Trafficking Of Transferrin Receptor Research Articles

Human transferrin receptor can mediate SARS-CoV-2 infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been detected in almost all organs of coronavirus disease-19 patients, although some organs do not express angiotensin-converting enzyme-2 (ACE2), a known receptor of SARS-CoV-2, implying the presence of alternative receptors and/or co-receptors. Here, we show that the ubiquitously distributed human transferrin receptor (TfR), which binds to diferric transferrin to traffic between membrane and endosome for the iron delivery cycle, can ACE2-independently mediate SARS-CoV-2 infection. Human, not mouse TfR, interacts with Spike protein with a high affinity (KD ~2.95 nM) to mediate SARS-CoV-2 endocytosis. TfR knock-down (TfR-deficiency is lethal) and overexpression inhibit and promote SARS-CoV-2 infection, respectively. Humanized TfR expression enables SARS-CoV-2 infection in baby hamster kidney cells and C57 mice, which are known to be insusceptible to the virus infection. Soluble TfR, Tf, designed peptides blocking TfR-Spike interaction and anti-TfR antibody show significant anti-COVID-19 effects in cell and monkey models. Collectively, this report indicates that TfR is a receptor/co-receptor of SARS-CoV-2 mediating SARS-CoV-2 entry and infectivity by likely using the TfR trafficking pathway.

Theft of Host Transferrin Receptor-1 by Toxoplasma gondii is required for infection.

Nutrient acquisition by apicomplexan parasites is essential to drive their intracellular replication, yet the mechanisms that underpin essential nutrient acquisition are not defined. Using the apicomplexan model Toxoplasma gondii , we show that host cell proteins including the transferrin receptor 1, transferrin, ferritin heavy and light chains, and clathrin light chain are robustly taken up by tachyzoites. Tachyzoite acquisition of host cell protein was not related to host cell type or parasite virulence phenotypes. Bradyzoites possessed little capacity to acquire host cell proteins consistent with the cyst wall representing a barrier to host cell protein cargo. Increased trafficking of host cell transferrin receptor 1 and transferrin to endolysosomes boosted tachyzoite acquisition of host proteins and growth rate. Theft of host transferrin 1 and transferrin did not significantly affect iron levels in the tachyzoite. This study provides insight into essential functions associated with parasite theft of host iron sequestration and storage proteins.

Insights into cargo sorting by SNX32 and its role in neurite outgrowth.

Sorting nexins (SNX) are a family of proteins containing the Phox homology domain, which shows a preferential endo-membrane association and regulates cargo sorting processes. Here, we established that SNX32, an SNX-BAR (Bin/Amphiphysin/Rvs) sub-family member associates with SNX4 via its BAR domain and the residues A226, Q259, E256, R366 of SNX32, and Y258, S448 of SNX4 that lie at the interface of these two SNX proteins mediate this association. SNX32, via its PX domain, interacts with the transferrin receptor (TfR) and Cation-Independent Mannose-6-Phosphate Receptor (CIMPR), and the conserved F131 in its PX domain is important in stabilizing these interactions. Silencing of SNX32 leads to a defect in intracellular trafficking of TfR and CIMPR. Further, using SILAC-based differential proteomics of the wild-type and the mutant SNX32, impaired in cargo binding, we identified Basigin (BSG), an immunoglobulin superfamily member, as a potential interactor of SNX32 in SHSY5Y cells. We then demonstrated that SNX32 binds to BSG through its PX domain and facilitates its trafficking to the cell surface. In neuroglial cell lines, silencing of SNX32 leads to defects in neuronal differentiation. Moreover, abrogation in lactate transport in the SNX32-depleted cells led us to propose that SNX32 may contribute to maintaining the neuroglial coordination via its role in BSG trafficking and the associated monocarboxylate transporter activity. Taken together, our study showed that SNX32 mediates the trafficking of specific cargo molecules along distinct pathways.

Apicobasal transferrin receptor localization and trafficking in brain capillary endothelial cells

The detailed mechanisms by which the transferrin receptor (TfR) and associated ligands traffic across brain capillary endothelial cells (BECs) of the CNS-protective blood–brain barrier constitute an important knowledge gap within maintenance and regulation of brain iron homeostasis. This knowledge gap also presents a major obstacle in research aiming to develop strategies for efficient receptor-mediated drug delivery to the brain. While TfR-mediated trafficking from blood to brain have been widely studied, investigation of TfR-mediated trafficking from brain to blood has been limited. In this study we investigated TfR distribution on the apical and basal plasma membranes of BECs using expansion microscopy, enabling sufficient resolution to separate the cellular plasma membranes of these morphological flat cells, and verifying both apical and basal TfR membrane domain localization. Using immunofluorescence-based transcellular transport studies, we delineated endosomal sorting of TfR endocytosed from the apical and basal membrane, respectively, as well as bi-directional TfR transcellular transport capability. The findings indicate different intracellular sorting mechanisms of TfR, depending on the apicobasal trafficking direction across the BBB, with the highest transcytosis capacity in the brain-to-blood direction. These results are of high importance for the current understanding of brain iron homeostasis. Also, the high level of TfR trafficking from the basal to apical membrane of BECs potentially explains the low transcytosis which are observed for the TfR-targeted therapeutics to the brain parenchyma.

Phosphorylation of REPS1 at Ser709 by RSK attenuates the recycling of transferrin receptor

RalBP1 associated EPS domain containing 1 (REPS1) is conserved from Drosophila to humans and implicated in the endocytic system. However, an exact role of REPS1 remains largely unknown. Here, we demonstrated that mitogen activated protein kinase kinase (MEK)-p90 ribosomal S6 Kinase (RSK) signaling pathway directly phosphorylated REPS1 at Ser709 upon stimulation by epidermal growth factor (EGF) and amino acid. While REPS2 is known to be involved in the endocytosis of EGF receptor (EGFR), REPS1 knockout (KO) cells did not show any defect in the endocytosis of EGFR. However, in the REPS1 KO cells and the KO cells reconstituted with a non-phosphorylatable REPS1 (REPS1 S709A), the recycling of transferrin receptor (TfR) was attenuated compared to the cells reconstituted with wild type REPS1. Collectively, we suggested that the phosphorylation of REPS1 at S709 by RSK may have a role of the trafficking of TfR.

Unconventional endocytosis and trafficking of transferrin receptor induced by iron.

The posttranslational regulation of transferrin receptor (TfR1) is largely unknown. We investigated whether iron availability affects TfR1 endocytic cycle and protein stability in HepG2 hepatoma cells exposed to ferric ammonium citrate (FAC). NH4Cl and bafilomycin A1, but not the proteasomal inhibitor MG132, prevented the FAC-mediated decrease in TfR1 protein levels, thus indicating lysosomal involvement. Knockdown experiments showed that TfR1 lysosomal degradation is independent of 1) endocytosis mediated by the clathrin adaptor AP2; 2) Tf, which was suggested to facilitate TfR1 internalization; 3) H-ferritin; and 4) MARCH8, previously implicated in TfR1 degradation. Notably, FAC decreased the number of TfR1 molecules at the cell surface and increased the Tf endocytic rate. Colocalization experiments confirmed that, upon FAC treatment, TfR1 was endocytosed in an AP2- and Tf-independent pathway and trafficked to the lysosome for degradation. This unconventional endocytic regulatory mechanism aimed at reducing surface TfR1 may represent an additional posttranslational control to prevent iron overload. Our results show that iron is a key regulator of the trafficking of TfR1, which has been widely used to study endocytosis, often not considering its function in iron homeostasis.

Complex Rab4-Mediated Regulation of Endosomal Size and EGFR Activation.

Early sorting endosomes are responsible for the trafficking and function of transferrin receptor (TfR) and EGFR. These receptors play important roles in iron uptake and signaling and are critical for breast cancer development. However, the role of morphology, receptor composition, and signaling of early endosomes in breast cancer remains poorly understood. A novel population of enlarged early endosomes was identified in breast cancer cells and tumor xenografts but not in noncancerous MCF10A cells. Quantitative analysis of endosomal morphology, cargo sorting, EGFR activation, and Rab GTPase regulation was performed using super-resolution and confocal microscopy followed by 3D rendering. MDA-MB-231 breast cancer cells have fewer, but larger EEA1-positive early endosomes compared with MCF10A cells. Live-cell imaging indicated dysregulated cargo sorting, because EGF and Tf traffic together via enlarged endosomes in MDA-MB-231, but not in MCF10A. Large EEA1-positive MDA-MB-231 endosomes exhibited prolonged and increased EGF-induced activation of EGFR upon phosphorylation at tyrosine-1068 (EGFR-p1068). Rab4A overexpression in MCF10A cells produced EEA1-positive enlarged endosomes that displayed prolonged and amplified EGF-induced EGFR-p1068 activation. Knockdown of Rab4A lead to increased endosomal size in MCF10A, but not in MDA-MB-231 cells. Nevertheless, Rab4A knockdown resulted in enhanced EGF-induced activation of EGFR-p1068 in MDA-MB-231 as well as downstream signaling in MCF10A cells. Altogether, this extensive characterization of early endosomes in breast cancer cells has identified a Rab4-modulated enlarged early endosomal compartment as the site of prolonged and increased EGFR activation. IMPLICATIONS: Enlarged early endosomes play a Rab4-modulated role in regulation of EGFR activation in breast cancer cells.

Leucine zipper transcription factor-like 1 binds adaptor protein complex-1 and 2 and participates in trafficking of transferrin receptor 1.

LZTFL1 participates in immune synapse formation, ciliogenesis, and the localization of ciliary proteins, and knockout of LZTFL1 induces abnormal distribution of heterotetrameric adaptor protein complex-1 (AP-1) in the Lztfl1-knockout mouse photoreceptor cells, suggesting that LZTFL1 is involved in intracellular transport. Here, we demonstrate that in vitro LZTFL1 directly binds to AP-1 and AP-2 and coimmunoprecipitates AP-1 and AP-2 from cell lysates. DxxFxxLxxxR motif of LZTFL1 is essential for these bindings, suggesting LZTFL1 has roles in AP-1 and AP-2-mediated protein trafficking. Since AP-1 and AP-2 are known to be involved in transferrin receptor 1 (TfR1) trafficking, the effect of LZTFL1 on TfR1 recycling was analyzed. TfR1, AP-1 and LZTFL1 from cell lysates could be coimmunoprecipitated. However, pull-down results indicate there is no direct interaction between TfR1 and LZTFL1, suggesting that LZTFL1 interaction with TfR1 is indirect through AP-1. We report the colocalization of LZTFL1 and AP-1, AP-1 and TfR1 as well as LZTFL1 and TfR1 in the perinuclear region (PNR) and the cytoplasm, suggesting a potential complex between LZTFL1, AP-1 and TfR1. The results from the disruption of adaptin recruitment with brefeldin A treatment suggested ADP-ribosylation factor-dependent localization of LZFL1 and AP-1 in the PNR. Knockdown of AP-1 reduces the level of LZTFL1 in the PNR, suggesting that AP-1 plays a role in LZTFL1 trafficking. Knockout of LZTFL1 reduces the cell surface level and the rate of internalization of TfR1, leading to a decrease of transferrin uptake, efflux, and internalization. However, knockout of LZTFL1 did not affect the cell surface levels of epidermal growth factor receptor and cation-independent mannose 6-phosphate receptor, indicating that LZTFL1 specifically regulates the cell surface level of TfR1. These data support a novel role of LZTFL1 in regulating the cell surface TfR1 level by interacting with AP-1 and AP-2.

Heterogeneity of Transferrin receptor expression, distribution and transferrin uptake in breast cancer cells in 2D and 3D spheroid cultures

Alterations in the expression of proteins involved in iron homeostasis have been detected in cancer cells, and their levels appear to correlate inversely with patient survival. Thus, elucidating the mechanisms underlying defective iron transport is crucial to understand the faulty iron homeostasis that is typical of cancer cells. Rapidly proliferating cancer cells show increased expression of proteins that support iron import, ex. transferrin receptor (TfR), as well as decreased levels of proteins involved in iron export, ex. ferroportin. TfR (CD71 or p90) delivers iron into cells via the binding of iron‐loaded transferrin (holo‐Tf) and subsequent endocytosis and recycling of TfR‐Tf complexes. Tf is a commonly used ligand for receptor mediated drug delivery and molecular imaging of breast tumors. However, TfR expression and Tf uptake tumor heterogeneity (Figure 1) is a crucial issue that limits our understanding of the role of defective iron transport in tumor progression as well as the use of Tf in targeted drug delivery and tumor imaging. Our long‐term goal is to develop novel imaging methodologies and biochemical approaches to establish, quantitate and modify TfR expression and Tf uptake heterogeneity using tumor xenografts and 3D cancer spheroids (Figure 1). Herein, novel approaches to modulate Tf uptake will be tested for their effect on the heterogeneity of Tf delivery across solid tumors. Previously, increasing the serum concentration of iron‐deleted Tf (apo‐Tf) has been shown to alter Tf saturation as well as the intracellular distribution and level of TfR expression in mice models of anemia, e.g. β‐Thalassemia. We currently hypothesize that increasing concentration of apo‐Tf will lead to a re‐distribution of TfR to the surface of the cells, as well as altered trafficking of iron in cancer cells. We will test the effect of apo‐Tf in TfR expression and distribution in human breast cancer 2D cell cultures, 3D cancer spheroids and tumor xenografts. Firstly, Tf uptake plate reader assays have shown that 24h incubation with apo‐Tf modulates Tf uptake differently in non‐cancerous breast epithelial cells (MCF10A) and in human breast cancer cells (MDA‐MB‐231 and T47D cells). Immunofluorescence imaging of non‐cancerous vs. breast cancer cells has shown that apo‐Tf treatment induces significant changes in the intracellular distribution of TfR and early endosomal marker EEA1. These results suggest that treatment with apo‐Tf acts via alteration of TfR‐Tf endocytic trafficking pathway. Similar experiments will be performed to determine how incubation with apo‐Tf affects TfR expression and Tf uptake heterogeneity in 3D cancer spheroids. Ability to potentially alter TfR trafficking in breast cancer cells is highly significant not only to alter iron homeostasis as a way to reduce cancer progression but also to optimize Tf‐mediated drug delivery approaches. Funding source: This work was supported by the National Institute of Health (NIH) R01 BRG CA207725.Support or Funding InformationFunding source: This work was supported by the National Institute of Health (NIH) R01 BRG CA207725.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

HRS-WASH axis governs actin-mediated endosomal recycling and cell invasion.

Transmembrane proteins in the sorting endosome are either recycled to their point of origin or destined for lysosomal degradation. Lysosomal sorting is mediated by interaction of ubiquitylated transmembrane proteins with the endosomal sorting complex required for transport (ESCRT) machinery. In this study, we uncover an alternative role for the ESCRT-0 component hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) in promoting the constitutive recycling of transmembrane proteins. We find that endosomal localization of the actin nucleating factor Wiscott-Aldrich syndrome protein and SCAR homologue (WASH) requires HRS, which occupies adjacent endosomal subdomains. Depletion of HRS results in defective constitutive recycling of epidermal growth factor receptor and the matrix metalloproteinase MT1-MMP, leading to their accumulation in internal compartments. We show that direct interactions with endosomal actin are required for efficient recycling and use a model system of chimeric transferrin receptor trafficking to show that an actin-binding motif can counteract an ubiquitin signal for lysosomal sorting. Directed receptor recycling is used by cancer cells to achieve invasive migration. Accordingly, abrogating HRS- and actin-dependent MT1-MMP recycling results in defective matrix degradation and invasion of triple-negative breast cancer cells.

Actin polymerization in the endosomal pathway, but not on the Coxiella-containing vacuole, is essential for pathogen growth

Coxiella burnetii is an intracellular bacterium that replicates within an expansive phagolysosome-like vacuole. Fusion between the Coxiella-containing vacuole (CCV) and late endosomes/multivesicular bodies requires Rab7, the HOPS tethering complex, and SNARE proteins, with actin also speculated to play a role. Here, we investigated the importance of actin in CCV fusion. Filamentous actin patches formed around the CCV membrane that were preferred sites of vesicular fusion. Accordingly, the mediators of endolysosomal fusion Rab7, VAMP7, and syntaxin 8 were concentrated in CCV actin patches. Generation of actin patches required C. burnetii type 4B secretion and host retromer function. Patches decorated with VPS29 and VPS35, components of the retromer, FAM21 and WASH, members of the WASH complex that engage the retromer, and Arp3, a component of the Arp2/3 complex that generates branched actin filaments. Depletion by siRNA of VPS35 or VPS29 reduced CCV actin patches and caused Rab7 to uniformly distribute in the CCV membrane. C. burnetii grew normally in VPS35 or VPS29 depleted cells, as well as WASH-knockout mouse embryo fibroblasts, where CCVs are devoid of actin patches. Endosome recycling to the plasma membrane and trans-Golgi of glucose transporter 1 (GLUT1) and cationic-independent mannose-6-phosphate receptor (CI-M6PR), respectively, was normal in infected cells. However, siRNA knockdown of retromer resulted in aberrant trafficking of GLUT1, but not CI-M6PR, suggesting canonical retrograde trafficking is unaffected by retromer disruption. Treatment with the specific Arp2/3 inhibitor CK-666 strongly inhibited CCV formation, an effect associated with altered endosomal trafficking of transferrin receptor. Collectively, our results show that CCV actin patches generated by retromer, WASH, and Arp2/3 are dispensable for CCV biogenesis and stability. However, Arp2/3-mediated production of actin filaments required for cargo transport within the endosomal system is required for CCV generation. These findings delineate which of the many actin related events that shape the endosomal compartment are important for CCV formation.

A specialized pathway for erythroid iron delivery through lysosomal trafficking of transferrin receptor 2

Erythroid progenitors are the largest consumers of iron in the human body. In these cells, a high flux of iron must reach the mitochondrial matrix to form sufficient heme to support hemoglobinization. Canonical erythroid iron trafficking occurs via the first transferrin receptor (TfR1)-mediated endocytosis of diferric-transferrin into recycling endosomes, where ferric iron is released, reduced, and exported to the cytosol via DMT1. However, mice lacking TfR1 or DMT1 demonstrate residual erythropoiesis, suggesting additional pathways for iron use. How iron moves from endosomes to mitochondria is incompletely understood, with both cytosolic chaperoning and "kiss and run" interorganelle transfer implicated. TfR2, in contrast to its paralog TfR1, has established roles in iron sensing, but not iron uptake. Recently, mice with marrow-selective TfR2 deficiency were found to exhibit microcytosis, suggesting TfR2 may also contribute to erythroid hemoglobinization. In this study, we identify alternative trafficking, in which TfR2 mediates lysosomal transferrin delivery. Imaging studies reveal an erythroid lineage-specific organelle arrangement consisting of a focal lysosomal cluster surrounded by a nest of mitochondria, with direct contacts between these 2 organelles. Erythroid TfR2 deficiency yields aberrant mitochondrial morphology, implicating TfR2-dependent transferrin trafficking in mitochondrial maintenance. Human TFR2 shares a lineage- and stage-specific expression pattern with MCOLN1, encoding a lysosomal iron channel, and MFN2, encoding a protein mediating organelle contacts. Functional studies reveal these latter factors to be involved in mitochondrial regulation and erythroid differentiation, with Mfn2 required for mitochondrial-lysosomal contacts. These findings identify a new pathway for erythroid iron trafficking involving TfR2-mediated lysosomal delivery followed by interorganelle transfer to mitochondria.

Controlling transferrin receptor trafficking with GPI-valence in bloodstream stage African trypanosomes.

Bloodstream-form African trypanosomes encode two structurally related glycosylphosphatidylinositol (GPI)-anchored proteins that are critical virulence factors, variant surface glycoprotein (VSG) for antigenic variation and transferrin receptor (TfR) for iron acquisition. Both are transcribed from the active telomeric expression site. VSG is a GPI2 homodimer; TfR is a GPI1 heterodimer of GPI-anchored ESAG6 and ESAG7. GPI-valence correlates with secretory progression and fate in bloodstream trypanosomes: VSG (GPI2) is a surface protein; truncated VSG (GPI0) is degraded in the lysosome; and native TfR (GPI1) localizes in the flagellar pocket. Tf:Fe starvation results in up-regulation and redistribution of TfR to the plasma membrane suggesting a saturable mechanism for flagellar pocket retention. However, because such surface TfR is non-functional for ligand binding we proposed that it represents GPI2 ESAG6 homodimers that are unable to bind transferrin—thereby mimicking native VSG. We now exploit a novel RNAi system for simultaneous lethal silencing of all native TfR subunits and exclusive in-situ expression of RNAi-resistant TfR variants with valences of GPI0–2. Our results conform to the valence model: GPI0 ESAG7 homodimers traffick to the lysosome and GPI2 ESAG6 homodimers to the cell surface. However, when expressed alone ESAG6 is up-regulated ~7-fold, leaving the issue of saturable retention in the flagellar pocket in question. Therefore, we created an RNAi-resistant GPI2 TfR heterodimer by fusing the C-terminal domain of ESAG6 to ESAG7. Co-expression with ESAG6 generates a functional heterodimeric GPI2 TfR that restores Tf uptake and cell viability, and localizes to the cell surface, without overexpression. These results resolve the longstanding issue of TfR trafficking under over-expression and confirm GPI valence as a critical determinant of intracellular sorting in trypanosomes.

Mammalian farnesyltransferase α subunit regulates vacuolar protein sorting-associated protein 4A (Vps4A) – dependent intracellular trafficking through recycling endosomes

The protein farnesyltransferase (FTase) mediates posttranslational modification of proteins with isoprenoid lipids. FTase is a heterodimer and although the β subunit harbors the active site, it requires the α subunit for its activity. Here we explore the other functions of the FTase α subunit in addition to its established role in protein prenylation. We found that in the absence of the β subunit, the α subunit of FTase forms a stable autonomous dimeric structure in solution. We identify interactors of FTase α using mass spectrometry, followed by rapid in vitro analysis using the Leishmania tarentolae cell – free system. Vps4A was validated for direct binding to the FTase α subunit both in vitro and in vivo. Analysis of the interaction with Vps4A in Hek 293 cells demonstrated that FTase α controls trafficking of transferrin receptor upstream of this protein. These results point to the existence of previously undetected biological functions of the FTase α subunit that includes control of intracellular membrane trafficking.

Ultrastructural Morphometry Points to a New Role for LAMTOR2 in Regulating the Endo/Lysosomal System

The late endosomal adaptor protein LAMTOR2/p14 is essential for tissue homeostasis by controlling MAPK and mTOR signaling, which in turn regulate cell growth and proliferation, migration and spreading. Moreover, LAMTOR2 critically controls architecture and function of the endocytic system, including epidermal growth factor receptor (EGFR) degradation in lysosomes, positioning of late endosomes and defense against intracellular pathogens. Here we describe the multifaceted ultrastructural phenotype of the endo/lysosomal system of LAMTOR2-deficient mouse embryonic fibroblasts. Quantitative (immuno-)electron microscopy of cryo-fixed samples revealed significantly reduced numbers of recycling tubules emanating from maturing multivesicular bodies (MVB). Instead, a distinct halo of vesicles surrounded MVB, tentatively interpreted as detached, jammed recycling tubules. These morphological changes in LAMTOR2-deficient cells correlated with the presence of growth factors (e.g. EGF), but were similarly induced in control cells by inactivating mTOR. Furthermore, proper transferrin receptor trafficking and recycling were apparently dependent on an intact LAMTOR complex. Finally, a severe imbalance in the relative proportions of endo/lysosomes was found in LAMTOR2-deficient cells, resulting from increased amounts of mature MVB and (autophago)lysosomes. These observations suggest that the LAMTOR/Ragulator complex is required not only for maintaining the homeostasis of endo/lysosomal subpopulations but also contributes to the proper formation of MVB-recycling tubules, and regulation of membrane/cargo recycling from MVB.

Distinct effects of Disabled-2 on transferrin uptake in different cell types and culture conditions.

Iron uptake by the transferrin (Tf)-transferrin receptor (TfR) complex is critical for erythroid differentiation. The mechanisms of TfR trafficking have been examined, but the adaptor proteins involved in this process are not fully elucidated. We have investigated the role of the adaptor protein, Disabled-2 (Dab2), in erythroid differentiation and Tf uptake in the cells of hematopoietic lineage. Dab2 was upregulated in a time-dependent manner during erythroid differentiation of mouse embryonic stem cells and human K562 erythroleukemic cells. Attenuating Dab2 expression in K562 cells diminished TfR internalization and increased surface levels of TfR concomitantly with a decrease in Tf uptake and erythroid differentiation. Dab2 regulated Tf uptake of the suspended, but not adherent, cultures of K562 cells. In contrast, Dab2 is not involved in TfR trafficking in the HeLa cells with epithelial origin. These differential effects are Dab2-specific because attenuating the expression of adaptor protein 2 μ subunit inhibited the uptake of Tf regardless of culture condition. We offer novel insight of Dab2 function in iron uptake and TfR internalization for the suspended culture of hematopoietic lineage cells.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Transferrin receptor (TfR) trafficking determines brain uptake of TfR antibody affinity variants

Antibodies to transferrin receptor (TfR) have potential use for therapeutic entry into the brain. We have shown that bispecific antibodies against TfR and β-secretase (BACE1 [β-amyloid cleaving enzyme-1]) traverse the blood-brain barrier (BBB) and effectively reduce brain amyloid β levels. We found that optimizing anti-TfR affinity improves brain exposure and BACE1 inhibition. Here we probe the cellular basis of this improvement and explore whether TfR antibody affinity alters the intracellular trafficking of TfR. Comparing high- and low-affinity TfR bispecific antibodies in vivo, we found that high-affinity binding to TfR caused a dose-dependent reduction of brain TfR levels. In vitro live imaging and colocalization experiments revealed that high-affinity TfR bispecific antibodies facilitated the trafficking of TfR to lysosomes and thus induced the degradation of TfR, an observation which was further confirmed in vivo. Importantly, high-affinity anti-TfR dosing induced reductions in brain TfR levels, which significantly decreased brain exposure to a second dose of low-affinity anti-TfR bispecific. Thus, high-affinity anti-TfR alters TfR trafficking, which dramatically impacts the capacity for TfR to mediate BBB transcytosis.

Reggies/flotillins interact with Rab11a and SNX4 at the tubulovesicular recycling compartment and function in transferrin receptor and E-cadherin trafficking

The lipid raft proteins reggie-1 and -2 (flotillins) are implicated in membrane protein trafficking but exactly how has been elusive. We find that reggie-1 and -2 associate with the Rab11a, SNX4, and EHD1-decorated tubulovesicular recycling compartment in HeLa cells and that reggie-1 directly interacts with Rab11a and SNX4. Short hairpin RNA-mediated down-regulation of reggie-1 (and -2) in HeLa cells reduces association of Rab11a with tubular structures and impairs recycling of the transferrin-transferrin receptor (TfR) complex to the plasma membrane. Overexpression of constitutively active Rab11a rescues TfR recycling in reggie-deficient HeLa cells. Similarly, in a Ca(2+) switch assay in reggie-depleted A431 cells, internalized E-cadherin is not efficiently recycled to the plasma membrane upon Ca(2+) repletion. E-cadherin recycling is rescued, however, by overexpression of constitutively active Rab11a or SNX4 in reggie-deficient A431 cells. This suggests that the function of reggie-1 in sorting and recycling occurs in association with Rab11a and SNX4. Of interest, impaired recycling in reggie-deficient cells leads to de novo E-cadherin biosynthesis and cell contact reformation, showing that cells have ways to compensate the loss of reggies. Together our results identify reggie-1 as a regulator of the Rab11a/SNX4-controlled sorting and recycling pathway, which is, like reggies, evolutionarily conserved.

Optineurin mediates negative regulation of Rab8 function by TBC1D17, a GTPase activating protein

Rab GTPases regulate various membrane trafficking pathways but the mechanisms by which GTPase-activating proteins recognise specific Rabs are not clear. Rab8 is involved in controlling several trafficking processes, including the trafficking of transferrin receptor from the early endosome to the recycling endosome. Here, we provide evidence to show that TBC1D17, a Rab GTPase-activating protein, through its catalytic activity, regulates Rab8-mediated endocytic trafficking of transferrin receptor. Optineurin, a Rab8-binding effector protein, mediates the interaction and colocalisation of TBC1D17 with Rab8. A non-catalytic region of TBC1D17 is required for direct interaction with optineurin. Co-expression of Rab8, but not other Rabs tested, rescues the inhibition of transferrin receptor trafficking by TBC1D17. The activated GTP-bound form of Rab8 is localised to the tubules emanating from the endocytic recycling compartment. Through its catalytic activity, TBC1D17 inhibits recruitment of Rab8 to the tubules and reduces colocalisation of transferrin receptor and Rab8. Knockdown of optineurin or TBC1D17 results in enhanced recruitment of Rab8 to the tubules. A glaucoma-associated mutant of optineurin, E50K, causes enhanced inhibition of Rab8 by TBC1D17, resulting in defective endocytic recycling of transferrin receptor. Our results show that TBC1D17, through its interaction with optineurin, regulates Rab8-mediated endocytic recycling of transferrin receptor and recruitment of Rab8 to the endocytic recycling tubules. We describe a mechanism of regulating a Rab GTPase by an effector protein (optineurin) that acts as an adaptor to bring together a Rab (Rab8) and its GTPase-activating protein (TBC1D17).