) neurons and orexigenic agouti-related peptide (AgRP) neurons require intact BBSome. Targeted disruption of the BBSome by Bbs1 gene deletion in POMC or AgRP neurons increases body weight and adiposity. We demonstrate that obesity in mice lacking the Bbs1 gene in POMC neurons is associated with hyperphagia. Mechanistically, we present evidence implicating the BBSome in the trafficking of G protein-coupled neuropeptide Y Y2 receptor (NPY2R) and serotonin 5-hydroxytryptamine (HT)2C receptor (5-HT2CR) to cilia and plasma membrane, respectively. Consistent with this, loss of the BBSome reduced cell surface expression of the 5-HT2CR, interfered with serotonin-evoked increase in intracellular calcium and membrane potential, and blunted the anorectic and weight-reducing responses evoked by the 5-HT2cR agonist, lorcaserin. Finally, we show that disruption of the BBSome causes the 5-HT2CR to be stalled in the late endosome. Our results demonstrate the significance of the hypothalamic BBSome for the control of energy balance through regulation of trafficking of important metabolic receptors.

Amyloidosis causes downregulation of SorLA, SorCS1 and SorCS3 expression in mice.

Accumulation of β-amyloid peptide (Aβ) is regarded as a primary cause of Alzheimer's disease (AD). Aβ is derived by sequential cleavage of the amyloid precursor protein (APP). Alterations in the subcellular targeting of APP are thought to affect the degree of Aβ production. Sorting receptors, such as SorLA, convey subcellular targeting of APP. Dysfunction of SorLA, and likely of the related receptors SorCS1 and SorCS3, cause AD. Nevertheless, disease progression could also provoke altered expression of the receptors. Here, we assessed if Aβ plaque formation promotes altered expression of SorLA, SorCS1 and SorCS3. We analyzed transcript levels during aging and after amyloidosis in brain areas characterized by early amyloid plaque formation in an AD mouse model (APPPS1) and wild types. We observed stable expression levels during aging (1-12 months). After plaque formation, SorCS1 and SorLA expression were markedly reduced in the frontal cerebral cortex and to a minor extent in the hippocampus, whereas SorCS3 expression was solely reduced in the frontal cerebral cortex. Our results indicate that disease progression, associated with Aβ accumulation, can negatively regulate expression of the receptors.

Phosphatidylinositol 4,5-bisphosphate controls Rab7 and PLEKHM1 membrane cycling during autophagosome-lysosome fusion.

The small GTPase Rab7 is a key organizer of receptor sorting and lysosomal degradation by recruiting of a variety of effectors depending on its GDP/GTP-bound state. However, molecular mechanisms that trigger Rab7 inactivation remain elusive. Here we find that, among the endosomal pools, Rab7-positive compartments possess the highest level of PI4P, which is primarily produced by PI4K2A kinase. Acute conversion of this endosomal PI4P to PI(4,5)P2 causes Rab7 dissociation from late endosomes and releases a regulator of autophagosome-lysosome fusion, PLEKHM1, from the membrane. Rab7 effectors Vps35 and RILP are not affected by acute PI(4,5)P2 production. Deletion of PI4K2A greatly reduces PIP5Kγ-mediated PI(4,5)P2 production in Rab7-positive endosomes leading to impaired Rab7 inactivation and increased number of LC3-positive structures with defective autophagosome-lysosome fusion. These results reveal a late endosomal PI4P-PI(4,5)P2 -dependent regulatory loop that impacts autophagosome flux by affecting Rab7 cycling and PLEKHM1 association.

SorCS2 Controls Functional Expression of Amino Acid Transporter EAAT3 and Protects Neurons from Oxidative Stress and Epilepsy-Induced Pathology

SummaryVPS10P domain receptors emerge as central regulators of intracellular protein sorting in neurons with relevance for various brain pathologies. Here, we identified a role for the family member SorCS2 in protection of neurons from oxidative stress and epilepsy-induced cell death. We show that SorCS2 acts as sorting receptor that sustains cell surface expression of the neuronal amino acid transporter EAAT3 to facilitate import of cysteine, required for synthesis of the reactive oxygen species scavenger glutathione. Lack of SorCS2 causes depletion of EAAT3 from the plasma membrane and impairs neuronal cysteine uptake. As a consequence, SorCS2-deficient mice exhibit oxidative brain damage that coincides with enhanced neuronal cell death and increased mortality during epilepsy. Our findings highlight a protective role for SorCS2 in neuronal stress response and provide a possible explanation for upregulation of this receptor seen in surviving neurons of the human epileptic brain.

Regulation of G protein-coupled receptor signaling by plasma membrane organization and endocytosis.

The trafficking of G protein coupled-receptors (GPCRs) is one of the most exciting areas in cell biology because of recent advances demonstrating that GPCR signaling is spatially encoded. GPCRs, acting in a diverse array of physiological systems, can have differential signaling consequences depending on their subcellular localization. At the plasma membrane, GPCR organization could fine-tune the initial stages of receptor signaling by determining the magnitude of signaling and the type of effectors to which receptors can couple. This organization is mediated by the lipid composition of the plasma membrane, receptor-receptor interactions, and receptor interactions with intracellular scaffolding proteins. GPCR organization is subsequently changed by ligand binding and the regulated endocytosis of these receptors. Activated GPCRs can modulate the dynamics of their own endocytosis through changing clathrin-coated pit dynamics, and through the scaffolding adaptor protein β-arrestin. This endocytic regulation has signaling consequences, predominantly through modulation of the MAPK cascade. This review explores what is known about receptor sorting at the plasma membrane, protein partners that control receptor endocytosis, and the ways in which receptor sorting at the plasma membrane regulates downstream trafficking and signaling.

SH3YL1 cooperates with ESCRT-I in the sorting and degradation of the EGF receptor.

Ubiquitinated membrane proteins such as epidermal growth factor receptor (EGFR) are delivered to early endosomes and then sorted to lysosomes via multivesicular bodies (MVBs) for degradation. The regulatory mechanism underlying formation of intralumenal vesicles en route to generation of MVBs is not fully understood. In this study, we found that SH3YL1, a phosphoinositide-binding protein, had a vesicular localization pattern overlapping with internalized EGF in endosomes in the degradative pathway. Deficiency of SH3YL1 prevents EGF trafficking from early to late endosomes and inhibits degradation of EGFR. Moreover, we show that SH3YL1 mediates EGFR sorting into MVBs in a manner dependent on its C-terminal SH3 domain, which is necessary for the interaction with an ESCRT-I component, Vps37B. Taken together, our observations reveal an indispensable role of SH3YL1 in MVB sorting and EGFR degradation mediated by ESCRT complexes.

Clathrin adaptor GGA1 modulates myogenesis of C2C12 myoblasts.

During myogenesis, myogenic stem cells undergo several sequential events, including cell division, migration, and cell-cell fusion, leading to the formation of multinuclear myotubes, which are the precursors of myofibers. To understand the molecular mechanisms underlying these complex processes, an RNA interference-based gene depletion approach was used. Golgi associated, gamma adaptin ear containing, ARF binding protein 1 (GGA1), a Golgi-resident monomeric clathrin adaptor, was found to be required for the process of myotube formation in C2C12 cells, a cultured murine myoblast cell line. Gga1 mRNA expression was upregulated during myogenesis, and Gga1 depletion prevented the formation of large multi-nucleated myotubes. Moreover, inhibition of lysosomal proteases in Gga1 knockdown myoblasts increased the amount of insulin receptor, suggesting that GGA1 is involved in the cell surface expression and sorting of the insulin receptor. These results suggested that GGA1 plays a significant role in the formation and maturation of myotubes by targeting the insulin receptor to the cell surface to establish functionally mature myofibers.

A novel function for the ER retention signals in the C-terminus of kainate receptor subunit, GluK5

Classically, endoplasmic reticulum (ER) retention signals in secreted integral membrane proteins impose the requirement to assemble with other cognate subunits to form functional assemblies before they can exit the ER. We report that GluK5 has two ER retention signals in its cytoplasmic C-terminus: an arginine-based signal and a di-leucine motif previously thought to be an endocytic motif. GluK5 assembles with GluK2, but surprisingly GluK2 association does little to block the ER retention signals. We find instead that the ER retention signals are blocked by two proteins involved in intracellular trafficking, SAP97 and CASK. We show that SAP97, in the presence of CASK and the receptor complex, assumes an extended conformation. In the extended conformation, SAP97 makes its SH3 and GuK domains available to bind and sterically mask the ER retention signals in the GluK5 C-terminus. SAP97 and CASK are also necessary for sorting receptor cargoes into the local dendritic secretory pathway in neurons. We show that the ER retention signals of GluK5 play a vital role in sorting the receptor complex in the local dendritic secretory pathway in neurons. These data suggest a new role for ER retention signals in trafficking integral membrane proteins in neurons. SignificanceWe present evidence that the ER retention signals in the kainate receptors containing GluK5 impose a requirement for sorting into local dendritic secretory pathways in neurons, as opposed to traversing the somatic Golgi apparatus. There are two ER retention signals in the C-terminus of GluK5. We show that both are blocked by physical association with SAP97 and CASK. The SH3 and GuK domains of SAP97, in the presence of CASK, bind directly to each ER retention signal and form a complex. These results support an entirely new function for ER retention signals in the C-termini of neuronal receptors, such as NMDA and kainate receptors, and define a mechanism for selective entry of receptors into local secretory pathways.

Roles for ER:endosome membrane contact sites in ligand-stimulated intraluminal vesicle formation.

Multivesicular endosomes/bodies (MVBs) sort membrane proteins between recycling and degradative pathways. Segregation of membrane proteins onto intraluminal vesicles (ILVs) of MVBs removes them from the recycling pathway and facilitates their degradation following fusion of MVBs with lysosomes. Sorting of many cargos onto ILVs depends on the ESCRT (Endosomal Sorting Complex Required for Transport) machinery, although ESCRT-independent mechanisms also exist. In mammalian cells, efficient sorting of ligand-stimulated epidermal growth factor receptors onto ILVs also depends on the tyrosine phosphatase, PTP1B, an ER-localised enzyme that interacts with endosomal targets at membrane contacts between MVBs and the ER. This review focuses on the potential roles played by ER:MVB membrane contact sites in regulating ESCRT-dependent ILV formation.

Comparative proteomics analysis of whitetop (Lepidium draba L.) seedlings in response to exogenous glucose

In this research, a comparative proteomics approach was conducted to understand the physiological processes behind the sulforaphane formation in whitetop seedlings in response to exogenous glucose. Initially, 5-day-old whitetop seedlings were elicited by different concentrations (0, 166, 250, 277, 360 mM) of glucose for 72 h. According to the results, sulforaphane formation was influenced in a dose-dependent manner by glucose, and was maximized with the concentrations of 166 and 250 mM. Consequently, 2-dimensional gel electrophoresis was performed on the 166 mM glucose-elicited seedlings and it was shown that 25 protein spots were differentially expressed between glucose-elicited seedlings and control. Two hypothetical (were down-regulated) and 9 unique proteins (44% and 56% up- and down-regulated, respectively) were identified based on the Mass spectrometry analysis. According to the functional classification of the unique proteins, photosynthetic, chaperone, energy metabolism, signaling and sorting related proteins are marked in response to the glucose elicitation. This is the first report to successfully identify the Abscisic acid receptor PYR1-like and sorting nexin 1 isoform X1 by proteomics technique. In addition, the role of the sorting nexin 1 isoform X1 in the glucose-elicited whitetop seedling is reported for the first time.