Tetherin/BST2, a physiologically and therapeutically relevant regulator of platelet receptor signalling.

The reactivity of platelets, which play a key role in the pathogenesis of atherothrombosis, is tightly regulated. The integral membrane protein tetherin/bone marrow stromal antigen-2 (BST-2) regulates membrane organization, altering both lipid and protein distribution within the plasma membrane. Because membrane microdomains have an established role in platelet receptor biology, we sought to characterize the physiological relevance of tetherin/BST-2 in those cells. To characterize the potential importance of tetherin/BST-2 to platelet function, we used tetherin/BST-2-/- murine platelets. In the mice, we found enhanced function and signaling downstream of a subset of membrane microdomain-expressing receptors, including the P2Y12, TP thromboxane, thrombin, and GPVI receptors. Preliminary studies in humans have revealed that treatment with interferon-α (IFN-α), which upregulates platelet tetherin/BST-2 expression, also reduces adenosine diphosphate-stimulated platelet receptor function and reactivity. A more comprehensive understanding of how tetherin/BST-2 negatively regulates receptor function was provided in cell line experiments, where we focused on the therapeutically relevant P2Y12 receptor (P2Y12R). Tetherin/BST-2 expression reduced both P2Y12R activation and trafficking, which was accompanied by reduced receptor lateral mobility specifically within membrane microdomains. In fluorescence lifetime imaging-Förster resonance energy transfer (FLIM-FRET)-based experiments, agonist stimulation reduced basal association between P2Y12R and tetherin/BST-2. Notably, the glycosylphosphatidylinositol (GPI) anchor of tetherin/BST-2 was required for both receptor interaction and observed functional effects. In summary, we established, for the first time, a fundamental role of the ubiquitously expressed protein tetherin/BST-2 in negatively regulating membrane microdomain-expressed platelet receptor function.

Modifying Self-Assembled Peptide Cages To Control Internalization into Mammalian Cells.

Nanoparticles can be used to transport a variety of biological cargoes into eukaryotic cells. Polypeptides provide a versatile material for constructing such systems. Previously, we have assembled nanoscale peptide cages (SAGEs) from de novo designed coiled-coil modules. Here, we show that the modules can be extended with short charged peptides to alter endocytosis of the assembled SAGE particles by cultured human cells in a tunable fashion. First, we find that the peptide extensions affect coiled-coil stability predictably: N-terminal polylysine and C-terminal polyglutamate tags are destabilizing; whereas, the reversed arrangements have little impact. Second, the cationic assembled particles are internalized faster and to greater extents by cells than the parent SAGEs. By contrast, anionic decorations markedly inhibit both aspects of uptake. These studies highlight how the modular SAGE system facilitates rational peptide design to fine-tune the bioactivity of nanoparticles, which should allow engineering of tailored cell-delivery vehicles.

Tetherin is an exosomal tether.

Exosomes are extracellular vesicles that are released when endosomes fuse with the plasma membrane. They have been implicated in various functions in both health and disease, including intercellular communication, antigen presentation, prion transmission, and tumour cell metastasis. Here we show that inactivating the vacuolar ATPase in HeLa cells causes a dramatic increase in the production of exosomes, which display endocytosed tracers, cholesterol, and CD63. The exosomes remain clustered on the cell surface, similar to retroviruses, which are attached to the plasma membrane by tetherin. To determine whether tetherin also attaches exosomes, we knocked it out and found a 4-fold reduction in plasma membrane-associated exosomes, with a concomitant increase in exosomes discharged into the medium. This phenotype could be rescued by wild-type tetherin but not tetherin lacking its GPI anchor. We propose that tetherin may play a key role in exosome fate, determining whether they participate in long-range or short-range interactions.

Microtubules depolymerization caused by the CK1 inhibitor IC261 may be not mediated by CK1 blockage.

The ubiquitously expressed serine/threonine specific casein kinase 1 (CK1) family plays important roles in the regulation of various physiological processes. Small-molecule inhibitors, such as the CK1δ/ε selectively inhibitor IC261, have been used to antagonize CK1 phosphorylation events in cells in many studies. Here we present data to show that, similarly to the microtubule destabilizing agent nocodazole, IC261 depolymerizes microtubules in interphase cells. IC261 treatment of interphase cells affects the morphology of the TGN and Golgi apparatus as well as the localization of CK1δ, which co-localizes with COPI positive membranes. IC261-induced depolymerization of microtubules is rapid, reversible and can be antagonized by pre-treatment of cells with taxol. At lower concentrations of IC261, mitotic spindle microtubule dynamics are affected; this leads to cell cycle arrest and, depending on the cellular background, to apoptosis in a dose-dependent manner. In addition, FACS analysis revealed that IC261 could induce apoptosis independent of cell cycle arrest. In summary this study provides additional and valuable information about various IC261-induced effects that could be caused by microtubule depolymerization rather than by inhibition of CK1. Data from studies that have used IC261 as an inhibitor of CK1 should be interpreted in light of these observations.

The cytosolic N-terminus of CD317/tetherin is a membrane microdomain exclusion motif.

The integral membrane protein CD317/tetherin has been associated with a plethora of biological processes, including restriction of enveloped virus release, regulation of B cell growth, and organisation of membrane microdomains. CD317 possesses both a conventional transmembrane (TM) domain and a glycophosphatidylinositol (GPI) anchor. We confirm that the GPI anchor is essential for CD317 to associate with membrane microdomains, and that the TM domain of CD44 is unable to rescue proper microdomain association of a ΔGPI-CD317 construct. Additionally, we demonstrate that the cytosolic amino terminal region of CD317 can function as a 'microdomain-excluding' motif, when heterologously expressed as part of a reporter construct. Finally, we show that two recently described isoforms of CD317 do not differ in their affinity for membrane microdomains. Together, these data help further our understanding of the fundamental cell biology governing membrane microdomain association of CD317.

Expression of HIV-1 Vpu leads to loss of the viral restriction factor CD317/Tetherin from lipid rafts and its enhanced lysosomal degradation.

CD317/tetherin (aka BST2 or HM1.24 antigen) is an interferon inducible membrane protein present in regions of the lipid bilayer enriched in sphingolipids and cholesterol (often termed lipid rafts). It has been implicated in an eclectic mix of cellular processes including, most notably, the retention of fully formed viral particles at the surface of cells infected with HIV and other enveloped viruses. Expression of the HIV viral accessory protein Vpu has been shown to lead to intracellular sequestration and degradation of tetherin, thereby counteracting the inhibition of viral release. There is evidence that tetherin interacts directly with Vpu, but it remains unclear where in the cell this interaction occurs or if Vpu expression affects the lipid raft localisation of tetherin. We have addressed these points using biochemical and cell imaging approaches focused on endogenous rather than ectopically over-expressed tetherin. We find i) no evidence for an interaction between Vpu and endogenous tetherin at the cell surface, ii) the vast majority of endogenous tetherin that is at the cell surface in control cells is in lipid rafts, iii) internalised tetherin is present in non-raft fractions, iv) expression of Vpu in cells expressing endogenous tetherin leads to the loss of tetherin from lipid rafts, v) internalised tetherin enters early endosomes, and late endosomes, in both control cells and cells expressing Vpu, but the proportion of tetherin molecules destined for degradation rather than recycling is increased in cells expressing Vpu vi) lysosomes are the primary site for degradation of endogenous tetherin in cells expressing Vpu. Our studies underlie the importance of studying endogenous tetherin and let us propose a model in which Vpu intercepts newly internalised tetherin and diverts it for lysosomal destruction rather than recycling to the cell surface.

Herpes simplex virus 1 counteracts tetherin restriction via its virion host shutoff activity.

The interferon-inducible membrane protein tetherin (Bst-2, or CD317) is an antiviral factor that inhibits enveloped virus release by cross-linking newly formed virus particles to the producing cell. The majority of viruses that are sensitive to tetherin restriction appear to be those that acquire their envelopes at the plasma membrane, although many viruses, including herpesviruses, envelope at intracellular membranes, and the effect of tetherin on such viruses has been less well studied. We investigated the tetherin sensitivity and possible countermeasures of herpes simplex virus 1 (HSV-1). We found that overexpression of tetherin inhibits HSV-1 release and that HSV-1 efficiently depletes tetherin from infected cells. We further show that the virion host shutoff protein (Vhs) is important for depletion of tetherin mRNA and protein and that removal of tetherin compensates for defects in replication and release of a Vhs-null virus. Vhs is known to be important for HSV-1 to evade the innate immune response in vivo. Taken together, our data suggest that tetherin has antiviral activity toward HSV-1 and that the removal of tetherin by Vhs is important for the efficient replication and dissemination of HSV-1.

CD317/tetherin is an organiser of membrane microdomains.

The integral membrane protein tetherin has been associated with an eclectic mix of cellular processes, including restricting the release of a range of enveloped viruses from infected cells. The unusual topology of tetherin (it possesses both a conventional transmembrane domain and a glycosylphosphatidylinositol anchor), its localisation to membrane microdomains (lipid rafts) and the fact that its cytosolic domain can be linked (indirectly) to the actin cytoskeleton, led us to speculate that tetherin might form a 'tethered picket fence' and thereby play a role in the organisation of lipid rafts. We now show that knocking down expression of tetherin leads to changes in the distribution of lipid raft-localised proteins and changes in the organisation of lipids in the plasma membrane. These changes can be reversed by re-expression of wild-type tetherin, but not by any of a range of tetherin-based constructs, indicating that no individual feature of the tetherin sequence is dispensable in the context of its lipid raft organising function.

IP3 3-kinase opposes NGF driven neurite outgrowth.

The inositol (1,4,5) trisphosphate 3-kinases comprise a family of enzymes (A, B, and C) that phosphorylate the calcium mobilising molecule inositol (1,4,5) trisphosphate (IP(3)) to generate inositol (1,3,4,5) tetrakisphosphate. This molecule can function as a second messenger, but its roles are not completely understood. The A isoform of inositol (1,4,5) trisphosphate 3-kinase localises to filamentous actin within dendritic spines in the hippocampus and is implicated in the regulation of spine morphology and long term potentiation, however the mechanisms through which it signals in neuronal cells are not completely understood. We have used NGF driven neurite outgrowth from PC12 cells as a platform to examine the impact of signaling via inositol (1,4,5) trisphosphate 3-kinase activity in a neuronal cell. We have found that the catalytic activity of the enzyme opposes neurite outgrowth, whilst pharmacological inhibition of inositol (1,4,5) trisphosphate 3-kinase leads to a significant increase in neurite outgrowth, and we show that the reduction in neurite outgrowth in response to inositol (1,4,5) trisphosphate 3-kinase activity correlates with reduced ERK activity as determined by western blotting using phosphorylation-specific antibodies. Our findings suggest a novel neuronal signaling pathway linking metabolism of IP(3) to signaling via ERK.

Release of filamentous and spherical influenza A virus is not restricted by tetherin.

The cellular protein tetherin is thought to act as a 'leash' that anchors many enveloped viruses to the plasma membrane and prevents their release. We found that replication of multiple strains of influenza A virus was generally insensitive to alteration of tetherin levels, as assessed by output titre or scanning electron microscopy of cell-associated virions. This included human, swine, avian and equine isolates, strains that form filamentous or spherical particles and viruses that lack the M2 or NS1 proteins. Levels of cell-surface tetherin were not reduced by influenza infection, but tetherin and the viral haemagglutinin co-localized on the plasma membrane. However, tetherin could not be detected in filamentous virions, suggesting that influenza may possess a mechanism to exclude it from virions. Overall, if influenza does encode a specific antagonist of tetherin, it is not M2 or NS1 and we find no evidence for a role in host range specificity.

HIV-1 antagonism of CD317 is species specific and involves Vpu-mediated proteasomal degradation of the restriction factor.

Mammals encode proteins that inhibit viral replication at the cellular level. In turn, certain viruses have evolved genes that can functionally counteract these intrinsic restrictions. Human CD317 (BST-2/HM1.24/tetherin) is a restriction factor that blocks release of human immunodeficiency virus type 1 (HIV-1) from the cell surface and can be overcome by HIV-1 Vpu. Here, we show that mouse and rat CD317 potently inhibit HIV-1 release but are resistant to Vpu. Interspecies chimeras reveal that the rodent-specific resistance and human-specific sensitivity to Vpu antagonism involve all three major structural domains of CD317. To promote virus release, Vpu depletes cellular pools of human CD317, but not of the rodent orthologs, by accelerating its degradation via the 20S proteasome. Thus, HIV-1 Vpu suppresses the expression of the CD317 antiviral factor in human cells, and the species-specific resistance to this suppression may guide the development of small animal models of HIV infection.

A CD317/tetherin-RICH2 complex plays a critical role in the organization of the subapical actin cytoskeleton in polarized epithelial cells.

CD317/tetherin is a lipid raft-associated integral membrane protein with a novel topology. It has a short N-terminal cytosolic domain, a conventional transmembrane domain, and a C-terminal glycosyl-phosphatidylinositol anchor. We now show that CD317 is expressed at the apical surface of polarized epithelial cells, where it interacts indirectly with the underlying actin cytoskeleton. CD317 is linked to the apical actin network via the proteins RICH2, EBP50, and ezrin. Knocking down expression of either CD317 or RICH2 gives rise to the same phenotype: a loss of the apical actin network with concomitant loss of apical microvilli, an increase in actin bundles at the basal surface, and a reduction in cell height without any loss of tight junctions, transepithelial resistance, or the polarized targeting of apical and basolateral membrane proteins. Thus, CD317 provides a physical link between lipid rafts and the apical actin network in polarized epithelial cells and is crucial for the maintenance of microvilli in such cells.

Clathrin-mediated endocytosis of a lipid-raft-associated protein is mediated through a dual tyrosine motif.

We have previously shown that the integral membrane protein CD317 has both a conventional transmembrane domain near its N-terminus and a C-terminal glycosyl-phosphatidylinositol (GPI) anchor. With the possible exception of a minor topological variant of the prion protein, there remain no other convincing examples of a mammalian protein with such a topology. CD317 is localised to cholesterol-rich lipid microdomains ('lipid rafts') in the plasma membrane and is internalised from the cell surface for delivery to a juxta-nuclear compartment (most probably the TGN). We have now investigated the mechanism by which CD317 is internalised and find that this raft-associated integral membrane protein is internalised through a clathrin-dependent pathway, internalisation is dependent upon a novel dual-tyrosine-based motif in the cytosolic domain of CD317, the cytosolic domain of CD317 can interact with the mu subunits of the AP2 and AP1 adaptor complexes, interaction with AP1 is required for delivery of CD317 back to the TGN, and removal of the GPI anchor from CD317 reduces the efficiency of CD317 internalisation. Collectively, these data indicate that CD317 is internalised and delivered back to the TGN by the sequential action of AP2 and AP1 adaptor complexes and that, surprisingly, the clathrin-mediated internalisation of CD317 occurs more efficiently if CD317 is localised to lipid rafts.

Casein kinase 1 delta (CK1delta) interacts with the SNARE associated protein snapin.

In this study we identified snapin as an interaction partner of the CK1 isoform delta (CK1delta) in the yeast two-hybrid system and localized the interacting domains of both proteins. The interaction of CK1delta with snapin was confirmed by co-immunoprecipitation. Snapin was phosphorylated by CK1delta in vitro. Both proteins localized in close proximity in the perinuclear region, wherein snapin was found to associate with membranes of the Golgi apparatus. The identification of snapin as a new substrate of CK1delta points towards a possible function for CK1delta in modulating snapin specific functions.

Regulation of CK2 activity by phosphatidylinositol phosphates.

The process of clathrin-coated vesicle (CCV) formation/disassembly involves numerous proteins that act cooperatively. Phosphorylation is an important regulatory mechanism governing protein interactions in CCVs, and many of the core and accessory proteins of the CCV machinery are reversibly phosphorylated in vivo. CK2 is highly enriched in CCVs and is capable of phosphorylating a number of peripheral membrane proteins involved in the process of clathrin-mediated endocytosis. At least some of these phosphorylation events have been shown to be inhibitory for CCV assembly, and CK2 has been shown to be inactive when associated with intact CCVs. Here we show that CCV membranes inhibit CK2 activity even after incubation in trypsin, indicating that a component of the lipid bilayer may be the inhibitory factor. Consistent with this, we showed that liposomes containing phosphatidylinositol phosphates inhibit the activity of CK2 and that CK2 binds to those liposomes. Notably, liposomes containing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)), a component of CCVs, bind CK2 and inhibit its activity. Furthermore, we showed that the binding of CK2 to PtdIns(4,5)P(2)-containing liposomes is via the active site of CK2, thus providing a molecular explanation for the inhibition of CK2 activity when it is bound to PtdIns(4,5)P(2)-containing liposomes. Thus CK2 is inactive in CCVs because of the fact that it is bound to the CCV membrane via an interaction between PtdIns(4,5)P(2) in the CCV membrane and the active site in CK2.

Hippocalcin functions as a calcium sensor in hippocampal LTD.

It is not fully understood how NMDAR-dependent LTD causes Ca(2+)-dependent endocytosis of AMPARs. Here we show that the neuronal Ca(2+) sensor hippocalcin binds the beta2-adaptin subunit of the AP2 adaptor complex and that along with GluR2 these coimmunoprecipitate in a Ca(2+)-sensitive manner. Infusion of a truncated mutant of hippocalcin (HIP(2-72)) that lacks the Ca(2+) binding domains prevents synaptically evoked LTD but has no effect on LTP. These data indicate that the AP2-hippocalcin complex acts as a Ca(2+) sensor that couples NMDAR-dependent activation to regulated endocytosis of AMPARs during LTD.

Identification and subcellular distribution of endogenous Ins(1,4,5)P(3) 3-kinase B in mouse tissues.

Inositol 1,4,5-trisphosphate 3-kinase (IP(3)-3K) catalyses the phosphorylation of inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate. cDNAs encoding three mammalian isoforms have been reported and referred to as IP(3)-3KA, IP(3)-3KB, and IP(3)-3KC. IP(3)-3KB is particularly sensitive to proteolysis at the N-terminus, a mechanism known to generate active fragments of lower molecular mass. Endogenous IP(3)-3KB has therefore not been formally identified in tissues. We have probed a series of murine tissues with an antibody directed against the C-terminus of IP(3)-3KB and used IP(3)-3KB deficient mouse tissues as negative controls. IP(3)-3KB was shown to be particularly well expressed in brain, lung, and thymus with molecular masses of 110-120kDa. The identification of the native IP(3)-3KB by Western blotting for the first time will facilitate further studies of regulation of its activity by specific proteases and/or phosphorylation.

Ins(1,4,5)P3 metabolism and the family of IP3-3Kinases.

The release of Ca2+ from intracellular stores is triggered by the second messenger inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3). The regulation of this process is critically important for cellular homeostasis. Ins(1,4,5)P3 is rapidly metabolised, either to inositol (1,4)-bisphosphate (Ins(1,4)P2) by inositol polyphosphate 5-phosphatases or to inositol (1,3,4,5)-tetrakisphosphate (Ins(1,3,4,5)P4) by one of a family of inositol (1,4,5)P3 3-kinases (IP3-3Ks). Three isoforms of IP3-3K have now been identified in mammals; they have a conserved C-terminal catalytic domain, but divergent N-termini. This review discusses the metabolism of Ins(1,4,5)P3, compares the IP3-3K isoforms and addresses potential mechanisms by which their activity might be regulated.

Regions of human kidney anion exchanger 1 (kAE1) required for basolateral targeting of kAE1 in polarised kidney cells: mis-targeting explains dominant renal tubular acidosis (dRTA).

Distal renal tubular acidosis (dRTA) is characterised by defective acid secretion by kidney alpha-intercalated cells. Some dominantly inherited forms of dRTA result from anion exchanger 1 (AE1) mutations. We have developed a stably transfected cell model for the expression of human kidney AE1 (kAE1) and mutant kAE1 proteins in MDCKI cells. Normal kAE1 was delivered to the plasma membrane of non-polarised cells and to the basolateral membrane of polarised cells. The AE1 N-glycan was processed to a complex form. Surprisingly, expression of kAE1 increased the permeability of the paracellular barrier of polarised MDCKI monolayers. All dominant dRTA mutations examined altered the targeting of kAE1 in MDCKI cells. The mutant proteins kAE1(R589H), kAE1(S613F) and kAE1(R901Stop) were retained in the ER in non-polarised cells, but the kAE1(R901Stop) protein was also present in late endosomes/lysosomes. The complex N-glycan of kAE1(R901Stop) was larger than that of normal kAE1. In polarised cells, the mutant kAE1(R901Stop) was mis-targeted to the apical membrane, while the kAE1(R589H) and kAE1(S613F) mutants did not reach the cell surface. These results demonstrate that dominant dRTA mutations cause aberrant targeting of kAE1 in polarised kidney cells and provide an explanation for the origin of dominant dRTA. Our data also demonstrate that the 11 C-terminal residues of kAE1 contain a tyrosine-dependent basolateral targeting signal that is not recognised by mu 1B-containing AP-1 adaptor complexes. In the absence of the N-terminus of kAE1, the C-terminus was not sufficient to localise kAE1 to the basolateral membrane. These results suggest that a determinant within the kAE1 N-terminus co-operates with the C-terminus for kAE1 basolateral localisation.