Adenovirus RIDbeta subunit contains a tyrosine residue that is critical for RID-mediated receptor internalization and inhibition of Fas- and TRAIL-induced apoptosis

The adenovirus-encoded receptor internalization and degradation (RID) protein (previously named E3-10.4K/14.5K), which is composed of RIDalpha and RIDbeta subunits, down-regulates a number of cell surface receptors in the tumor necrosis factor (TNF) receptor superfamily, namely Fas, TRAIL receptor 1, and TRAIL receptor 2. Down-regulation of these "death" receptors protects adenovirus-infected cells from apoptosis induced by the death receptor ligands Fas ligand and TRAIL. RID also down-regulates certain tyrosine kinase cell surface receptors, especially the epidermal growth factor receptor (EGFR). RID-mediated Fas and EGFR down-regulation occurs via endocytosis of the receptors into endosomes followed by transport to and degradation within lysosomes. However, the molecular interactions underlying this function of RID are unknown. To investigate the molecular determinants of RIDbeta that are involved in receptor down-regulation, mutations within the cytoplasmic tail of RIDbeta were constructed and the mutant proteins were analyzed for their capacity to internalize and degrade Fas and EGFR and to protect cells from death receptor ligand-induced apoptosis. The results demonstrated the critical nature of a tyrosine residue near the RIDbeta C terminus; mutation of this residue to alanine abolished RID function. Mutating the tyrosine to phenylalanine did not abolish the function of RID, arguing that phosphorylation of the tyrosine is not required for function. These data suggest that this tyrosine residue forms part of a tyrosine-based sorting signal (Yxxphi). Additional mutations that target another potential sorting motif and several possible protein-protein interaction motifs had no discernible effect on RID function. It was also demonstrated that mutation of serine 116 to alanine eliminated phosphorylation of RIDbeta but did not affect any of the functions of RID that were examined. These results suggest a model in which the tyrosine-based sorting signal in RID plays a role in RID's ability to down-regulate receptors.     

Differential role of actin,clathrin, and dynamin in Fc gamma receptor-mediated endocytosis and phagocytosis

Clustering of macrophage Fc gamma receptors by multimeric immunoglobulin complexes leads to their internalization. Formation of small aggregates leads to endocytosis, whereas large particulate complexes induce phagocytosis. In RAW-264.7 macrophages, Fc gamma receptor endocytosis was found to be dependent on clathrin and dynamin and insensitive to cytochalasin. Clathrin also associates with nascent phagosomes, and earlier observations suggested that it plays an essential role in phagosome formation. However, we find that phagocytosis of IgG-coated large (> or =3 microm) particles was unaffected by inhibition of dynamin or by reducing the expression of clathrin using antisense mRNA but was eliminated by cytochalasin, implying a distinct mechanism dependent on actin assembly. The uptake of smaller particles (< or =1 microm) was only partially blocked by cytochalasin. Remarkably, the cytochalasin-resistant component was also insensitive to dominant-negative dynamin I and to clathrin antisense mRNA, implying the existence of a third internalization mechanism, independent of actin, dynamin, and clathrin. The uptake of small particles occurred by a process distinct from fluid phase pinocytosis, because it was not inhibited by dominant-negative Rab5. The insensitivity of phagocytosis to dominant-negative dynamin I enabled us to test the role of dynamin in phagosomal maturation. Although internalization of receptors from the plasma membrane was virtually eliminated by the K44A and S45N mutants of dynamin I, clearance of transferrin receptors and of CD18 from maturing phagosomes was unaffected by these mutants. This implies that removal of receptors from the phagosomal membrane occurs by a mechanism that is different from the one mediating internalization of the same receptors at the plasma membrane. These results imply that, contrary to prevailing notions, normal dynamin and clathrin function is not required for phagocytosis and reveal the existence of a component of phagocytosis that is independent of actin and Rab5.     

Phosphorylation of the medium chain subunit of the AP-2 adaptor complex does not influence its interaction with the tyrosine based internalisation motif of TGN38

Tyrosine based motifs conforming to the consensus YXXphi (where phi represents a bulky hydrophobic residue) have been shown to interact with the medium chain subunit of clathrin adaptor complexes. These medium chains are targets for phosphorylation by a kinase activity associated with clathrin coated vesicles. We have used the clathrin coated vesicle associated kinase activity to specifically phosphorylate a soluble recombinant fusion protein of mu2, the medium chain subunit of the plasma membrane associated adaptor protein complex AP-2. We have tested whether this phosphorylation has any effect on the interaction of mu2 with the tyrosine based motif containing protein, TGN38, that has previously been shown to interact with mu2. Phosphorylation of mu2 was shown to have no significant effect on the in vitro interaction of mu2 with the cytosolic domain of TGN38, indicating that reversible phosphorylation of mu2 does not play a role in regulating its direct interaction with tyrosine based internalisation motifs. In addition, although a casein kinase II-like activity has been shown to be associated with clathrin coated vesicles, we show that mu2 is not phosphorylated by casein kinase II implying that another kinase activity is present in clathrin coated vesicles. Furthermore the kinase activity associated with clathrin coated vesicles was shown to be capable of phosphorylating dynamin 1. Phosphorylation of dynamin 1 has previously been shown to regulate its interaction with other proteins involved in clathrin mediated endocytosis.     

Functional comparison of the role of dynamin 2 splice variants on GLUT-4 endocytosis in 3T3L1 adipocytes

Previously, we reported that expression of a dominant-interfering neuronal-specific dynamin 1 (K44A/dynamin 1) inhibited the plasma membrane internalization of GLUT-4 in 3T3L1 adipocytes (15). To investigate the role of the ubiquitously expressed isoform of dynamin, dynamin 2, on adipocyte GLUT-4 internalization, and to determine whether dynamin splice variants have functional specificity, we expressed each of the four dynamin 2 isoforms (aa, ab, ba, and bb) as either wild-type proteins or GTPase-defective mutants. When expressed as enhanced green fluorescent protein (EGFP) fusions, these isoforms were found to have overlapping subcellular distributions being localized throughout the cell cytoplasm, on punctate vesicles and in a perinuclear compartment. This distribution was qualitatively similar to that of endogenous dynamin 2 and overlapped with GLUT-4 in the basal state. Expression of wild-type dynamin 2 isoforms had no effect on the basal or insulin-stimulated distribution of GLUT-4; however, expression of the dominant-interfering dynamin 2 mutants inhibited GLUT-4 endocytosis. These data demonstrate that dynamin 2 is required for GLUT-4 endocytosis in 3T3L1 adipocytes and suggest that, relative to GLUT-4 trafficking, the dynamin 2 splice variants have overlapping functions and are probably not responsible for mediating distinct GLUT-4 budding events.     

Dynamin and Rab5a-dependent trafficking and signaling of the neurokinin 1 receptor

Understanding the molecular mechanisms of agonist-induced trafficking of G-protein-coupled receptors is important because of the essential role of trafficking in signal transduction. We examined the role of the GTPases dynamin 1 and Rab5a in substance P (SP)-induced trafficking and signaling of the neurokinin 1 receptor (NK1R), an important mediator of pain, depression, and inflammation, by studying transfected cells and enteric neurons that naturally express the NK1R. In unstimulated cells, the NK1R colocalized with dynamin at the plasma membrane, and Rab5a was detected in endosomes. SP induced translocation of the receptor into endosomes containing Rab5a immediately beneath the plasma membrane and then in a perinuclear location. Expression of the dominant negative mutants dynamin 1 K44E and Rab5aS34N inhibited endocytosis of SP by 45 and 32%, respectively. Dynamin K44E caused membrane retention of the NK1R, whereas Rab5aS34N also impeded the translocation of the receptor from superficially located to perinuclear endosomes. Both dynamin K44E and Rab5aS34N strongly inhibited resensitization of SP-induced Ca(2+) mobilization by 60 and 85%, respectively, but had no effect on NK1R desensitization. Dynamin K44E but not Rab5aS34N markedly reduced SP-induced phosphorylation of extracellular signal regulated kinases 1 and 2. Thus, dynamin mediates the formation of endosomes containing the NK1R, and Rab5a mediates both endosomal formation and their translocation from a superficial to a perinuclear location. Dynamin and Rab5a-dependent trafficking is essential for NK1R resensitization but is not necessary for desensitization of signaling. Dynamin-dependent but not Rab5a-dependent trafficking is required for coupling of the NK1R to the mitogen-activated protein kinase cascade. These processes may regulate the nociceptive, depressive, and proinflammatory effects of SP.     

Redundant and Distinct Functions for Dynamin-1 and Dynamin-2 Isoforms

A role for dynamin in clathrin-mediated endocytosis is now well established. However, mammals express three closely related, tissue-specific dynamin isoforms, each with multiple splice variants. Thus, an important question is whether these isoforms and splice variants function in vesicle formation from distinct intracellular organelles. There are conflicting data as to a role for dynamin-2 in vesicle budding from the TGN. To resolve this issue, we compared the effects of overexpression of dominant-negative mutants of dynamin-1 (the neuronal isoform) and dynamin-2 (the ubiquitously expressed isoform) on endocytic and biosynthetic membrane trafficking in HeLa cells and polarized MDCK cells. Both dyn1(K44A) and dyn2(K44A) were potent inhibitors of receptor-mediated endocytosis; however neither mutant directly affected other membrane trafficking events, including transport mediated by four distinct classes of vesicles budding from the TGN. Dyn2(K44A) more potently inhibited receptor-mediated endocytosis than dyn1(K44A) in HeLa cells and at the basolateral surface of MDCK cells. In contrast, dyn1(K44A) more potently inhibited endocytosis at the apical surface of MDCK cells. The two dynamin isoforms have redundant functions in endocytic vesicle formation, but can be targeted to and function differentially at subdomains of the plasma membrane.     

Increased GTP-binding to dynamin II does not stimulate receptor-mediated endocytosis

Regarding the molecular mechanism of dynamin in receptor-mediated endocytosis, GTPase activity of dynamin has been thought to have a critical role in endocytic vesicle internalization. However, a recent report suggested that GTP-binding to dynamin itself activates the dynamin to recruit molecular machinery necessary for endocytosis. In this study, to investigate the role of GTP binding to dynamin II, we generated two mutant dynamin II constructs: G38V and K44E. G38V, its GTP binding site might be mainly occupied by GTP caused by reduced GTPase activity, and K44E mutant, its GTP binding site might be vacant, caused by its decreased affinity for GTP and GDP. From the analysis of the ratio of GTP vs GDP bound to dynamin, we confirmed these properties. To test the effect of these mutant dynamins on endocytosis, we performed flow cytometry and confocal immunofluorescence analysis and found that these two mutants have inhibitory effect on transferrin-induced endocytosis. Whereas fluorescent transferrin was completely internalized in wild-type (WT) dynamin II expressing cells, no intracellular accumulation of fluorescent transferrin was found in the cells overexpressing K44E and G38V mutant. Interestingly, the amount of GTP bound to K44E was increased when endocytosis was induced than that bound to WT. The present results suggested that the GTPase activity of dynamin II is required for formation of endocytic vesicle and GTP-binding to dynamin II per se is not sufficient for stimulating endocytosis.     

Inhibition of Clathrin-Mediated Endocytosis Selectively Attenuates Specific Insulin Receptor Signal Transduction Pathways

To examine the role of clathrin-dependent insulin receptor internalization in insulin-stimulated signal transduction events, we expressed a dominant-interfering mutant of dynamin (K44A/dynamin) by using a recombinant adenovirus in the H4IIE hepatoma and 3T3L1 adipocyte cell lines. Expression of K44A/dynamin inhibited endocytosis of the insulin receptor as determined by both cell surface radioligand binding and trypsin protection analysis. The inhibition of the insulin receptor endocytosis had no effect on either the extent of insulin receptor autophosphorylation or insulin receptor substrate 1 (IRS1) tyrosine phosphorylation. In contrast, expression of K44A/dynamin partially inhibited insulin-stimulated Shc tyrosine phosphorylation and activation of the mitogen-activated protein kinases ERK1 and -2. Although there was an approximately 50% decrease in the insulin-stimulated activation of the phosphatidylinositol 3-kinase associated with IRS1, insulin-stimulated Akt kinase phosphorylation and activation were unaffected. The expression of K44A/dynamin increased the basal rate of amino acid transport, which was additive with the effect of insulin but had no effect on the basal or insulin-stimulated DNA synthesis. In 3T3L1 adipocytes, expression of K44A/dynamin increased the basal rate of glucose uptake, glycogen synthesis, and lipogenesis without any significant effect on insulin stimulation. Together, these data demonstrate that the acute actions of insulin are largely independent of insulin receptor endocytosis and are initiated by activation of the plasma membrane-localized insulin receptor.     

CIN85 associates with TNF receptor 1 via Src and modulates TNF-alpha-induced apoptosis

CIN85 is a multidomain protein that associates with receptors carrying tyrosine kinase domains. Here we report that it is also a component of the signaling complex associated with tumor necrosis factor receptor 1 (TNFR1), which lacks a tyrosine kinase domain. This was established by showing that CIN85 was co-precipitated with TNFR1, TRADD, cIAP-1 and TARF1/2, but not with FADD, RIP, caspase-8 or TRAF6. However, CIN85 did not bind directly to the cytoplasmic domain of TNFR1 (TNFR1-CYT) but to Src family kinases, Cbl and the p85alpha subunit of phosphatidylinositol 3-kinase (PI3-K p85alpha). Src bound directly to TNFR1-CYT, but Cbl and PI3-K p85alpha did not. A human cell line ectopically expressing CIN85 was 10 times more susceptible to TNF-alpha-induced apoptosis than control cells, which expressed identical levels of TNFR1 on their surface. However, the susceptibility of these two cell lines to CD95-induced apoptosis was the same. The three SH3 domains of CIN85 were essential for this increased susceptibility to apoptosis and its proline-rich regions were also required for maximal effect. TNF-alpha treatment recruited CIN85 to the TNFR1 signaling complex. Taken together, these results indicate that CIN85 associates with TNFR1 via Src and modulates TNF-alpha-induced apoptosis.     

Herpes simplex virus 1 precludes replenishment of the short-lived receptor of tumor necrosis factor alpha by virion host shutoff-dependent degradation of its mRNA

Cell function is tightly regulated by surface receptors. Earlier reports showed that herpes simplex virus 1 regulates by diverse mechanisms the presentation of antigenic peptides, downregulates the signaling pathways associated with receptor tyrosine kinases, and posttranslationally modifies members of the Src family of protein kinases. Here we report that the receptor for tumor necrosis factor alpha (TNF-R1) rapidly disappears from both the cell surface and total cell lysates in cells infected with wild-type virus or a variety of mutants but not in cells infected with the mutant DeltaU(L)41, which lacks the U(L)41 gene, the virion host shutoff gene. The half-life of TNF-R1 appears to be less than 30 min in both mock-infected and infected cells. The disappearance of TNF-R1 correlates with the disappearance of cytoplasmic TNF-R1 mRNA in wild-type-virus-infected cells. The results suggest that by degrading the TNFR1 mRNA, the virus precludes the replenishment of naturally decaying TNF-R1.     

Entry of newly synthesized GLUT4 into the insulin-responsive storage compartment is GGA dependent

Following biosynthesis, both GLUT1 and VSV-G proteins appear rapidly (2-3 h) at the plasma membrane, whereas GLUT4 is retained in intracellular membrane compartments and does not display any significant insulin responsiveness until 6-9 h. Surprisingly, the acquisition of insulin responsiveness did not require plasma membrane endocytosis, as expression of a dominant-interfering dynamin mutant (Dyn/K44A) had no effect on the insulin-stimulated GLUT4 translocation. Furthermore, expression of endocytosis-defective GLUT4 mutants or continuous surface labeling with an exofacial specific antibody demonstrated that GLUT4 did not transit the cell surface prior to the acquisition of insulin responsiveness. The expression of a dominant-interfering GGA mutant (VHS-GAT) had no effect on the trafficking of newly synthesized GLUT1 or VSV-G protein to the plasma membrane, but completely blocked the insulin-stimulated translocation of newly synthesized GLUT4. Furthermore, in vitro budding of GLUT4 vesicles but not GLUT1 or the transferrin receptor was inhibited by VHS-GAT. Together, these data demonstrate that following biosynthesis, GLUT4 directly sorts and traffics to the insulin-responsive storage compartment through a specific GGA-sensitive process.     

Cowpox virus exploits the endoplasmic reticulum retention pathway to inhibit MHC class I transport to the cell surface

Major histocompatibility complex (MHC) class I molecules assemble with peptides in the ER lumen and are transported via Golgi to the plasma membrane for recognition by T cells. Inhibiting MHC assembly, transport, and surface expression are common viral strategies of evading immune recognition. Cowpox virus, a clinically relevant orthopoxvirus, downregulates MHC class I expression on infected cells. However, the viral protein(s) and mechanisms responsible are unknown. We identify CPXV203 as a cowpox virus protein that associates with fully assembled MHC class I molecules and blocks their transport through the Golgi. A C-terminal KTEL motif in CPXV203 closely resembles the canonical ER retention motif KDEL and is required for CPXV203 function, indicating that a physiologic pathway is exploited to retain MHC class I in the ER. This viral mechanism for MHC class I downregulation may explain virulence differences between clinical isolates of orthopoxviruses.     

Rapid agonist-induced desensitization and internalization of the A(2B) adenosine receptor is mediated by a serine residue close to the COOH terminus

The G(s)-coupled rat A(2B) adenosine receptor (A(2B)-AR) was epitope-tagged at the NH(2) terminus with hemagglutinin (HA) and subjected to progressive deletions or point mutations of the COOH terminus in order to determine regions of the receptor that contribute to agonist-induced desensitization and internalization. When expressed stably in Chinese hamster ovary cells, a mutant receptor in which the final 2 amino acids were deleted, the Leu(330)-stop mutant, underwent rapid agonist-induced desensitization and internalization as did the wild type (WT) receptor. However, the Phe(328) and the Gln(325)-stop mutants were resistant to rapid agonist-induced desensitization and internalization. Co-expression of arrestin-2-green fluorescent protein (arrestin-2-GFP) with WT receptor or Leu(330)-stop mutant resulted in rapid translocation of arrestin-2-GFP from cytosol to membrane upon agonist addition. On the other hand, agonist activation of the Phe(328)-stop or Gln(325)-stop mutant did not result in translocation of arrestin-2-GFP from cytosol. A COOH terminus point mutant, S329G, was also unable to undergo rapid agonist-induced desensitization and internalization, indicating that Ser(329) is a critical residue for these processes. A further deletion mutant (Ser(326)-stop) unexpectedly underwent rapid agonist-induced desensitization and internalization. However, activation of this mutant did not promote translocation of arrestin-2-GFP from cytosol to membrane. In addition, whereas WT receptor internalization was markedly inhibited by co-expression of dominant negative mutants of arrestin-2 (arrestin-2-(319-418)), dynamin (dynamin K44A), or Eps-15 (EDelta95-295), Ser(326)-stop receptor internalization was only inhibited by dominant negative mutant dynamin. Taken together these results indicate that Ser(329), close to the COOH terminus of the rat A(2B)-AR, is critical for the rapid agonist-induced desensitization and internalization of the receptor. However, deletion of the COOH terminus also uncovers a motif that is able to redirect internalization of the receptor to an arrestin- and clathrin-independent pathway.     

Germline mutations in the extracellular domains of the 55 kDa TNF receptor, TNFR1, define a family of dominantly inherited autoinflammatory syndromes

Autosomal dominant periodic fever syndromes are characterized by unexplained episodes of fever and severe localized inflammation. In seven affected families, we found six different missense mutations of the 55 kDa tumor necrosis factor receptor (TNFR1), five of which disrupt conserved extracellular disulfide bonds. Soluble plasma TNFR1 levels in patients were approximately half normal. Leukocytes bearing a C52F mutation showed increased membrane TNFR1 and reduced receptor cleavage following stimulation. We propose that the autoinflammatory phenotype results from impaired downregulation of membrane TNFR1 and diminished shedding of potentially antagonistic soluble receptor. TNFR1-associated periodic syndromes (TRAPS) establish an important class of mutations in TNF receptors. Detailed analysis of one such mutation suggests impaired cytokine receptor clearance as a novel mechanism of disease.     

Clathrin-dependent APP endocytosis and Aβ secretion are highly sensitive to the level of plasma membrane cholesterol

Several lines of evidence support a strong relationship between cholesterol and Alzheimer's disease pathogenesis. Membrane cholesterol is known to modulate amyloid precursor protein (APP) endocytosis and amyloid-β (Aβ) secretion. Here we show in a human cell line model of endocytosis (HEK293 cells) that cholesterol exerts these effects in a dose-dependent and linear manner, over a wide range of concentrations (-40% to + 40% variations of plasma membrane cholesterol induced by methyl-beta-cyclodextrin (MBCD) and MBCD-cholesterol complex respectively). We found that the gradual effect of cholesterol is inhibited by small interference RNA-mediated downregulation of clathrin. Modulation of clathrin-mediated APP endocytosis by cholesterol was further demonstrated using mutants of proteins involved in the formation of early endosomes (dynamin2, Eps15 and Rab5). Importantly we show that membrane proteins other than APP are not affected by cholesterol to the same extent. Indeed clathrin-dependent endocytosis of transferrin and cannabinoid1 receptors as well as internalization of surface proteins labelled with a biotin derivative (sulfo-NHS-SS-biotin) were not sensitive to variations of plasma membrane cholesterol from -40% to 40%. In conclusion clathrin-dependent APP endocytosis appears to be very sensitive to the levels of membrane cholesterol. These results suggest that cholesterol increase in AD could be responsible for the enhanced internalization of clathrin-, dynamin2-, Eps15- and Rab5-dependent endocytosis of APP and the ensuing overproduction of Aβ.     

Binding of amyloidogenic transthyretin to the plasma membrane alters membrane fluidity and induces neurotoxicity

Transthyretin (TTR) can deposit as amyloid in the peripheral nervous system and induce a peripheral neuropathy. We examined the mechanism of TTR amyloid neurotoxicity on SH-SY5Y neuroblastoma cells. Wild-type (WT) TTR and two amyloidogenic mutants (V30M and L55P) were expressed in Escherichia coli. Incubation (aging) of WT TTR at 37 degrees C for 1 week caused no significant aggregation. However, there was a significant increase in the extent of amyloid fibril formation after the amyloidogenic mutants had been aged. L55P TTR aggregated more readily than V30M TTR. Both amyloidogenic mutants were neurotoxic after aging. The order of neurotoxicity was as follows: L55P > V30M > WT. As binding of amyloid proteins to the plasma membrane may cause cytotoxicity, we studied the binding of TTR to a plasma membrane-enriched preparation from SH-SY5Y cells by surface plasmon resonance. All three forms bound to the plasma membrane through electrostatic interactions. The binding of the amyloidogenic mutants was increased by aging. The amount of binding correlated closely with the amount of aggregation and with the cytotoxicity of each form. As membrane fluidity can influence cell viability, we also examined the effect of TTR on membrane fluidity using a fluorescence anisotropy method. Binding of the amyloidogenic TTR mutants increased membrane fluidity, and once again, the order of potency was as follows: L55P > V30M > WT. These results demonstrate that TTR can bind to the plasma membrane and cause a change in membrane fluidity. Altered membrane fluidity may be the cause of the neurotoxicity.