Endocytic Motif Research Articles

Long term rescue of Alzheimer's deficits in vivo by one-time gene-editing of App C-terminus.

Gene-editing technologies promise to create a new class of therapeutics that can achieve permanent correction with a single intervention. Besides eliminating mutant alleles in familial disease, gene-editing can also be used to favorably manipulate upstream pathophysiologic events and alter disease-course in wider patient populations, but few such feasible therapeutic avenues have been reported. Here we use CRISPR-Cas9 to edit the last exon of amyloid precursor protein (App), relevant for Alzheimer's disease (AD). Our strategy effectively eliminates an endocytic (YENPTY) motif at APP C-terminus, while preserving the N-terminus and compensatory APP-homologues. This manipulation favorably alters events along the amyloid-pathway - inhibiting toxic APP-β-cleavage fragments (including Aβ) and upregulating neuroprotective APP-α-cleavage products. AAV-driven editing ameliorates neuropathologic, electrophysiologic, and behavioral deficits in an AD knockin mouse model. Effects persist for many months, and no abnormalities are seen in WT mice even after germline App-editing; underlining overall efficacy and safety. Pathologic alterations in the glial-transcriptome of App-KI mice, as seen by single nuclei RNA-sequencing (sNuc-Seq), are also normalized by App C-terminus editing. Our strategy takes advantage of innate transcriptional rules that render terminal exons insensitive to nonsense-decay, and the upstream manipulation is expected to be effective for all forms of AD. These studies offer a path for a one-time disease-modifying treatment for AD.

TXNIP-mediated crosstalk between oxidative stress and glucose metabolism.

Thioredoxin-interacting protein (TXNIP) has emerged as a key player in cancer and diabetes since it targets thioredoxin (TRX)-mediated redox regulation and glucose transporter (GLUT)-mediated metabolism. TXNIP consists of two arrestin (ARR, N-ARR and C-ARR) domains at its amino-terminus and two PPxY (PY) motifs and a di-leucine (LL) motif for endocytosis at its carboxyl-terminus. Here, we report that TXNIP shuffles between TRX and GLUTs to regulate homeostasis of intracellular oxidative stress and glucose metabolism. While TXNIP functions as a gatekeeper of TRX by default, it robustly interacted with class I GLUTs through its C-ARR domain upon increase of intracellular reactive oxygen species. This interaction prompted the surface expression downregulation and lysosomal degradation of GLUTs by its carboxyl-terminal LL endocytic signaling motif to attenuate glucose uptake. Consequently, TXNIP expression significantly limited glucose uptake, leading to the suppression of glycolysis, hexosamine biosynthesis, and the pentose phosphate pathway. Our findings establish a fundamental link between ROS and glucose metabolism through TXNIP and provide a promising target for the drug development against GLUT-related metabolic disorders.

Lysosome-targeting chimeras containing an endocytic signaling motif trigger endocytosis and lysosomal degradation of cell-surface proteins.

Lysosome-targeting degradation technologies have emerged as a promising therapeutic strategy for the selective depletion of target extracellular and cell-surface proteins by harnessing a cell-surface effector protein such as lysosome-targeting receptors (LTRs) or transmembrane E3 ligases that direct lysosomal degradation. We recently developed a lysosome-targeting degradation platform termed signal-mediated lysosome-targeting chimeras (SignalTACs) that functions independently of an LTR or E3 ligase; these are engineered fusion proteins comprising a target binder, a cell-penetrating peptide (CPP), and a lysosomal sorting signal motif (P1). Herein, we present the next-generation SignalTACs containing a single endocytic signal that bypasses the need for a CPP. We demonstrate that the fusion with a 10-amino acid endocytic signaling peptide (P3) derived from the cation-independent mannose-6-phosphate receptor (CI-M6PR) induces robust internalization and lysosomal degradation of the target protein. The P3-based SignalTAC exhibited enhanced antitumor efficacy compared to the parent antibody. We envision that the fusion of the endocytic signaling peptide P3 to a target binder may allow the construction of an effective degrader for membrane-associated targets. Furthermore, mechanistic studies identified different drivers for the activities of the P3- and P1-based SignalTACs, which is expected to provide crucial insights toward the harnessing of the intrinsic signaling pathways to direct protein trafficking and degradation.

OR03-05 Cinacalcet Acts As A Spatially Biased Allosteric Modulator To Enhance CaSR Signaling From Endosomal Membranes

Abstract Disclosure: R. Wyatt: None. C.M. Gorvin: None. The calcium-sensing receptor (CaSR) is a G protein-coupled receptor that plays a fundamental role in extracellular calcium homeostasis by activating Gαq/11 and Gαi/o signaling to regulate parathyroid hormone (PTH) release. CaSR can also continue signaling once internalised (known as sustained signaling) to elicit responses that are distinct from those at the plasma membrane. Loss-of-function mutations of CaSR cause familial hypocalciuric hypercalcemia type 1 (FHH1), while Arg15 mutations in the adaptor protein-2 σ-subunit (AP2σ) cause FHH3, by impairing CaSR internalisation and disrupting sustained signaling. However, the sustained signaling pathway has been incompletely defined and the effects of allosteric modulators on the process are unknown. Here we examined CaSR membrane trafficking and G protein activation in different endomembrane compartments using bystander BRET and HILO advanced imaging in HEK293 cells. This showed that internalised CaSR is targeted to Rab5-positive early endosomes and Rab4-positive early-to-recycling membranes, and not to degradation pathways as previously hypothesised. CaSR exclusively recruits active Gq/11 to early endosome membranes to drive sustained signals that produce distinct transcriptional profiles than those from plasma membranes. We next mutated a C-terminal region within CaSR predicted to form a dileucine endocytic motif that interacts with AP2σ. NanoBiT assays confirmed that the CaSR-dileucine mutant disrupted interactions between CaSR and AP2σ to the same magnitude as AP2σ-Arg15 mutations. Moreover, this CaSR-dileucine mutant impaired CaSR internalisation and reduced Gq/11 sustained signals from Rab5 and Rab4 endosomes to similar levels to those observed in cells expressing the FHH3-associated AP2σ-Arg15 mutant. Gi/o and G12/13 responses from plasma membranes were not affected by the CaSR-dileucine mutant. Finally, we assessed whether the CaSR positive allosteric modulator, cinacalcet, affects sustained signalling. When exposed to cinacalcet CaSR significantly increased the recruitment of active Gq/11 to early endosomes, resulting in a significant increase in signaling, while signals from the plasma membrane were not different to vehicle-treated cells. In summary, we have demonstrated that CaSR is targeted to an early-to-recycling endosomal pathway, which could explain how CaSR responds rapidly to fluctutations in serum calcium to maintain calcium homeostasis. Moreover, we have defined the endocytic motif within the CaSR required for sustained signaling and shown that cinacalcet is a spatially-biased allosteric modulator of the CaSR that preferentially enhances signaling from endosomal membranes. Thus, FHH3-associated AP2σ mutations, CaSR dileucine mutants and cinacalcet can be used as tools to better understand CaSR spatiotemporal signaling and define its physiological significance. Presentation: Thursday, June 15, 2023

Endocytosis and Endocytic Motifs across the Connexin Gene Family.

Proteins fated to be internalized by clathrin-mediated endocytosis require an endocytic motif, where AP-2 or another adaptor protein can bind and recruit clathrin. Tyrosine and di-leucine-based sorting signals are such canonical motifs. Connexin 43 (Cx43) has three canonical tyrosine-based endocytic motifs, two of which have been previously shown to recruit clathrin and mediate its endocytosis. In addition, di-leucine-based motifs have been characterized in the Cx32 C-terminal domain and shown to mediate its endocytosis. Here, we examined the amino acid sequences of all 21 human connexins to identify endocytic motifs across the connexin gene family. We find that although there is limited conservation of endocytic motifs between connexins, 14 of the 21 human connexins contain one or more canonical tyrosine or di-leucine-based endocytic motif in their C-terminal or intracellular loop domain. Three connexins contain non-canonical (modified) di-leucine motifs. However, four connexins (Cx25, Cx26, Cx31, and Cx40.1) do not harbor any recognizable endocytic motif. Interestingly, live cell time-lapse imaging of different GFP-tagged connexins that either contain or do not contain recognizable endocytic motifs readily undergo endocytosis, forming clearly identifiable annular gap junctions when expressed in HeLa cells. How connexins without defined endocytic motifs are endocytosed is currently not known. Our results demonstrate that an array of endocytic motifs exists in the connexin gene family. Further analysis will establish whether the sites we identified in this in silico analysis are legitimate endocytic motifs.

Engineering phosphatidylinositol-4,5-bisphosphate model membranes enriched in endocytic cargo: A neutron reflectometry, AFM and QCM-D structural study

The combination of in vitro models of biological membranes based on solid-supported lipid bilayers (SLBs) and of surface sensitive techniques, such as neutron reflectometry (NR), atomic force microscopy (AFM) and quartz crystal microbalance with dissipation monitoring (QCM-D), is well suited to provide quantitative information about molecular level interactions and lipid spatial distributions. In this work, cellular plasma membranes have been mimicked by designing complex SLB, containing phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) lipids as well as incorporating synthetic lipo-peptides that simulate the cytoplasmic tails of transmembrane proteins. The QCM-D results revealed that the adsorption and fusion kinetics of PtdIns4,5P2 are highly dependent of Mg2+. Additionally, it was shown that increasing concentrations of PtdIns4,5P2 leads to the formation of SLBs with higher homogeneity. The presence of PtdIns4,5P2 clusters was visualized by AFM. NR provided important insights about the structural organization of the various components within the SLB, highlighting that the leaflet symmetry of these SLBs is broken by the presence of CD4-derived cargo peptides. Finally, we foresee our study to be a starting point for more sophisticated in vitro models of biological membranes with the incorporation of inositol phospholipids and synthetic endocytic motifs.

Rhizobia induce SYMRK endocytosis in Phaseolus vulgaris root hair cells

Main conclusionPvSYMRK-EGFP undergoes constitutive and rhizobia-induced endocytosis, which rely on the phosphorylation status of T589, the endocytic YXXØ motif and the kinase activity of the receptor.Legume-rhizobia nodulation is a complex developmental process. It initiates when the rhizobia-produced Nod factors are perceived by specific LysM receptors present in the root hair apical membrane. Consequently, SYMRK (Symbiosis Receptor-like Kinase) becomes active in the root hair and triggers an extensive signaling network essential for the infection process and nodule organogenesis. Despite its relevant functions, the underlying cellular mechanisms involved in SYMRK signaling activity remain poorly characterized. In this study, we demonstrated that PvSYMRK-EGFP undergoes constitutive and rhizobia-induced endocytosis. We found that in uninoculated roots, PvSYMRK-EGFP is mainly associated with the plasma membrane, although intracellular puncta labelled with PvSymRK-EGFP were also observed in root hair and nonhair-epidermal cells. Inoculation with Rhizobium etli producing Nod factors induces in the root hair a redistribution of PvSYMRK-EGFP from the plasma membrane to intracellular puncta. In accordance, deletion of the endocytic motif YXXØ (YKTL) and treatment with the endocytosis inhibitors ikarugamycin (IKA) and tyrphostin A23 (TyrA23), as well as brefeldin A (BFA), drastically reduced the density of intracellular PvSYMRK-EGFP puncta. A similar effect was observed in the phosphorylation-deficient (T589A) and kinase-dead (K618E) mutants of PvSYMRK-EGFP, implying these structural features are positive regulators of PvSYMRK-EGFP endocytosis. Our findings lead us to postulate that rhizobia-induced endocytosis of SYMRK modulates the duration and amplitude of the SYMRK-dependent signaling pathway.

Acidic dileucine motifs in the cylindrical inclusion protein of turnip mosaic virus are crucial for endosomal targeting and viral replication

Previously we reported that the multifunctional cylindrical inclusion (CI) protein of turnip mosaic virus (TuMV) is targeted to endosomes through the interaction with the medium subunit of adaptor protein complex 2 (AP2β), which is essential for viral infection. Although several functionally important regions in the CI have been identified, little is known about the determinant(s) for endosomal trafficking. The CI protein contains seven conserved acidic dileucine motifs [(D/E)XXXL(L/I)] typical of endocytic sorting signals recognized by AP2β. Here, we selected five motifs for further study and identified that they all were located in the regions of CI interacting with AP2β. Coimmunoprecipitation assays revealed that alanine substitutions in the each of these acidic dileucine motifs decreased binding with AP2β. Moreover, these CI mutants also showed decreased accumulation of punctate bodies, which enter endocytic‐tracking styryl‐stained endosomes. The mutations were then introduced into a full‐length infectious clone of TuMV, and each mutant had reduced viral replication and systemic infection. The data suggest that the acidic dileucine motifs in CI are indispensable for interacting with AP2β for efficient viral replication. This study provides new insights into the role of endocytic sorting motifs in the intracellular movement of viral proteins for replication.

A MET-PTPRK kinase-phosphatase rheostat controls ZNRF3 and Wnt signaling.

Zinc and ring finger 3 (ZNRF3) is a transmembrane E3 ubiquitin ligase that targets Wnt receptors for ubiquitination and lysosomal degradation. Previously, we showed that dephosphorylation of an endocytic tyrosine motif (4Y motif) in ZNRF3 by protein tyrosine phosphatase receptor-type kappa (PTPRK) promotes ZNRF3 internalization and Wnt receptor degradation (Chang et al 2020). However, a responsible protein tyrosine kinase(s) (PTK) phosphorylating the 4Y motif remained elusive. Here we identify the proto-oncogene MET (mesenchymal-epithelial transition factor) as a 4Y kinase. MET binds to ZNRF3 and induces 4Y phosphorylation, stimulated by the MET ligand HGF (hepatocyte growth factor, scatter factor). HGF-MET signaling reduces ZNRF3-dependent Wnt receptor degradation thereby enhancing Wnt/β-catenin signaling. Conversely, depletion or pharmacological inhibition of MET promotes the internalization of ZNRF3 and Wnt receptor degradation. We conclude that HGF-MET signaling phosphorylates- and PTPRK dephosphorylates ZNRF3 to regulate ZNRF3 internalization, functioning as a rheostat for Wnt signaling that may offer novel opportunities for therapeutic intervention.

Endocytic Motif on a Biotin-Tagged HIV-1 Env Modulates the Co-Transfer of Env and Gag during Cell-to-Cell Transmission.

During HIV-1 transmission through T cell virological synapses, the recruitment of the envelope (Env) glycoprotein to the site of cell–cell contact is important for adhesion and for packaging onto nascent virus particles which assemble at the site. Live imaging studies in CD4 T cells have captured the rapid recruitment of the viral structural protein Gag to VSs. We explored the role of endocytic trafficking of Env initiated by a membrane proximal tyrosine motif during HIV transfer into target cells and examined the factors that allow Gag and Env to be transferred together across the synapse. To facilitate tracking of Env in live cells, we adapted an Env tagging method and introduced a biotin acceptor peptide (BAP) into the V4 loop of Env gp120, enabling sensitive fluorescent tracking of V4-biotinylated Env. The BAP-tagged and biotinylated HIVs were replication-competent in cell-free and cell-to-cell infection assays. Live cell fluorescent imaging experiments showed rapid internalized cell surface Env on infected cells. Cell–cell transfer experiments conducted with the Env endocytosis mutant (Y712A) showed increased transfer of Env. Paradoxically, this increase in Env transfer was associated with significantly reduced Gag transfer into target cells, when compared to viral transfer associated with WT Env. This Y712A Env mutant also exhibited an altered Gag/biotin Env fluorescence ratio during transfer that correlated with decreased productive cell-to-cell infection. These results may suggest that the internalization of Env into recycling pools plays an important role in the coordinated transfer of Gag and Env across the VS, which optimizes productive infection in target cells.

Endocytosis of BRASSINOSTEROID INSENSITIVE1 Is Partly Driven by a Canonical Tyr-Based Motif.

Clathrin-mediated endocytosis (CME) and its core endocytic machinery are evolutionarily conserved across all eukaryotes. In mammals, the heterotetrameric adaptor protein complex-2 (AP-2) sorts plasma membrane (PM) cargoes into vesicles via the recognition of motifs based on Tyr or di-Leu in their cytoplasmic tails. However, in plants, very little is known about how PM proteins are sorted for CME and whether similar motifs are required. In Arabidopsis (Arabidopsis thaliana), the brassinosteroid (BR) receptor BR INSENSITIVE1 (BRI1) undergoes endocytosis, which depends on clathrin and AP-2. Here, we demonstrate that BRI1 binds directly to the medium AP-2 subunit (AP2M). The cytoplasmic domain of BRI1 contains five putative canonical surface-exposed Tyr-based endocytic motifs. The Tyr-to-Phe substitution in Y898KAI reduced BRI1 internalization without affecting its kinase activity. Consistently, plants carrying the BRI1Y898F mutation were hypersensitive to BRs. Our study demonstrates that AP-2-dependent internalization of PM proteins via the recognition of functional Tyr motifs also operates in plants.

Three YXXL Sequences of a Bovine Leukemia Virus Transmembrane Protein are Independently Required for Fusion Activity by Controlling Expression on the Cell Membrane.

Bovine leukemia virus (BLV), which is closely related to human T-cell leukemia viruses, is the causative agent of enzootic bovine leukosis, the most common neoplastic disease of cattle. The transmembrane subunit of the BLV envelope glycoprotein, gp30, contains three completely conserved YXXL sequences that fit an endocytic sorting motif. The two N-terminal YXXL sequences are reportedly critical for viral infection. However, their actual function in the viral life cycle remains undetermined. Here, we identified the novel roles of each YXXL sequence. Syncytia formation ability was upregulated by a single mutation of the tyrosine (Tyr) residue in any of the three YXXL sequences, indicating that each YXXL sequence is independently able to regulate the fusion event. The alteration resulted from significantly high expression of gp51 on the cell surface, thereby decreasing the amount of gp51 in early endosomes and further revealing that the three YXXL sequences are independently required for internalization of the envelope (Env) protein, following transport to the cell surface. Moreover, the 2nd and 3rd YXXL sequences contributed to Env protein incorporation into the virion by functionally distinct mechanisms. Our findings provide new insights regarding the three YXXL sequences toward the BLV viral life cycle and for developing new anti-BLV drugs.

Ifitm3 Is Essential for PI(3,4,5)P3-Dependent B-Cell Activation and Leukemogenesis

Background: The interferon-inducible transmembrane protein Ifitm3 (also known as Fragilis) plays an important role in primordial germ cell specification and functions as critical antiviral effector preventing fusion of virion-containing vesicles with endosomal membranes. Results: Here, we identified Ifitm3 as a biomarker of poor clinical outcome in patients with various B-cell malignancies. We found that higher IFITM3 mRNA levels at the time of diagnosis represents a strong predictor of poor clinical outcome for children (COG P9906; P=0.011; n=207) and adults (ECOG E2993; P=0.017; n=55) with B-ALL. Interestingly, IFITM3 is a transcriptional target and strongly repressed by IKZF1 (Ikaros) a potent tumor suppressor in B-ALL and high IFITM3 mRNA levels represents a biomarker for patients with IKZF1-deletion. To study the function of Ifitm3 in a model for human pre-B ALL, pre-B cells from Ifitm3-/- mice were transformed with BCR-ABL1 or oncogenic NRASG12D. Strikingly, deletion of Ifitm3 resulted in destabilization of lipid rafts, loss of CD19 surface expression and loss of PI3K signaling. Ifitm3-/- leukemia cells could not sustain oncogenic signaling from BCR-ABL1 or oncogenic NRASG12D and failed to initiate fatal leukemia in transplant recipient mice. These changes were paralleled by G0/1 cell cycle arrest (P<0.001), loss of colony formation capacity (P=0.0004) and increased propensity to apoptosis. In mechanistic studies, we identified N-terminal phosphorylation at endocytic motif (20YEML23) by Src-kinases induced Ifitm3 cell surface accumulation during normal activation or oncogenic transformation of B-cells. In a lipid-binding assay in vitro, recombinant IFITM3 directly bound to PI(3,4,5)P3 but not any other phospholipids. In the cell membrane, therefore, IFITM3 functioned as a scaffold for PI(3,4,5)P3 and was essential for Src-kinase and PI3K signaling, as well as lipid raft formation and surface expression of multiple raft-associated receptors. Conversely, inducible overexpression of IKZF1 transcriptionally silenced IFITM3, resulting in loss of IFITM3 expression, reduction of lipid rafts and impairment of Src-kinase signaling. In the absence of Ifitm3, resting B-cell populations developed normally. However, consistent with defective Src-kinase and PI3K signaling, Ifitm3-/- B-cells failed to form germinal centers and to give rise to antigen-specific humoral immune responses. Likewise, Ifitm3-/- B-cell precursors were resistant to malignant transformation and lacked the ability to initiate BCR-ABL1- and NRASG12D-driven leukemia. Conversely, an Ifitm3-phosphomimetic of Src-kinase phosphorylation induced constitutive cell membrane localization, triggered oncogenic Src-PI3K signaling and initiated overt leukemia in pre-malignant B-cells. Conclusions: We conclude that Src-kinase-mediated phosphorylation of Ifitm3 induces a dynamic switch from antiviral effector functions in endosomes to lipid raft signaling at the cell membrane. While membrane-bound Ifitm3 is critical for normal B-cell receptor signaling and antigen-specific B-cell responses, its role as signal amplifier can be leveraged by multiple oncogenes for malignant B-cell transformation. Figure Disclosures Nix: UCSF: Patents & Royalties. Chen:Genovel Biotech Corp: Other: scientific founder and Chairman. Wiita:UCSF: Patents & Royalties; Indapta Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Protocol Intelligence: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Characterization of the BspA and Pmp protein family of trichomonads

BackgroundTrichomonas vaginalis is a human-infecting trichomonad and as such the best studied and the only for which the full genome sequence is available considering its parasitic lifestyle, T. vaginalis encodes an unusually high number of proteins. Many gene families are massively expanded and some genes are speculated to have been acquired from prokaryotic sources. Among the latter are two gene families that harbour domains which share similarity with proteins of Bacteroidales/Spirochaetales and Chlamydiales: the BspA and the Pmp proteins, respectively.ResultsWe sequenced the transcriptomes of five trichomonad species and screened for the presence of BspA and Pmp domain-containing proteins and characterized individual candidate proteins from both families in T. vaginalis. Here, we demonstrate that (i) BspA and Pmp domain-containing proteins are universal to trichomonads, but specifically expanded in T. vaginalis; (ii) in line with a concurrent expansion of the endocytic machinery, there is a high number of BspA and Pmp proteins which carry C-terminal endocytic motifs; and (iii) both families traffic through the ER and have the ability to increase adhesion performance in a non-virulent T. vaginalis strain and Tetratrichomonas gallinarum by a so far unknown mechanism.ConclusionsOur results initiate the functional characterization of these two broadly distributed protein families and help to better understand the origin and evolution of BspA and Pmp domains in trichomonads.

The human nephrin Y1139RSL motif is essential for podocyte foot process organization and slit diaphragm formation during glomerular development

Nephrin is an immunoglobulin-type cell-adhesion molecule with a key role in the glomerular interpodocyte slit diaphragm. Mutations in the nephrin gene are associated with defects in the slit diaphragm, leading to early-onset nephrotic syndrome, typically resistant to treatment. Although the endocytic trafficking of nephrin is essential for the assembly of the slit diaphragm, nephrin's specific endocytic motifs remain unknown. To search for endocytic motifs, here we performed a multisequence alignment of nephrin and identified a canonical YXXØ-type motif, Y1139RSL, in the nephrin cytoplasmic tail, expressed only in primates. Using site-directed mutagenesis, various biochemical methods, single-plane illumination microscopy, a human podocyte line, and a human nephrin-expressing zebrafish model, we found that Y1139RSL is a novel endocytic motif and a structural element for clathrin-mediated nephrin endocytosis that functions as a phosphorylation-sensitive signal. We observed that Y1139RSL motif-mediated endocytosis helps to localize nephrin to specialized plasma membrane domains in podocytes and is essential for normal foot process organization into a functional slit diaphragm between neighboring foot processes in zebrafish. The importance of nephrin Y1139RSL for healthy podocyte development was supported by population-level analyses of genetic variations at this motif, revealing that such variations are very rare, suggesting that mutations in this motif have autosomal-recessive negative effects on kidney health. These findings expand our understanding of the mechanism underlying nephrin endocytosis and may lead to improved diagnostic tools or therapeutic strategies for managing early-onset, treatment-resistant nephrotic syndrome.

The MAP1B Binding Domain of Nav1.6 Is Required for Stable Expression at the Axon Initial Segment.

Nav1.6 (SCN8A) is a major voltage-gated sodium channel in the mammalian CNS, and is highly concentrated at the axon initial segment (AIS). As previously demonstrated, the microtubule associated protein MAP1B binds the cytoplasmic N terminus of Nav1.6, and this interaction is disrupted by the mutation p.VAVP(77-80)AAAA. We now demonstrate that this mutation results in WT expression levels on the somatic surface but reduced surface expression at the AIS of cultured rat embryonic hippocampal neurons from both sexes. The mutation of the MAP1B binding domain did not impair vesicular trafficking and preferential delivery of Nav1.6 to the AIS; nor was the diffusion of AIS inserted channels altered relative to WT. However, the reduced AIS surface expression of the MAP1B mutant was restored to WT levels by inhibiting endocytosis with Dynasore, indicating that compartment-specific endocytosis was responsible for the lack of AIS accumulation. Interestingly, the lack of AIS targeting resulted in an elevated percentage of persistent current, suggesting that this late current originates predominantly in the soma. No differences in the voltage dependence of activation or inactivation were detected in the MAP1B binding mutant relative to WT channel. We hypothesize that MAP1B binding to the WT Nav1.6 masks an endocytic motif, thus allowing long-term stability on the AIS surface. This work identifies a critical and important new role for MAP1B in the regulation of neuronal excitability and adds to our understanding of AIS maintenance and plasticity, in addition to identifying new target residues for pathogenic mutations of SCN8ASIGNIFICANCE STATEMENT Nav1.6 is a major voltage-gated sodium channel in human brain, where it regulates neuronal activity due to its localization at the axon initial segment (AIS). Nav1.6 mutations cause epilepsy, intellectual disability, and movement disorders. In the present work, we show that loss of interaction with MAP1B within the Nav1.6 N terminus reduces the steady-state abundance of Nav1.6 at the AIS. The effect is due to increased Nav1.6 endocytosis at this neuronal compartment rather than a failure of forward trafficking to the AIS. This work confirms a new biological role of MAP1B in the regulation of sodium channel localization and will contribute to future analysis of patient mutations in the cytoplasmic N terminus of Nav1.6.

An extracellular histidine-containing motif in the zinc transporter ZIP4 plays a role in zinc sensing and zinc-induced endocytosis in mammalian cells

Zinc is an essential trace element that serves as a cofactor for enzymes in critical biochemical processes and also plays a structural role in numerous proteins. Zinc transporter ZIP4 (ZIP4) is a zinc importer required for dietary zinc uptake in the intestine and other cell types. Studies in cultured cells have reported that zinc stimulates the endocytosis of plasma membrane-localized ZIP4 protein, resulting in reduced cellular zinc uptake. Thus, zinc-regulated trafficking of ZIP4 is a key means for regulating cellular zinc homeostasis, but the underlying mechanisms are not well understood. In this study, we used mutational analysis, immunoblotting, HEK293 cells, and immunofluorescence microscopy to identify a histidine-containing motif (398HTH) in the first extracellular loop that is required for high sensitivity to low zinc concentrations in a zinc-induced endocytic response of mouse ZIP4 (mZIP4). Moreover, using synthetic peptides with selective substitutions and truncated mZIP4 variants, we provide evidence that histidine residues in this motif coordinate a zinc ion in mZIP4 homodimers at the plasma membrane. These findings suggest that 398HTH is an important zinc-sensing motif for eliciting high-affinity zinc-stimulated endocytosis of mZIP4 and provide insight into cellular mechanisms for regulating cellular zinc homeostasis in mammalian cells.

IFITM3-Mediated Regulation of Cell Membrane Dynamics Is Essential for Malignant B-Cell Transformation

Background & Hypothesis: B cell receptor (BCR) signaling and oncogenic tyrosine kinases that mimic BCR-signaling in B-lineage leukemia and lymphoma depend on assembly of membrane proximal signaling complexes. Signalosomes in normal BCR- and oncogene (e.g. BCR-ABL1, RAS-pathway lesions) signal transduction are recruited to phospholipid anchors in lipid rafts. The robustness of these complexes depends on cholesterol accumulation in lipid rafts. Here we identified the interferon-induced transmembrane protein IFITM3 as a central regulator of cholesterol in lipid rafts.Results: IFITM3 is mostly localized to endosomal compartments. By antagonizing VAP-A and oxysterol-binding protein 1 (OSBP1), IFITM3 promotes cholesterol accumulation and solidifies the endosomal membrane. This mechanism is particular important in anti-viral immunity, to “trap”intraluminal viral particles for lysosomal degradation. In B-cells, IFITM3 can translocate to the cell membrane and form a complex with the BCR and its co-receptors CD19, CD81 and CD21. While the functional significance of membrane expression of IFITM3 on B-cells was not known, we found that higher IFITM3 mRNA levels at the time of diagnosis represents a strong predictor of poor clinical outcome for children (COG P9906; P=0.006; n=207) and adults (ECOG E2993; P=0.014; n=215) with B-ALL. In addition, higher than median IFITM3 mRNA levels at the time of diagnosis were associated with a higher risk of relapse and positive MRD status at the end of induction chemotherapy in B-ALL and other B-cell malignancies. Interestingly, IFITM3 is a transcriptional target and strongly repressed by IKZF1 (Ikaros) a potent tumor suppressor in B- ALL and high IFITM3 mRNA levels represents a biomarker for patients with IKZF1-deletion.While its membrane-topology can vary in different cell types, we found that IFITM3 functions as a dual-pass transmembrane protein in tight association with CD19 and the Iga and Igb signaling chains of the BCR in B-ALL and B-cell lymphoma cells. To study the function of Ifitm3 in a model for human pre-B ALL, pre-B cells from Ifitm3-/- mice were transformed with BCR-ABL1 or oncogenic NRASG12D. Strikingly, deletion of IFITM3 resulted in destabilization of lipid rafts, loss of CD19 surface expression and loss of PI3K signaling. Ifitm3-/- leukemia cells could not sustain oncogenic signaling from BCR-ABL1 or oncogenic NRASG12D and failed to initiate fatal leukemia in transplant recipient mice. These changes were paralleled by G0/1 cell cycle arrest (P<0.001), loss of colony formation capacity (P=0.0004) and increased propensity to apoptosis.In mechanistic studies, we identified type II transmembrane topology for IFITM3 at plasma membrane with extracellular C and intracellular N terminus which interacted with CD19, LYN, SYK, PI3K and AKT (see schematic, left). Disruption of endocytic motif (20YEML23) by substitution of Tyr20 to Phe induced IFITM3 gain of function and forced accumulation of IFITM3 on the cell membrane, constitutive CD19-PI3K signaling, intracellular calcium mobilization, homotypic cellular aggregation and massively increased proliferation of pre-B ALL cells (see schematic, right). Conversely, inducible overexpression of IKZF1 transcriptionally silenced IFITM3, resulting in loss of IFITM3 expression, reduction of lipid rafts and impairment of membrane-associated oncogenic signaling. Through Filipin-based cholesterol staining, we found Ifitm3-/- pre-B cells have reduced levels of cholesterol in lipid rafts, which causes disruption of lipid rafts formation, as reflected by decreased levels of ganglioside GM1. Notably, the homeostatic cholesterol fluidity by presence of IFITM3 on plasma membrane was also required for initiation of B- and T cell receptor signaling in mature B- and T cell lymphoma to induce Ca2+ mobilization.Conclusions: These findings identify novel role of the viral immunity IFITM3 surface receptor as a central regulator of cell membrane cholesterol fluidity and critical mediator of sustained oncogenic tyrosine kinase (BCR-ABL1) and RAS (NRASG12D) signaling in B cell malignancies. In promoting cholesterol aggregates in lipid rafts, IFITM3 protects healthy individuals from potentially lethal viral infections, but also enables oncogenic signaling by providing a robust membrane scaffold for tyrosine kinase and RAS-pathway oncogenes. DisclosuresWiita:Sutro Biopharma: Research Funding; TeneoBio: Research Funding.

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.

Postsynaptic localization and regulation of AMPA receptors and Cav1.2 by β2 adrenergic receptor/PKA and Ca2+/CaMKII signaling.

The synapse transmits, processes, and stores data within its tiny space. Effective and specific signaling requires precise alignment of the relevant components. This review examines current insights into mechanisms of AMPAR and NMDAR localization by PSD-95 and their spatial distribution at postsynaptic sites to illuminate the structural and functional framework of postsynaptic signaling. It subsequently delineates how β2 adrenergic receptor (β2 AR) signaling via adenylyl cyclase and the cAMP-dependent protein kinase PKA is organized within nanodomains. Here, we discuss targeting of β2 AR, adenylyl cyclase, and PKA to defined signaling complexes at postsynaptic sites, i.e., AMPARs and the L-type Ca2+ channel Cav1.2, and other subcellular surface localizations, the role of Akinase anchor proteins, the physiological relevance of the spatial restriction of corresponding signaling, and their interplay with signal transduction by the Ca2+- and calmodulin-dependent kinase CaMKII How localized and specific signaling by cAMP occurs is a central cellular question. The dendritic spine constitutes an ideal paradigm for elucidating the dimensions of spatially restricted signaling because of their small size and defined protein composition.