Endocytosis Machinery Research Articles

Single Quantum Dot Tracking Reveals that an Individual Multivalent HIV-1 Tat Protein Transduction Domain Can Activate Machinery for Lateral Transport and Endocytosis

The mechanisms underlying the cellular entry of the HIV-1 Tat protein transduction domain (TatP) and the molecular information necessary to improve the transduction efficiency of TatP remain unclear due to the technical limitations for direct visualization of TatP's behavior in cells. Using confocal microscopy, total internal reflection fluorescence microscopy, and four-dimensional microscopy, we developed a single-molecule tracking assay for TatP labeled with quantum dots (QDs) to examine the kinetics of TatP initially and immediately before, at the beginning of, and immediately after entry into living cells. We report that even when the number of multivalent TatP (mTatP)-QDs bound to a cell was low, each single mTatP-QD first locally induced the cell's lateral transport machinery to move the mTatP-QD toward the center of the cell body upon cross-linking of heparan sulfate proteoglycans. The centripetal and lateral movements were linked to the integrity and flow of actomyosin and microtubules. Individual mTatP underwent lipid raft-mediated temporal confinement, followed by complete immobilization, which ultimately led to endocytotic internalization. However, bivalent TatP did not sufficiently promote either cell surface movement or internalization. Together, these findings provide clues regarding the mechanisms of TatP cell entry and indicate that increasing the valence of TatP on nanoparticles allows them to behave as cargo delivery nanomachines.

Abstract LB-240: A novel approach to detecting RNA expression in living breast cancer cells.

Abstract When studying the complexities of cancer biomarkers, the ability to monitor gene expression within live cells that have become cancerous provides better insight than examining fixed or lysed tissues. However, monitoring RNA within intact cells can prove to be challenging with current techniques, which involve complex RNA isolation procedures that compromise cell viability. Further, amplification methods can create false positives or erroneously inflate differences. Here we describe a probe-based detection method that allows for the detection of ErbB-2 and EGFR mRNA levels across live intact SK-BR-3 breast cancer cells without the need for transfection reagents. The detection methodology is based upon an oligonucleotide gold nanoparticle conjugate capable of detecting intracellular levels of mRNA and miRNA in living cells. The probes enter the cell by means of the cell's native endocytosis machinery. Upon binding to the complementary target RNA sequence, a fluorescent signal (flare) is released, which allows the cells to be detected on any fluorescence analysis platform. In contrast to traditional RNA detection methods, since our technique allows for detection within live cells without altering their gene expression, the same cells that were profiled for EGFR and ErbB-2 expression could be sorted based on their specific mRNA content and subsequently further studied. Stimulation or treatments could then be performed on the previously profiled cells and assessed using probes for additional RNA markers or more traditional techniques such as antibody staining, qRT-PCR, or Array profiling. For this purpose, we combined immunostaining for cell surface EGFR with fluorogenic nanoparticle-based detection of EGFR mRNA in 4 breast cancer cell lines known to range in EGFR expression levels, and observed significant correlation for the signals in two-color flow cytometry. Thus, detecting RNA expression levels in live cancer cells with the ability to utilize the same cells in downstream testing gives researchers the ability to perform experiments which were previously thought to be impossible. Citation Format: Don Weldon, Kevin Su, Alex Ko, Grace Johnston, Matthew Hsu, Lucas Armstrong. A novel approach to detecting RNA expression in living breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-240. doi:10.1158/1538-7445.AM2013-LB-240

Casein Kinase 2 Phosphorylation of Protein Kinase C and Casein Kinase 2 Substrate in Neurons (PACSIN) 1 Protein Regulates Neuronal Spine Formation

The PACSIN (protein kinase C and casein kinase 2 substrate in neurons) adapter proteins couple components of the clathrin-mediated endocytosis machinery with regulators of actin polymerization and thereby regulate the surface expression of specific receptors. The brain-specific PACSIN 1 is enriched at synapses and has been proposed to affect neuromorphogenesis and the formation and maturation of dendritic spines. In studies of how phosphorylation of PACSIN 1 contributes to neuronal function, we identified serine 358 as a specific site used by casein kinase 2 (CK2) in vitro and in vivo. Phosphorylated PACSIN 1 was found in neuronal cytosol and membrane fractions. This localization could be modulated by trophic factors such as brain-derived neurotrophic factor (BDNF). We further show that expression of a phospho-negative PACSIN 1 mutant, S358A, or inhibition of CK2 drastically reduces spine formation in neurons. We identified a novel protein complex containing the spine regulator Rac1, its GTPase-activating protein neuron-associated developmentally regulated protein (NADRIN), and PACSIN 1. CK2 phosphorylation of PACSIN 1 leads to a dissociation of the complex upon BDNF treatment and induces Rac1-dependent spine formation in dendrites of hippocampal neurons. These findings suggest that upon BDNF signaling PACSIN 1 is phosphorylated by CK2 which is essential for spine formation.

Cells must Accumulate Interleukin-4 Receptor Subunits within Cortical Signaling Endosomes to Drive Complex Formation and Signal Transduction

The cytokines Interleukin-4 (IL-4) and IL-13 provide cues for many important immune functions and their upregulation is associated with disorders like allergy and asthma. Signaling requires ligand mediated dimerization of single-pass transmembrane receptors. We recently established epithelial HEK293T cells as a model to characterize ectopically expressed IL-4R subunits with single- and dual-color fluorescence correlation spectroscopy [1]. Here we report an improved experimental setup employing hexahistidine specific dyes and demonstrate for the first time ligand-induced IL-4R complex formation in a native plasma membrane. Furthermore, we quantified the two-dimensional affinity constants for all three combinations of receptor dimers with 120 (IL-4:IL-4Rα/IL-13Rα1), 510 (IL-13:IL-13Rα1/IL-4Rα), and 825 (IL-4:IL-4Rα/IL-2Rγ) receptor molecules per μm2. However, considering physiological surface expression levels of several 100-1000 receptor molecules per cell, such low affinities challenge the traditional view that signaling productive complexes self-assemble in significant numbers in the plasma membrane.Instead, we mount several lines of evidence that signal transduction requires the accumulation of the receptor subunits within a novel class of early endosomes. These cortical signaling endosomes are stably anchored within the actin cortex just beneath the plasma membrane and carry markers of both the early sorting (EEA1, Rab5) and recycling compartments (Rab11). The IL-4R subunits show Rac1/Pak-dependent trafficking from the surface into these endosomes with a time constant of 6-9 min. Using fluorescence lifetime imaging / Forster resonance energy transfer (FLIM/FRET) microscopy, we could demonstrate ligand-dependent complex formation within the cortical signaling endosomes. Importantly, specific inhibition of the endocytosis machinery with drugs abrogates both receptor trafficking and phosphorylation of the downstream signal transducer STAT6. In summary, our findings suggest a unique thermodynamic function for endocytosis upstream of JAK/STAT pathway activation.[1] Weidemann, T., et al. (2011) Biophys. J.101: 2360-69.

Direct binding of Magi2 to the USH1G protein SANS links the periciliary USH protein network to endocytosis

The human Usher syndrome (USH) is the most common form of combined deaf-blindness. The encoded molecules are integrated into protein networks by scaffolds including the USH1G protein SANS (scaffold protein containing ankyrin repeats and SAM domain). Previous studies indicated SANS´ participation in vesicle transport and cargo handover at the periciliary region of photoreceptor cells. To decipher the precise cellular role of SANS, we searched for interacting partners. Therefore we adopted a yeast-2-hybrid screen of a retinal cDNA library using SANS´ C-terminus as bait. Amongst others we identified the MAGUK protein Magi2 (membrane-associated guanylate kinase inverted-2) as putative binding partner. Magi2 is known as scaffold protein involved in endocytosis at synapses. We confirmed Magi2 as direct interaction partner of SANS by complementary interaction assays. In addition, correlative light and electron microscopy showed partial co-localization of SANS and Magi2 in the periciliary region of photoreceptor cells. Furthermore, immunoelectron microscopy revealed the association of Magi2-SANS complex with membranous vesicles in this region. These vesicles may represent either vesicles related to ciliary transport or endocytotic vesicles. Preliminary data revealed endocytotic processes at the periciliary region of photoreceptor cells. In conclusion, we have identified Magi2 as component of the USH protein interactome. Present data indicate a role of SANS-Magi2 complex in endocytosis in the periciliary region of photoreceptor cells. These findings confirm the recently emphasized endocytotic function of the ciliary pocket in primary cilia. Furthermore, we provide first evidence for a molecular link of the USH protein interactome to the endocytosis machinery in photoreceptor cells.

Search for inhibitors of endocytosis

We discuss here the variety of approaches that have been taken to inhibit different forms of endocytosis. Typically, both non-specific and specific chemical inhibitors of endocytosis are tried in order to “classify” entry of a new plasma membrane protein into one of the various types of endocytosis. This classification can be confirmed through genetic approaches of protein depletion or overexpression of mutants of known endocytosis machinery components. Although some new compounds have been designed to be selective in biochemical assays, we caution investigators to be alert to the unintended consequences that sometimes arise when these compounds are applied to intact cells.

Sequestration of Sup35 by Aggregates of huntingtin Fragments Causes Toxicity of [PSI+] Yeast

Expression of huntingtin fragments with 103 glutamines (HttQ103) is toxic in yeast containing either the [PIN(+)] prion, which is the amyloid form of Rnq1, or [PSI(+)] prion, which is the amyloid form of Sup35. We find that HttQP103, which has a polyproline region at the C-terminal end of the polyQ repeat region, is significantly more toxic in [PSI(+)] yeast than in [PIN(+)], even though HttQP103 formed multiple aggregates in both [PSI(+)] and [PIN(+)] yeast. This toxicity was only observed in the strong [PSI(+)] variant, not the weak [PSI(+)] variant, which has more soluble Sup35 present than the strong variant. Furthermore, expression of the MC domains of Sup35, which retains the C-terminal domain of Sup35, but lacks the N-terminal prion domain, almost completely rescued HttQP103 toxicity, but was less effective in rescuing HttQ103 toxicity. Therefore, the toxicity of HttQP103 in yeast containing the [PSI(+)] prion is primarily due to sequestration of the essential protein, Sup35.

Characterization of two distinct modes of endophilin in clathrin-mediated endocytosis

Endophilin, one of the main accessory proteins involved in clathrin-mediated endocytosis, interacts with other endocytic proteins, such as dynamin, by its SH3 domain. We previously reported that voltage-gated Ca2+ channels are an integral part of the synaptic vesicle (SV) endocytosis machinery through their interaction with endophilin. Formation of the endophilin–channel complex is Ca2+ dependent. A glutamate residue, E264, in endophilin is part of the primary Ca2+ sensor for Ca2+-dependent formation of the channel–endophilin complex. We proposed that endophilin exists in two distinct modes (conformations), an open mode in the absence of Ca2+, and a closed mode in the presence of Ca2+. Binding of Ca2+ switches endophilin from its open mode to the closed mode, resulting in dissociation of endophilin from other proteins. The present study is aimed at understanding the functional roles of endophilin in its two different modes, by creating two endophilin mutants, E264A and E264R, to mimic endophilin in its permanent open mode and permanent closed mode respectively. Here, we show that these two modes of endophilin have different effects on how endophilin interacts with other proteins, such as dynamin or β1-adrenergic receptors. In living cells, endophilin in its permanent closed mode does not show obvious effects on agonist-induced internalization of β1-adrenergic receptors. Endophilin, when in its permanent open mode, enhances the short-term synaptic depression in cultured hippocampal neurons, due partly to its failure to dissociate from Ca2+ channels in the presence of Ca2+. Our results show that modal switching by Ca2+ allows endophilin to regulate, more effectively, the clathrin-mediated endocytosis of SV at the nerve terminal.

The role of Numb/Numblike in lung epithelium

During embryonic development, asymmetric cell division of stem cells plays an important role in generating distinct sibling cell fates. One of the factors involved in asymmetric cell division processes and cell fate determination is the membrane-associated adaptor protein Numb which is known to segregate apically as a cortical crescent in one of the two cleaved daughter cells. The ability of regulating cell fate determination is promoted by inhibition of Notch-signaling thus leading to premature differentiation and depletion of stem- or progenitor cells [1, 2]. Furthermore, there is evidence that both Numb and Notch are implicated in human tumors (non-small-cell lung carcinomas, NSCLCs). Class-1 NSCLCs show a loss of Numb expression while there is simultaneously an upregulated activation of Notch meaning that Numb might have a potential tumor suppressive function [3]. However, the role of Numb/Numblike during lung development has not been investigated so far. In this work we want to investigate if Numb and Numblike are critical factors concerning self-renewal and asymmetric cell division of lung stem cells as well as the complex developmental and differentiation processes of the lung. Therefore we have performed conditional knockout mice for Numb on a Numblike deficient background using the doxycycline inducible SP-CrtTA-tet-O-Cre (surfactant protein C) system which enables us to analyse potential Numb functions in pulmonary stem cell maintenance and/or tumor formation in the distal lung epithelium. SPCrtTApos/tetO-Crepos/Numblox-/-/Numblike-/- mutants are viable and exhibit a complete loss of Numb in the distal lung epithelium. Conditional deletion of Numb/Numblike in the distal lung epithelium under non-disease conditions leads to morphological changes of epithelia cells accompanied by a basal-to-apicolateral translocation of E-Cadherin. These changes indicate a potential function of Numb/Numblike in the lung epithelium polarity and integrity. We have also generated a mouse strain which will allow us to specifically elucidate changes in the transcriptome of conditional mutants by FACS sorting of S-PC positive Alveolar Type II cells (ATll). Due to the fact that Numb/Numblike are multifunctional adapter proteins we have established a SILAC (Stable isotope labeling in cells) based protein interaction screen in HEK 293T cells to identify novel Numb/Numblike interaction partners. In addition to previously described interaction partners of the endocytosis machinery we identified novel interaction partners of Numb/Numblike, which could be confirmed by Co-IP/Western Blotting. Literature: [1] Uemura, T., et al., numb, a gene required in determination of cell fate during sensory organ formation in Drosophila embryos. Cell, 1989. 58(2): p.349–60. [2] Cayouette, M. and M. Raff, Asymmetric segregation of Numb: a mechanism for neural specification from Drosophila to mammals. Nat Neurosci, 2002. 5(12): p.1265–9. [3] Westhoff, B., et al., Alterations of the Notch pathway in lung cancer. Proc Natl Acad Sci U S A, 2009. 106(52): p.22293–8.

Regulation of fibroblast growth factor receptor signalling and trafficking by Src and Eps8

Fibroblast growth factor receptors (FGFRs) mediate a wide spectrum of cellular responses that are crucial for development and wound healing. However, aberrant FGFR activity leads to cancer. Activated growth factor receptors undergo stimulated endocytosis, but can continue to signal along the endocytic pathway. Endocytic trafficking controls the duration and intensity of signalling, and growth factor receptor signalling can lead to modifications of trafficking pathways. We have developed live-cell imaging methods for studying FGFR dynamics to investigate mechanisms that coordinate the interplay between receptor trafficking and signal transduction. Activated FGFR enters the cell following recruitment to pre-formed clathrin-coated pits (CCPs). However, FGFR activation stimulates clathrin-mediated endocytosis; FGF treatment increases the number of CCPs, including those undergoing endocytosis, and this effect is mediated by Src and its phosphorylation target Eps8. Eps8 interacts with the clathrin-mediated endocytosis machinery and depletion of Eps8 inhibits FGFR trafficking and immediate Erk signalling. Once internalized, FGFR passes through peripheral early endosomes en route to recycling and degredative compartments, through an Src- and Eps8-dependent mechanism. Thus Eps8 functions as a key coordinator in the interplay between FGFR signalling and trafficking. This work provides the first detailed mechanistic analysis of growth factor receptor clustering at the cell surface through signal transduction and endocytic trafficking. As we have characterised the Src target Eps8 as a key regulator of FGFR signalling and trafficking, and identified the early endocytic system as the site of Eps8-mediated effects, this work provides novel mechanistic insight into the reciprocal regulation of growth factor receptor signalling and trafficking.

Cellular Uptake and Intracellular Trafficking of Antisense and siRNA Oligonucleotides

Significant progress is being made concerning the development of oligonucleotides as therapeutic agents. Studies with antisense, siRNA, and other forms of oligonucleotides have shown promise in cellular and animal models and in some clinical studies. Nonetheless, our understanding of how oligonucleotides function in cells and tissues is really quite limited. One major issue concerns the modes of uptake and intracellular trafficking of oligonucleotides, whether as "free" molecules or linked to various delivery moieties such as nanoparticles or targeting ligands. In this review, we examine the recent literature on oligonucleotide internalization and subcellular trafficking in the context of current insights into the basic machinery for endocytosis and intracellular vesicular traffic.

Abstract 1207: Molecular determinants of CXCR4 trafficking dictate the metastatic potential in breast cancer

Abstract The G-protein-coupled CXC chemokine receptor 4 (CXCR4) was shown to be a major determinant in organ-specific metastasis for various cancers. The C-terminus of the receptor contains phosphorylation/ubiquitination sites and residues that interact with adaptor proteins which couple to the endocytosis machinery. Post-endocytic events that lead to a decision between degradation and recycling are hitherto poorly understood. We used fluorescently tagged receptor molecules stably expressed in breast cancer cells to establish that mutations in the protein kinase C (PKC) site of CXCR4 affect not only the cellular equilibrium between ESCRT-mediated degradation and recycling pathways but also the invasive properties. Importantly, the obtained in vitro results excellently translate to a murine model of breast cancer, in which we found that mutation of the PKC motif influences the metastatic potential. Furthermore, by using fluorescence lifetime imaging (FLIM) to measure direct protein-protein interactions via Förster resonance energy transfer (FRET) we show for the first time that ligand-dependent dimerization of CXCR4 receptors is a crucial mechanistic step in the above processes. Our findings reveal a new mechanism whereby the penetrance of a mutated receptor that exhibits resistance to its desensitization and confers an enhanced cancer migratory phenotype, is reduced through the dimerization with its wildtype counterpart. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1207. doi:10.1158/1538-7445.AM2011-1207

Endocytosis Regulates Cell Soma Translocation and the Distribution of Adhesion Proteins in Migrating Neurons

Newborn neurons migrate from their birthplace to their final location to form a properly functioning nervous system. During these movements, young neurons must attach and subsequently detach from their substrate to facilitate migration, but little is known about the mechanisms cells use to release their attachments. We show that the machinery for clathrin-mediated endocytosis is positioned to regulate the distribution of adhesion proteins in a subcellular region just proximal to the neuronal cell body. Inhibiting clathrin or dynamin function impedes the movement of migrating neurons both in vitro and in vivo. Inhibiting dynamin function in vitro shifts the distribution of adhesion proteins to the rear of the cell. These results suggest that endocytosis may play a critical role in regulating substrate detachment to enable cell body translocation in migrating neurons.

HIV-1 Rev–binding protein accelerates cellular uptake of iron to drive Notch-induced T cell leukemogenesis in mice

Somatic activating mutations in Notch1 contribute to the pathogenesis of T cell acute lymphoblastic lymphoma (T-ALL), but how activated Notch1 signaling exerts this oncogenic effect is not completely understood. Here we identify HIV-1 Rev-binding protein (Hrb), a component of the clathrin-mediated endocytosis machinery, as a critical mediator of Notch-induced T-ALL development in mice. Hrb was found to be a direct transcriptional target of Notch1, and Hrb loss reduced the incidence or delayed the onset of T-ALL in mouse models in which activated Notch1 signaling either contributes to or drives leukemogenesis. Consistent with this observation, Hrb supported survival and proliferation of hematopoietic and T cell precursor cells in vitro. We demonstrated that Hrb accelerated the uptake of transferrin, which was required for upregulation of the T cell protooncogene p21. Indeed, iron-deficient mice developed Notch1-induced T-ALL substantially more slowly than control mice, further supporting a critical role for iron uptake during leukemogenesis. Taken together, these results reveal that Hrb is a critical Notch target gene that mediates lymphoblast transformation and disease progression via its ability to satisfy the enhanced demands of transformed lymphoblasts for iron. Further, our data suggest that Hrb may be targeted to improve current treatment or design novel therapies for human T-ALL patients.

Clathrin‐mediated endocytosis: What works for small, also works for big

Clathrin and the endocytosis machinery has recently been described as being required in mammalian cells for the internalization of large particles including pathogenic bacteria, fungi, and large viruses. These apparently unexpected observations, within the framework of the classical mechanisms for the formation of clathrin-coated vesicles, are now considered as examples of a new non-classical function of clathrin, which can promote the internalization of membrane domains associated to planar clathrin lattices. The role of actin downstream of clathrin seems to be critical for this still poorly characterized process. The historical frontier between endocytosis and phagocytosis is vanishing in the light of this new role for clathrin.

Specific Effects of Surface Amines on Polystyrene Nanoparticles in their Interactions with Mesenchymal Stem Cells

We have investigated the uptake of cationic polystyrene nanoparticles by mesenchymal stem cells (MSCs) using confocal fluorescence microscopy and flow cytometry. Two types of nanoparticles of about 100 nm diameter with similar zeta potentials were employed in this study, plain polystyrene (PS) nanoparticles and amino-functionalized polystyrene (NPS) nanoparticles, each carrying about 6000 amino groups on the surface. To assess the relative importance of specific endocytosis mechanisms, uptake was observed in the presence of the drugs dynasore and chlorpromazine. NPS nanoparticles were rapidly internalized and accumulated to a much higher level in MSCs than PS nanoparticles, predominantly via the main clathrin-mediated pathway. PS nanoparticles were internalized mainly via clathrin-independent endocytosis. The pronounced difference in the internalization behavior of PS and NPS nanoparticles points to specific interactions of the amino groups on the nanoparticle surface with the endocytosis machinery of the cells.

Accommodation of structural rearrangements in the huntingtin-interacting protein 1 coiled-coil domain

Huntingtin-interacting protein 1 (HIP1) is an important link between the actin cytoskeleton and clathrin-mediated endocytosis machinery. HIP1 has also been implicated in the pathogenesis of Huntington's disease. The binding of HIP1 to actin is regulated through an interaction with clathrin light chain. Clathrin light chain binds to a flexible coiled-coil domain in HIP1 and induces a compact state that is refractory to actin binding. To understand the mechanism of this conformational regulation, a high-resolution crystal structure of a stable fragment from the HIP1 coiled-coil domain was determined. The flexibility of the HIP1 coiled-coil region was evident from its variation from a previously determined structure of a similar region. A hydrogen-bond network and changes in coiled-coil monomer interaction suggest that the HIP1 coiled-coil domain is uniquely suited to allow conformational flexibility.

RNA Interference and Single Particle Tracking Analysis of Hepatitis C Virus Endocytosis

Hepatitis C virus (HCV) enters hepatocytes following a complex set of receptor interactions, culminating in internalization via clathrin-mediated endocytosis. However, aside from receptors, little is known about the cellular molecular requirements for infectious HCV entry. Therefore, we analyzed a siRNA library that targets 140 cellular membrane trafficking genes to identify host genes required for infectious HCV production and HCV pseudoparticle entry. This approach identified 16 host cofactors of HCV entry that function primarily in clathrin-mediated endocytosis, including components of the clathrin endocytosis machinery, actin polymerization, receptor internalization and sorting, and endosomal acidification. We next developed single particle tracking analysis of highly infectious fluorescent HCV particles to examine the co-trafficking of HCV virions with cellular cofactors of endocytosis. We observe multiple, sequential interactions of HCV virions with the actin cytoskeleton, including retraction along filopodia, actin nucleation during internalization, and migration of internalized particles along actin stress fibers. HCV co-localizes with clathrin and the ubiquitin ligase c-Cbl prior to internalization. Entering HCV particles are associated with the receptor molecules CD81 and the tight junction protein, claudin-1; however, HCV-claudin-1 interactions were not restricted to Huh-7.5 cell-cell junctions. Surprisingly, HCV internalization generally occurred outside of Huh-7.5 cell-cell junctions, which may reflect the poorly polarized nature of current HCV cell culture models. Following internalization, HCV particles transport with GFP-Rab5a positive endosomes, which is consistent with trafficking to the early endosome. This study presents technical advances for imaging HCV entry, in addition to identifying new host cofactors of HCV infection, some of which may be antiviral targets.

Protein kinase A regulation of P2X 4 receptors: Requirement for a specific motif in the C-terminus

The P2X purinergic receptor sub-family of ligand-gated ion channels are subject to protein kinase modulation. We have previously demonstrated that P2X 4R signaling can be positively regulated by increasing intracellular cAMP levels. The molecular mechanism underlying this effect was, however, unknown. The present study initially addressed whether protein kinase A (PKA) activation was required. Subsequently a mutational approach was utilized to determine which region of the receptor was required for this potentiation. In both DT-40 3KO and HEK-293 cells transiently expressing P2X 4R, forskolin treatment enhanced ATP-mediated signaling. Specific PKA inhibitors prevented the forskolin-induced enhancement of ATP-mediated inward currents in P2X 4R expressing HEK-293 cells. To define which region of the P2X 4R was required for the potentiation, mutations were generated in the cytoplasmic C-terminal tail. It was determined that a limited region of the C-terminus, consisting of a non-canonical tyrosine based sorting motif, was required for the effects of PKA. Of note, this region does not harbor any recognizable PKA phosphorylation motifs, and no direct phosphorylation of P2X 4R was detected, suggesting that PKA phosphorylation of an accessory protein interacts with the endocytosis motif in the C-terminus of the P2X 4R. In support of this notion, using Total Internal Reflection Fluorescence Microscopy (TIRF)\\ P2X 4-EGFP was shown to accumulate at/near the plasma membrane following forskolin treatment. In addition, disrupting the endocytosis machinery using a dominant-negative dynamin construct also prevented the PKA-mediated enhancement of ATP-stimulated Ca 2+ signals. Our results are consistent with a novel mechanism of P2XR regulation, whereby PKA activity, without directly phosphorylating P2X 4R, markedly enhances ATP-stimulated P2X 4R currents and hence cytosolic Ca 2+ signals. This may occur at least in part, by altering the trafficking of a population of P2X 4R present at the plasma membrane.

Participation of Tom1L1 in EGF-stimulated endocytosis of EGF receptor

Although many proteins have been shown to participate in ligand-stimulated endocytosis of EGF receptor (EGFR), the adaptor protein responsible for interaction of activated EGFR with endocytic machinery remains elusive. We show here that EGF stimulates transient tyrosine phosphorylation of Tom1L1 by the Src family kinases, resulting in transient interaction of Tom1L1 with the activated EGFR bridged by Grb2 and Shc. Cytosolic Tom1L1 is recruited onto the plasma membrane and subsequently redistributes into the early endosome. Mutant forms of Tom1L1 defective in Tyr-phosphorylation or interaction with Grb2 are incapable of interaction with EGFR. These mutants behave as dominant-negative mutants to inhibit endocytosis of EGFR. RNAi-mediated knockdown of Tom1L1 inhibits endocytosis of EGFR. The C-terminal tail of Tom1L1 contains a novel clathrin-interacting motif responsible for interaction with the C-terminal region of clathrin heavy chain, which is important for exogenous Tom1L1 to rescue endocytosis of EGFR in Tom1L1 knocked-down cells. These results suggest that EGF triggers a transient Grb2/Shc-mediated association of EGFR with Tyr-phosphorylated Tom1L1 to engage the endocytic machinery for endocytosis of the ligand–receptor complex.