E-cadherin negatively regulates neoplastic growth in non-small cell lung cancer: role of Rho GTPases.

Non-small cell lung cancers (NSCLC) that express the cell surface adhesion protein E-cadherin may carry a better prognosis than E-cadherin-negative tumors. Here, we found substantial inhibition of anchorage-independent growth in soft agar and cell migration in each of four NSCLC lines stably transfected with E-cadherin. The inhibitory effects were independent of the EGFR and beta-catenin/Wnt-signaling pathways. However, E-cadherin expression was associated with an adhesion-dependent reduction in the activity of Rho family proteins, RhoA in two lines and Cdc42 in the other two. The reduction of RhoA activity was dependent on DLC-1 Rho-GAP and p190 Rho-GAP and associated with an increase in a membrane-associated p190 Rho-GAP/p120 Ras-GAP complex. In parental cells with high levels of RhoA-GTP, siRNA-mediated knock-down of RhoA reduced cell migration and agar growth in a manner analogous to E-cadherin. In parental cells with high levels of Cdc42-GTP, transfection of a Cdc42 dominant-negative mutant reduced cell growth and migration similarly to cells expressing E-cadherin. Thus, E-cadherin can negatively regulate cell proliferation and migration in NSCLC by reducing the level of the predominant active form of Rho family protein, RhoA or Cdc42. These proteins can be considered downstream effectors of E-cadherin and might represent therapeutic targets in some NSCLC.

Putative ClC-2 chloride channel mediates inward rectification in Drosophila retinal photoreceptors.

We report that Drosophila retinal photoreceptors express inwardly rectifying chloride channels that seem to be orthologous to mammalian ClC-2 inward rectifier channels. We measured inwardly rectifying Cl(-) currents in photoreceptor plasma membranes: Hyperpolarization under whole-cell tight-seal voltage clamp induced inward Cl(-) currents; and hyperpolarization of voltage-clamped inside-out patches excised from plasma membrane induced Cl(-) currents that have a unitary channel conductance of approximately 3.7 pS. The channel was inhibited by 1 mM: Zn(2+) and by 1 mM: 9-anthracene, but was insensitive to DIDS. Its anion permeability sequence is Cl(-) = SCN(-)> Br(-)>> I(-), characteristic of ClC-2 channels. Exogenous polyunsaturated fatty acid, linolenic acid, enhanced or activated the inward rectifier Cl(-) currents in both whole-cell and excised patch-clamp recordings. Using RT-PCR, we found expression in Drosophila retina of a ClC-2 gene orthologous to mammalian ClC-2 channels. Antibodies to rat ClC-2 channels labeled Drosophila photoreceptor plasma membranes and synaptic regions. Our results provide evidence that the inward rectification in Drosophila retinal photoreceptors is mediated by ClC-2-like channels in the non-transducing (extra-rhabdomeral) plasma membrane, and that this inward rectification can be modulated by polyunsaturated fatty acid.

Positively charged termini of the L2 minor capsid protein are necessary for papillomavirus infection.

Coexpression of bovine papillomavirus L1 with L2 mutants lacking either eight N-terminal or nine C-terminal amino acids that encode positively charged domains resulted in wild-type levels of viral genome encapsidation. Despite wild-type binding to the cell surface, the resulting virions were noninfectious. An L2 mutant encoding a scrambled version of the nine C-terminal residues restored infectivity, in contrast to an L2 mutant encoding a scrambled version of the N-terminal residues.

Papillomavirus-like particles induce acute activation of dendritic cells.

The role of viral structural proteins in the initiation of adaptive immune responses is poorly understood. To address this issue, we focused on the effect of noninfectious papillomavirus-like particles (VLPs) on dendritic cell (DC) activation. We found that murine bone marrow-derived dendritic cells (BMDCs) effectively bound and rapidly internalized bovine papillomavirus VLPS: Exposure to fully assembled VLPs of bovine papillomavirus, human papillomavirus (HPV)16 or HPV18, but not to predominately disordered HPV16 capsomers, induced acute phenotypic maturation of BMDCS: Structurally similar polyomavirus VLPs bound to the DC surface and were internalized, but failed to induce maturation. DCs that had incorporated HPV16 VLPs produced proinflammatory cytokines IL-6 and TNF-alpha; however, the release of these cytokines was delayed relative to LPS activation. Production of IL-12p70 by VLP-exposed DCs required the addition of syngeneic T cells or rIFN-gamma. Finally, BMDCs pulsed with HPV16 VLPs induced Th1-dominated primary T cell responses in vitro. Our data provide evidence that DCs respond to intact papillomavirus capsids and that they play a central role in VLP-induced immunity. These results offer a mechanistic explanation for the striking ability of papillomavirus VLP-based vaccines to induce potent T and B cell responses even in the absence of adjuvant.

L1 interaction domains of papillomavirus l2 necessary for viral genome encapsidation.

BPHE-1 cells, which harbor 50 to 200 viral episomes, encapsidate viral genome and generate infectious bovine papillomavirus type 1 (BPV1) upon coexpression of capsid proteins L1 and L2 of BPV1, but not coexpression of BPV1 L1 and human papillomavirus type 16 (HPV16) L2. BPV1 L2 bound in vitro via its C-terminal 85 residues to purified L1 capsomers, but not with intact L1 virus-like particles in vitro. However, when the efficiency of BPV1 L1 coimmunoprecipitation with a series of BPV1 L2 deletion mutants was examined in vivo, the results suggested that residues 129 to 246 and 384 to 460 contain independent L1 interaction domains. An L2 mutant lacking the C-terminal L1 interaction domain was impaired for encapsidation of the viral genome. Coexpression of BPV1 L1 and a chimeric L2 protein composed of HPV16 L2 residues 1 to 98 fused to BPV1 L2 residues 99 to 469 generated infectious virions. However, inefficient encapsidation was seen when L1 was coexpressed with either BPV1 L2 with residues 91 to 246 deleted or with BPV1 L2 with residues 1 to 225 replaced with HPV16 L2. Impaired genome encapsidation did not correlate closely with impairment of the L2 proteins either to localize to promyelocytic leukemia oncogenic domains (PODs) or to induce localization of L1 or E2 to PODs. We conclude that the L1-binding domain located near the C terminus of L2 may bind L1 prior to completion of capsid assembly, and that both L1-binding domains of L2 are required for efficient encapsidation of the viral genome.

Coordinated gating of TRP-dependent channels in rhabdomeral membranes from Drosophila retinas.

Using a newly developed dissociation procedure, we isolated the specialized rhabdomeral membranes from Drosophila retinal photoreceptors. From these membranes, we have recorded spontaneous active currents in excised patch, voltage-clamp recordings. We observed rapid opening events that closely resembled those ascribed to one class of light-activated channels, TRP. All activity exhibited Ba(2+) permeability, little voltage dependence, and sensitivity to La(3+) block. Mutational analysis indicated that the spontaneous activity present in these membranes was TRP-dependent. Excised patches from wild-type rhabdomeral membranes exhibited a wide range of conductance amplitudes. In addition, large conductance events exhibited many conductance levels in the open state. Block of activity by La(3+) both developed and recovered in a stepwise manner. Our results indicate that TRP-dependent channels have a small unitary conductance and that many channels can be gated coordinately.

A novel member of the BTB/POZ family, PATZ, associates with the RNF4 RING finger protein and acts as a transcriptional repressor.

We have identified a novel human gene encoding a 59-kDa POZ-AT hook-zinc finger protein (PATZ) that interacts with RNF4, a mediator of androgen receptor activity, and acts as a transcriptional repressor. PATZ cDNA was isolated through a two-hybrid interaction screening using the RING finger protein RNF4 as a bait. In vitro and in vivo interaction between RNF4 and PATZ was demonstrated by protein-protein affinity chromatography and coimmunoprecipitation experiments. Such interaction occurred through a small region of PATZ containing an AT-hook DNA binding domain. Immunofluorescence staining and confocal microscopy showed that PATZ localizes in distinct punctate nuclear regions and colocalizes with RNF4. Functional analysis was performed by cotransfection assays: PATZ acted as a transcriptional repressor, whereas its partner RNF4 behaved as a transcriptional activator. When both proteins were overexpressed a strong repression of the basal transcription was observed, indicating that the association of PATZ with RNF4 switches activation to repression. In addition, RNF4 was also found to associate with HMGI(Y), a chromatin-modeling factor containing AT-hook domains.

Intracellular localization of proteasomal degradation of a viral antigen.

To better understand proteasomal degradation of nuclear proteins and viral antigens we studied mutated forms of influenza virus nucleoprotein (NP) that misfold and are rapidly degraded by proteasomes. In the presence of proteasome inhibitors, mutated NP (dNP) accumulates in highly insoluble ubiquitinated and nonubiquitinated species in nuclear substructures known as promyelocytic leukemia oncogenic domains (PODs) and the microtubule organizing center (MTOC). Immunofluorescence revealed that dNP recruits proteasomes and a selective assortment of molecular chaperones to both locales, and that a similar (though less dramatic) effect is induced by proteasome inhibitors in the absence of dNP expression. Biochemical evidence is consistent with the idea that dNP is delivered to PODs/MTOC in the absence of proteasome inhibitors. Restoring proteasome activity while blocking protein synthesis results in disappearance of dNP from PODs and the MTOC and the generation of a major histocompatibility complex class I-bound peptide derived from dNP but not NP. These findings demonstrate that PODs and the MTOC serve as sites of proteasomal degradation of misfolded dNP and probably cellular proteins as well, and imply that antigenic peptides are generated at one or both of these sites.

Rescue of excitation by inositol following Li(+)-induced block in Limulus ventral photoreceptors.

The phosphoinositide (PI) intracellular signaling pathway, which triggers Ca2+ release from intracellular stores, appears to be a central feature of phototransduction in most invertebrate species studied. Procedures designed to inhibit PI-pathway reactions cause suppression of excitation to dim lights. However, in Limulus photoreceptors, responses to bright stimuli are in fact enhanced by some of these procedures, suggesting that PI metabolism is not obligatory for light-induced excitation. Other studies, however, suggest that Ca2+ release is obligatory for excitation. We studied this issue by examining the effects of PI-pathway inhibitor, Li+, on electrophysiological responses to light in Limulus photoreceptors. Li+ is reported to cause depletion of intracellular PI-pathway intermediate, inositol; and it offers the pharmacological advantage that its block can be bypassed by introducing exogenous inositol. Introduction of Li+ caused a very slowly developing but complete suppression of responses to dim stimuli. In contrast, Li+ caused a rapidly developing but partial suppression of responses to bright stimuli. Li(+)-induced suppression was reversed by exogenous introduction of inositol. In addition, inositol prevented Li(+)-induced suppression of excitation. Li+ enhanced light adaptation (light-induced desensitization) but slowed response deactivation, indicating a difference in the processes underlying these phenomena. Li+ slowed dark adaptation, the recovery of sensitivity following light adaptation. All of these effects were prevented or rescued by extracellularly applied inositol, suggesting the presence of a transmembrane inositol transport system. The overall results suggest that PI-dependent signaling is central and obligatory for excitation in Limulus, at least for responses to dim to moderate illumination. The failure of Li+ to suppress bright light-induced excitation completely may be due to a failure of Li+ to block PI metabolism completely, as in other systems; however, it may point to a parallel, PI-independent excitation pathway possessing very low light sensitivity when PI metabolism is inhibited.

The papillomavirus minor capsid protein, L2, induces localization of the major capsid protein, L1, and the viral transcription/replication protein, E2, to PML oncogenic domains.

We have used immunofluorescent staining and confocal microscopy to examine the subcellular localization of structural and nonstructural bovine papillomavirus (BPV) proteins in cultured cells that produce infectious virions. When expressed separately, L1, the major capsid protein, showed a diffuse nuclear distribution while L2, the minor capsid protein, was found to localize to punctate nuclear regions identified as promonocytic leukemia protein (PML) oncogenic domains (PODs). Coexpression of L1 and L2 induced a relocation of L1 into the PODs, leading to the colocalization of L1 and L2. The effect of L2 expression on the distribution of the nonstructural viral proteins E1 and E2, which are required for maintenance of the genome and viral DNA synthesis, was also examined. The localization of the E1 protein was unaffected by L2 expression. However, the pattern of anti-E2 staining was dramatically altered in L2-expressing cells. Similar to L1, E2 was shifted from a dispersed nuclear locality into the PODs and colocalized with L2. The recruitment of full-length E2 by L2 occurred in the absence of other viral components. L2 was shown previously to be essential for the generation of infectious BPV. Our present results provide evidence for a role for L2 in the organization of virion components by recruiting them to a distinct nuclear domain. This L2-dependent colocalization probably serves as a mechanism to promote the assembly of papillomaviruses either by increasing the local concentration of virion constituents or by providing the physical architecture necessary for efficient packaging and assembly. The data also suggest a role for a nonstructural viral protein, E2, in virion assembly, specifically the recruitment of the viral genome to the sites of assembly, through its high-affinity interaction with specific sequences in the viral DNA.

Current issues in invertebrate phototransduction. Second messengers and ion conductances.

Investigation of phototransduction in invertebrate photoreceptors has revealed many physiological and biochemical features of fundamental biological importance. Nonetheless, no complete picture of phototransduction has yet emerged. In most known cases, invertebrate phototransduction involves polyphosphoinositide and cyclic GMP (cGMP) intracellular biochemical signaling pathways leading to opening of plasma membrane ion channels. Excitation is Ca(2+)-dependent, as are adaptive feedback processes that regulate sensitivity to light. Transduction takes place in specialized subcellular regions, rich in microvilli and closely apposed to submicrovillar membrane systems. Thus, excitation is a highly localized process. This article focuses on the intracellular biochemical signaling pathways and the ion channels involved in invertebrate phototransduction. The coupling of signaling cascades with channel activation is not understood for any invertebrate species. Although photoreceptors have features that are common to most or all known invertebrate species, each species exhibits unique characteristics. Comparative electrophysiological, biochemical, morphological, and molecular biological approaches to studying phototransduction in these species lead to fundamental insights into cellular signaling. Several current controversies and proposed phototransduction models are evaluated.

Direct delivery of exogenous MHC class I molecule-binding oligopeptides to the endoplasmic reticulum of viable cells.

After brief incubation of cells with fluorescein-conjugated peptides that bind major histocompatibility complex (MHC) class I molecules, peptides were detected within the endoplasmic reticulum (ER) by microscopy or by binding to radiolabeled class I molecules. ER delivery of a nonfluorescent peptide was demonstrated using a mAb highly specific for the peptide-class I molecule complex. ER localization of peptides: (i) required expression of appropriate class I molecules in the ER but not on the cell surface, (ii) was diminished by expression of TAP, the MHC-encoded cytosol to ER peptide transporter, and (iii) was blocked by pinocytosis inhibitors but not by brefeldin A. These findings demonstrate the existence of a pathway, likely vesicular in nature, that conveys small extracellular substances to the ER without traversing the Golgi complex or the cytosol. This pathway contributes to the loading of exogenous peptides to MHC class I molecules, but its evolutionary significance may lie in other cellular processes, such as maintaining ER homeostasis or signaling by extracellular substances.

Characterization of protein phosphatases type 1 and type 2A in Limulus nervous tissue: their light regulation in the lateral eye and evidence of involvement in the photoresponse.

The activities of both protein phosphatases and protein kinases are responsible for the transient changes in the levels of phosphorylation and probably the functions of protein intermediates involved in the biochemical and physiological mechanisms underlying the photoresponse in photoreceptor cells from both vertebrate and invertebrate organisms. Of the known protein serine/threonine phosphatases, various forms of type 1 (PP 1) and type 2A (PP 2A) protein phosphatases are responsible for dephosphorylating many of the known phosphoproteins including those involved in photoreceptor cell function. In this report, we provide biochemical evidence for both PP 1- and PP 2A-like activities in the visual and nonvisual tissue of the horseshoe crab, Limulus polyphemus, that membrane and soluble forms of both enzymes are present, and that the activities of both enzymes are greater in light- than in dark-adapted lateral eyes. These activities were characterized using glycogen phosphorylase a, a substrate for both PP 1 and PP 2A, and various protein phosphatase inhibitors, including okadaic acid. We also report that okadaic acid, at concentrations required to inhibit PP 1, inhibited physiological functions of photoreceptor cells from the ventral eye, causing a delayed reduction of the resting membrane, and slowing and reducing light responses.

The second messenger for visual excitation in invertebrate phototransduction.

Invertebrate visual transduction involves a second messenger cascade process that leads to an increase in membrane conductance. The identity of the second messenger that gates the light-dependent channels is presently a major focus of attention. Cyclic GMP, inositol trisphosphate and Ca2+ are the most likely candidates for being such a messenger in the species studied so far. Here we review the available evidence for each of these molecules.

The second messenger for visual excitation in invertebrate phototransduction

Invertebrate visual transduction involves a second messenger cascade process that leads to an increase in membrane conductance. The identity of the second messenger that gates the light-dependent channels is presently a major focus of attention. Cyclic GMP, inositol trisphosphate and Ca2+ are the most likely candidates for being such a messenger in the species studied so far. Here we review the available evidence for each of these molecules

Cyclic-GMP enhances light-induced excitation and induces membrane currents in Drosophila retinal photoreceptors.

Phototransduction in the Drosophila retina appears to require the phosphoinositide signaling cascade following receptor/G-protein activation. Subsequent opening of membrane cationic channels causes excitation. The biochemical events underlying channel opening and regulation of sensitivity remain largely unknown. Evidence is mounting that phototransduction in Drosophila and other invertebrate species may additionally involve the second messenger, cyclic-GMP (cGMP). We report that exogenous cGMP influenced Drosophila retinal phototransduction in two ways. In whole cell tight-seal voltage-clamp experiments, membrane permeant cGMP analog, 8-bromo-cyclic-GMP (8-Br-cGMP), induced membrane currents and dramatically enhanced light-induced currents. The currents induced by 8-Br-cGMP possessed reversal potentials similar to those induced by light. The magnitudes of cGMP-induced currents exhibited marked dependence on intensity of background illumination. Potential direct or modulatory roles of cGMP in Drosophila phototransduction are discussed.

Inhibitors of cyclic-GMP phosphodiesterase alter excitation of Limulus ventral photoreceptors in Ca(2+)-dependent fashion.

We have examined the hypothesis that Ca(2+)-dependent cyclic-GMP metabolism may play a role in visual transduction in Limulus photoreceptors. Although phosphoinositide hydrolysis is central to phototransduction and phosphoinositide-dependent Ca(2+)-mobilization seems to be required for transduction, the subsequent steps leading to ion channel gating (the immediate cause of excitation) are not understood. Channels normally opened in response to light can be opened in excised membrane patches by cGMP but not by Ca2+, suggesting that cGMP acts as a channel ligand in excitation. Using phosphodiesterase inhibitors, we investigated whether changes in cGMP metabolism could affect excitation. We report that zaprinast and IBMX increased the amplitudes and retarded the kinetics of physiological light responses. These effects were maximal for brightest stimuli. The effects were markedly enhanced in low Ca2+ conditions. In contrast, excitation induced by direct IP3-injection and by direct Ca(2+)-injection were inhibited. These observations suggest that PI-induced excitation is dependent on cGMP metabolism in a Ca(2+)-dependent manner, and they support the possibility that transduction involves modification of cGMP metabolism by Ca(2+)-release resulting from phosphoinositide hydrolysis.

Effect of TAP on the generation and intracellular trafficking of peptide-receptive major histocompatibility complex class I molecules.

Using a fluorescein-conjugated antigenic peptide, peptide-receptive H-2Kb MHC class I molecules were found throughout the secretory pathways of RMA cells and peptide transporter (TAP)-deficient derivative cells (RMA/S). RMA/S cells displayed higher levels of intracellular peptide-receptive molecules, while, surprisingly, RMA cells expressed 3- to 5-fold more cell surface peptide-receptive molecules. Metabolic radiolabeling of Kb-associated oligosaccharides with [1-3H]galactose demonstrated that despite a large difference in the fraction of Kb molecules in native conformation in detergent extracts, Kb transport rates from the trans-Golgi complex to the surfaces of RMA and RMA/S cells were similar. Thus, although considerable numbers of class I alpha chains reach the RMA/S cell surface, they are a less productive source of peptide-receptive molecules than class I molecules synthesized by TAP-expressing RMA cells, suggesting paradoxically that TAP functions to increase the amount of peptide-receptive molecules at the cell surface.

The Drosophila dgq gene encodes a G alpha protein that mediates phototransduction.

We examined the roles of the Drosophila Gq alpha proteins (DGq) in the phototransduction pathway. The DGq proteins immunolocalized to the ocelli and all eight retinular photoreceptor cell rhabdomeres. An affinity-purified anti-DGq alpha immunoglobulin blocked the light-dependent GTP hydrolysis activity associated with Drosophila head membranes in vitro, suggesting that rhodopsin stimulated DGq. Dominantly active DGq1 mutants exhibited a light-independent GTPase activity and abnormal electrophysiological light responses, such as reduced retinal sensitivity and slow response kinetics compared with wild-type flies. Dominant DGq2 mutants exhibited a light-independent GTPase activity with normal electrophysiological light responses. Retinas of double mutants of DGq1, but not DGq2, with the light-dependent retinal degeneration mutant rdgB degenerated even in the dark. DGq1 stimulation of rdgB retinal degeneration in the dark was norpA-dependent. These results indicate that DGq1 mediates the stimulation by light-activated rhodopsin of the norpA-encoded phospholipase C in the visual transduction cascade.

Pertussis toxin expression in Drosophila alters the visual response and blocks eating behaviour.

Pertussis toxin inactivates certain G-proteins by introducing an ADP-ribose group near the carboxyl-terminus of the alpha-subunit. The major pertussis toxin substrate in Drosophila tissues is Go alpha. We introduced a pertussis toxin gene under control of the hsp70 heat-shock promoter into the Drosophila genome. When heat-shocked, transformed flies produce active pertussis toxin which ADP-ribosylates endogenous Go alpha. Pertussis toxin is expressed in photoreceptors, in the lamina of the eye and in epithelial cells lining the gut. As expected from the absence of Go alpha in photoreceptors, pertussis toxin does not affect the photoreceptor component of the Drosophila visual response. However, it abolishes light on- and off-transients in the electroretinogram. These transients normally arise from the lamina, a tissue where Go alpha transcripts have been detected. Pertussis toxin expression also blocks embryonic development and shortens the lifetime of adult Drosophila. Following heat-shock, transformed adults are active, but they fail to take up nutrients because they stop eating. High energy metabolites are significantly depleted shortly after pertussis toxin expression is induced and the flies die within 48 h.