Trans Interactions Research Articles

Actin-Delimited Adhesion-Independent Clustering of E-Cadherin Forms the Nanoscale Building Blocks of Adherens Junctions

E-cadherin is the major adhesion receptor in epithelial adherens junctions, which connect cells to form tissues and are essential for morphogenesis and homeostasis. The mechanism by which E-cadherin monomers cluster and become organized in adherens junctions remains poorly understood. Here, using superresolution microscopy techniques in combination with structure-informed functional mutations, we found that loosely organized clusters of approximately five E-cadherin molecules that form independently of cis or trans interactions, and that are delimited by the cortical F-actin meshwork, are the precursors of trans-ligated adhesive clusters that make up the adherens junction. The density of E-cadherin clusters was wide ranged, and notably, we could detect densities consistent with the crystal lattice structure at the core of adhesive clusters, which were dependent on extracellular domain interactions. Thus, our results elucidate the nanoscale architecture of adherens junctions, as well as the molecular mechanisms driving its assembly.

Aggregation of Claudins and Formation of Tight Junctions: A Coarse-Grained Molecular Dynamics Study

Tight junctions are dynamic structures that consist of a number of membrane proteins and their cytoplasmic counterparts. Claudins are one the major components of tight junctions that control the transport of ions and small molecules in paracellular pathways. The recently solved crystallographic structure of claudin-15 shows a membrane protein consisting of four transmembrane helices (TM1-TM4) and a large extracellular domain (ESC). Claudin monomers polymerize in membrane to form tight junction strands, which are connected to similar strands in neighboring cells and glue two cells together. We have used the structure of claudin-15 to investigate the polymerization and assembly of claudins in lipid membranes with molecular dynamics simulations. The simulations revealed that the linear arrangement of claudins as observed in the crystal is not stable in the membrane. Individual claudins rotate in the membrane to form new contacts between TM3 and ECS resulting in a curved configuration with a diameter of 100A. To obtain more insight about the side-by-side interaction of claudins in the membrane, 16 claudins were placed in a lipid bilayer in a 4x4 matrix arrangement and their diffusion was studied in coarse-grained simulations. In these simulations, claudins form linear configurations consisting of 2-8 claudins stabilized by interactions between TM2 or TM3 segments and ECS. These results show that the side-by-side interaction of claudins in the membrane occurs through two interfaces. The presence of two interaction surfaces might in fact be a key factor in stabilization of long and flexible tight junction strands in the cell. In addition, head-to-head interaction of claudins across two neighboring cells, which is essential for tight junction formation, was studied. In the presence of trans interactions, claudins show a slower dynamics in each membrane and form shorter strands.

Molecular Mechanism for Hypertensive Renal Disease: Differential Regulation of Chromogranin A Expression at 3'-Untranslated Region Polymorphism C+87T by MicroRNA-107.

Chromogranin A (CHGA) is coreleased with catecholamines from secretory vesicles in adrenal medulla and sympathetic axons. Genetic variation in the CHGA 3'-region has been associated with autonomic control of circulation, hypertension, and hypertensive nephropathy, and the CHGA 3'-untranslated region (3'-UTR) variant C+87T (rs7610) displayed peak associations with these traits in humans. Here, we explored the molecular mechanisms underlying these associations. C+87T occurred in a microRNA-107 (miR-107) motif (match: T>C), and CHGA mRNA expression varied inversely with miR-107 abundance. In cells transfected with chimeric luciferase/CHGA 3'-UTR reporters encoding either the T allele or the C allele, changes in miR-107 expression levels had much greater effects on expression of the T allele. Cotransfection experiments with hsa-miR-107 oligonucleotides and eukaryotic CHGA plasmids produced similar results. Notably, an in vitro CHGA transcription/translation experiment revealed that changes in hsa-miR-107 expression altered expression of the T allele variant only. Mice with targeted ablation of Chga exhibited greater eGFR. Using BAC transgenesis, we created a mouse model with a humanized CHGA locus (T/T genotype at C+87T), in which treatment with a hsa-miR-107 inhibitor yielded prolonged falls in SBP/DBP compared with wild-type mice. We conclude that the CHGA 3'-UTR C+87T disrupts an miR-107 motif, with differential effects on CHGA expression, and that a cis:trans (mRNA:miR) interaction regulates the association of CHGA with BP and hypertensive nephropathy. These results indicate new strategies for probing autonomic circulatory control and ultimately, susceptibility to hypertensive renal sequelae.

Cis and trans internucleosomal interactions of H3 and H4 tails in tetranucleosomes.

Chromatin structure and the transcriptional state of a gene can be modulated by modifications made on H3 and H4 tails of histones. Elucidating the internucleosomal interactions of these tails is vital to understanding epigenetic regulation. Differentiation between cis (intra-nucleosomal) and trans (inter-nucleosomal) interactions is often difficult with conventional techniques since H3 and H4 tails are flexible. The distinction, however, is important because these interactions model short- and long-range chromatin interactions respectively and have different bearings in biological processes. Combining FCS and PCH analysis, we can decouple the contribution of histone tails to cis and trans effects. A Mg(2+) gradient was employed to facilitate compaction and oligomerization of tetranucleosomes with H3 and/or H4 tail truncations. H3 tails were found to play a multifunctional role and exhibit the ability to partake in both attractive cis and trans interactions simultaneously. H4 tails partake in attractive cis and repulsive trans interactions at low [Mg(2+)]. These interactions are diminished at higher [Mg(2+)]. Simultaneous H3 and H4 tail truncation inhibited array oligomerization but maintained local array compaction at relatively high [Mg(2+)]. The established experimental approach can be extended to study the detailed molecular interactions mediated by epigenetic modification of flexible histone tails.

CEACAM1 regulates TIM-3-mediated tolerance and exhaustion.

T-cell immunoglobulin domain and mucin domain-3 (TIM-3, also known as HAVCR2) is an activation-induced inhibitory molecule involved in tolerance and shown to induce T-cell exhaustion in chronic viral infection and cancers. Under some conditions, TIM-3 expression has also been shown to be stimulatory. Considering that TIM-3, like cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), is being targeted for cancer immunotherapy, it is important to identify the circumstances under which TIM-3 can inhibit and activate T-cell responses. Here we show that TIM-3 is co-expressed and forms a heterodimer with carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), another well-known molecule expressed on activated T cells and involved in T-cell inhibition. Biochemical, biophysical and X-ray crystallography studies show that the membrane-distal immunoglobulin-variable (IgV)-like amino-terminal domain of each is crucial to these interactions. The presence of CEACAM1 endows TIM-3 with inhibitory function. CEACAM1 facilitates the maturation and cell surface expression of TIM-3 by forming a heterodimeric interaction in cis through the highly related membrane-distal N-terminal domains of each molecule. CEACAM1 and TIM-3 also bind in trans through their N-terminal domains. Both cis and trans interactions between CEACAM1 and TIM-3 determine the tolerance-inducing function of TIM-3. In a mouse adoptive transfer colitis model, CEACAM1-deficient T cells are hyper-inflammatory with reduced cell surface expression of TIM-3 and regulatory cytokines, and this is restored by T-cell-specific CEACAM1 expression. During chronic viral infection and in a tumour environment, CEACAM1 and TIM-3 mark exhausted T cells. Co-blockade of CEACAM1 and TIM-3 leads to enhancement of anti-tumour immune responses with improved elimination of tumours in mouse colorectal cancer models. Thus, CEACAM1 serves as a heterophilic ligand for TIM-3 that is required for its ability to mediate T-cell inhibition, and this interaction has a crucial role in regulating autoimmunity and anti-tumour immunity.

Fringe proteins modulate Notch-ligand cis and trans interactions to specify signaling states.

The Notch signaling pathway consists of multiple types of receptors and ligands, whose interactions can be tuned by Fringe glycosyltransferases. A major challenge is to determine how these components control the specificity and directionality of Notch signaling in developmental contexts. Here, we analyzed same-cell (cis) Notch-ligand interactions for Notch1, Dll1, and Jag1, and their dependence on Fringe protein expression in mammalian cells. We found that Dll1 and Jag1 can cis-inhibit Notch1, and Fringe proteins modulate these interactions in a way that parallels their effects on trans interactions. Fringe similarly modulated Notch-ligand cis interactions during Drosophila development. Based on these and previously identified interactions, we show how the design of the Notch signaling pathway leads to a restricted repertoire of signaling states that promote heterotypic signaling between distinct cell types, providing insight into the design principles of the Notch signaling system, and the specific developmental process of Drosophila dorsal-ventral boundary formation.

Semaphorin signalling during development.

Semaphorins are secreted and membrane-associated proteins that regulate many different developmental processes, including neural circuit assembly, bone formation and angiogenesis. Trans and cis interactions between semaphorins and their multimeric receptors trigger intracellular signal transduction networks that regulate cytoskeletal dynamics and influence cell shape, differentiation, motility and survival. Here and in the accompanying poster we provide an overview of the molecular biology of semaphorin signalling within the context of specific cell and developmental processes, highlighting the mechanisms that act to fine-tune, diversify and spatiotemporally control the effects of semaphorins.

Trans-regulation of RNA-binding protein motifs by microRNA.

The wide array of vital functions that RNA performs is dependent on its ability to dynamically fold into different structures in response to intracellular and extracellular changes. RNA-binding proteins regulate much of this activity by targeting specific RNA structures or motifs. One of these structures, the 3-way RNA junction, is characteristically found in ribosomal RNA and results from the RNA folding in cis, to produce three separate helices that meet around a central unpaired region. Here we demonstrate that 3-way junctions can also form in trans as a result of the binding of microRNAs in an unconventional manner with mRNA by splinting two non-contiguous regions together. This may be used to reinforce the base of a stem-loop motif being targeted by an RNA-binding protein. Trans interactions between non-coding RNA and mRNA may be used to control the post-transcriptional regulatory code and suggests a possible role for some of the recently described transcripts of unknown function expressed from the human genome.

Myristoylation Restricts Orientation of the GRASP Domain on Membranes and Promotes Membrane Tethering

The mammalian Golgi reassembly stacking protein (GRASP) proteins are Golgi-localized homotypic membrane tethers that organize Golgi stacks into a long, contiguous ribbon-like structure. It is unknown how GRASPs undergo trans pairing given that cis interactions between the proteins in the plane of the membrane are intrinsically favored. To test the hypothesis that myristoylation of the self-interacting GRASP domain restricts its orientation on the membrane to favor trans pairing, we established an in vitro assay that recapitulates GRASP-dependent membrane tethering and used neutron reflection under similar conditions to determine the orientation of the GRASP domain. In vivo, the membrane association of GRASP proteins is conferred by the simultaneous insertion of an N-terminal myristic acid and binding to a Golgi-associated binding partner. In our assay, the latter contact was replaced using a C-terminal hexa-His moiety, which bound to Ni(2+)-conjugated lipids incorporated into a substrate-supported bilayer lipid membrane. Nonmyristoylated protein lacked a fixed orientation on the membrane and inefficiently tethered liposomes. In contrast, myristoylated GRASP promoted tethering and exhibited a unique membrane complex. Thus, myristoylation restricts the membrane orientation of the GRASP domain favoring interactions in trans for membrane tethering.

Regulation of chromogranin A by hsa‐miR‐107 in hypertension and nephropathy (1136.19)

Chromogranin A (CHGA) is a major protein in secretory vesicles of neuroendocrine cells, co‐released with catecholamines. Genetic variation in the CHGA 3’‐UTR region is associated with hypertensive nephropathy. This study documents the biological mechanism underlying the genetic association of marker and trait.Methods and Results: An African‐American population case/control study showed that individuals carrying the CHGA 3’‐UTR variant C+87T (rs7610) haplotype are likely to develop end stage renal disease. Computational analysis showed that the variant is in a micro‐RNA (hsa‐miR‐107) recognition motif, with a superior match for the risk (T) allele.In vitro expression studies revealed that CHGA mRNA expression is inversely dependent on miR‐107 abundance in cells and tissues. The effects of miR‐107 were seen with transfected chimeric luciferase/CHGA 3’‐UTR (C+87 versus +87T) reporters in cell lines and corroborated in studies of coupled in vitro transcription/translation of each 3’‐UTR/C+87T allele in CHGA cDNA.In vivo, we exploited mice with a “humanized”CHGA locus (T/T homozygotes) generated by BAC transgenesis. These mice treated with miR‐107 antagomir yielded prolonged, significant falls in SBP/DBP compared to the wild‐type mice.Conclusion: The 3’‐UTRmRNA:miRNA‐107 (cis:trans) interaction plays a role in the clinical manifestations of the CHGA 3’‐UTR associations with BP and nephropathy.Grant Funding Source: Supported by National Institutes of Health (SMV & DTO'C)

Molecular mechanism of Rac1 regulation of endothelial permeability (1095.4)

The RhoGTPase Rac1 plays a critical role in regulating endothelial junctional permeability; however, the causal relationship between Rac1 activity at adherens junctions (AJs) and stability of Vascular Endothelial (VE)‐cadherin adhesion, the main adhesive complex of AJs, is not well understood. Here using photo‐activatable (PA)‐Rac1 and VE‐cadherin‐Dendra2 reporter, we studied Rac1‐mediated VE‐cadherin dynamics. We observed that activation of Rac1 reduced VE‐cadherin dissociation from AJs but had no effect on VE‐cadherin association. The net result of Rac1 activation was to promote steady‐state accumulation of VE‐cadherin at AJs by increasing the affinity of VE‐cadherin homotypic interaction. Inhibition of VE‐cadherin‐mediated adhesion with a peptide that prevents formation of VE‐cadherin‐trans interaction abolished Rac1‐mediated effect on VE‐cadherin kinetics, suggesting that stabilization of trans interaction is required for Rac1 to re‐anneal the AJ barrier. Importantly, Rac1 counterbalanced RhoA activity and decreased the mechanical tension applied to VE‐cadherin adhesion as measured using FRET biosensor. Similar to Rac1 activation, inhibition of Rho kinase (ROCK) at AJs, by un‐caging ROCK inhibitor in UV‐dependent manner, led to accumulation of VE‐cadherin at AJs via stabilization of VE‐cadherin‐trans interaction. Our findings elucidate the important molecular functions of Rac1 in promoting AJ re‐annealing through stabilizing VE‐cadherin‐trans interaction.Grant Funding Source: Supported by AHA 13PRE14670018 and NIH R01 HL045638‐24

Comprehensive Cysteine-scanning Mutagenesis Reveals Claudin-2 Pore-lining Residues with Different Intrapore Locations

The first extracellular loop (ECL1) of claudins forms paracellular pores in the tight junction that determine ion permselectivity. We aimed to map the pore-lining residues of claudin-2 by comprehensive cysteine-scanning mutagenesis of ECL1. We screened 45 cysteine mutations within the ECL1 by expression in polyclonal Madin-Darby canine kidney II Tet-Off cells and found nine mutants that displayed a significant decrease of conductance after treatment with the thiol-reactive reagent 2-(trimethylammonium)ethyl methanethiosulfonate, indicating the location of candidate pore-lining residues. Next, we stably expressed these candidates in monoclonal Madin-Darby canine kidney I Tet-Off cells and exposed them to thiol-reactive reagents. The maximum degree of inhibition of conductance, size selectivity of degree of inhibition, and size dependence of the kinetics of reaction were used to deduce the location of residues within the pore. Our data support the following sequence of pore-lining residues located from the narrowest to the widest part of the pore: Ser(68), Ser(47), Thr(62)/Ile(66), Thr(56), Thr(32)/Gly(45), and Met(52). The paracellular pore appears to primarily be lined by polar side chains, as expected for a predominantly aqueous environment. Furthermore, our results strongly suggest the existence of a continuous sequence of residues in the ECL1 centered around Asp(65)-Ser(68) that form a major part of the lining of the pore.

Protrusion‐guided extracellular vesicles mediate CD30 trans‐signalling in the microenvironment of Hodgkin's lymphoma

Classical Hodgkin's lymphoma (cHL)-affected lymphoid tissue contains only a few malignant Hodgkin and Reed-Sternberg (HRS) cells, which are disseminated within a massive infiltrate of reactive cells. In particular, the innate immune infiltrate is deemed to support tumour growth by direct cell-cell interaction. Since they are rarely found in close proximity to the malignant cells in situ, we investigated whether cHL-derived extracellular vesicles might substitute for a direct cell-cell contact. We studied the crosstalk of the transmembrane proteins CD30 and CD30 ligand (CD30L) because they are selectively expressed on HRS and innate immune cells, respectively. Here, we showed that HRS cells released both the ectodomain as a soluble molecule (sCD30) and the entire receptor on the surface of extracellular vesicles. The vesicle diameter was 40-800 nm, as determined by cryo- and immune electron microscopy. In addition to CD30, typical extracellular vesicle markers were detected by mass spectrometry and flow cytometry, including tetraspanins, flotillins, heat shock proteins and adhesion molecules. In contrast to sCD30, vesicles caused a CD30-dependent release of interleukin-8 in CD30L(+) eosinophil-like EoL-1 cells and primary granulocytes from healthy donors, underscoring the functionality of CD30 on vesicles. In extracellular matrix (ECM)-embedded culture of HRS cells, a network of actin and tubulin-based protrusions guided CD30(+) vesicles into the micro-environment. This network targeted CD30(+) vesicles towards distant immune cells and caused a robust polarization of CD30L. Confocal laser scanning microscopy of 30 µm sections showed a CD30 vesicle-containing network also in cHL-affected lymphoid tissue of both mixed-cellularity and nodular sclerosing subtypes. This network might facilitate the communication between distant cell types in cHL tissue and allow a functional CD30-CD30L interaction in trans. The tubulin backbone of the network may provide a target for the therapy of cHL with antitubulin-based CD30 antibody constructs.

The Neuroligins and Their Ligands: from Structure to Function at the Synapse

The neuroligins are cell adhesion proteins whose extracellular domain belongs to the α/β-hydrolase fold family of proteins, mainly containing enzymes and exemplified by acetylcholinesterase. The ectodomain of postsynaptic neuroligins interacts through a calcium ion with the ectodomain of presynaptic neurexins to form flexible trans-synaptic associations characterized by selectivity for neuroligin or neurexin subtypes. This heterophilic interaction, essential for synaptic differentiation, maturation, and maintenance, is regulated by gene selection, alternative mRNA splicing, and posttranslational modifications. Mutations leading to deficiencies in the expression, folding, maturation, and binding properties of either partner are associated with autism spectrum disorders. The currently available structural and functional data illustrate how these two families of cell adhesion molecules bridge the synaptic cleft to participate in synapse plasticity and support its dynamic nature. Neuroligin partners distinct from the neurexins, and which may undergo either trans or cis interaction, have also been described, and tridimensional structures of some of them are available. Our study emphasizes the partnership versatility of the neuroligin ectodomain associated with molecular flexibility and alternative binding sites, proposes homology models of the structurally non-characterized neuroligin partners, and exemplifies the large structural variability at the surface of the α/β-hydrolase fold subunit. This study also provides new insights into possible surface binding sites associated with non-catalytic properties of the acetylcholinesterase subunit.

SNARE and regulatory proteins induce local membrane protrusions to prime docked vesicles for fast calcium‐triggered fusion

Synaptic vesicles fuse with the plasma membrane in response to Ca(2+) influx, thereby releasing neurotransmitters into the synaptic cleft. The protein machinery that mediates this process, consisting of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and regulatory proteins, is well known, but the mechanisms by which these proteins prime synaptic membranes for fusion are debated. In this study, we applied large-scale, automated cryo-electron tomography to image an in vitro system that reconstitutes synaptic fusion. Our findings suggest that upon docking and priming of vesicles for fast Ca(2)(+)-triggered fusion, SNARE proteins act in concert with regulatory proteins to induce a local protrusion in the plasma membrane, directed towards the primed vesicle. The SNAREs and regulatory proteins thereby stabilize the membrane in a high-energy state from which the activation energy for fusion is profoundly reduced, allowing synchronous and instantaneous fusion upon release of the complexin clamp.

The composition of EphB2 clusters determines the strength in the cellular repulsion response

Trans interactions of erythropoietin-producing human hepatocellular (Eph) receptors with their membrane-bound ephrin ligands generate higher-order clusters that can form extended signaling arrays. The functional relevance of the cluster size for repulsive signaling is not understood. We used chemical dimerizers and fluorescence anisotropy to generate and visualize specific EphB2 cluster species in living cells. We find that cell collapse responses are induced by small-sized EphB2 clusters, suggesting that extended EphB2 arrays are dispensable and that EphB2 activation follows an ON-OFF switch with EphB2 dimers being inactive and trimers and tetramers being fully functional. Moreover, the strength of the collapse response is determined by the abundance of multimers over dimers within a cluster population: the more dimers are present, the weaker the response. Finally, we show that the C-terminal modules of EphB2 have negative regulatory effects on ephrin-induced clustering. These results shed new light on the mechanism and regulation of EphB2 activation and provide a model on how Eph signaling translates into graded cellular responses.

Catalytic reduction of N2 to NH3 by an Fe-N2 complex featuring a C-atom anchor.

While recent spectroscopic studies have established the presence of an interstitial carbon atom at the center of the iron-molybdenum cofactor (FeMoco) of MoFe-nitrogenase, its role is unknown. We have pursued Fe-N2 model chemistry to explore a hypothesis whereby this C-atom (previously denoted as a light X-atom) may provide a flexible trans interaction with an Fe center to expose an Fe-N2 binding site. In this context, we now report on Fe complexes of a new tris(phosphino)alkyl (CP(iPr)3) ligand featuring an axial carbon donor. It is established that the iron center in this scaffold binds dinitrogen trans to the C(alkyl)-atom anchor in three distinct and structurally characterized oxidation states. Fe-C(alkyl) lengthening is observed upon reduction, reflective of significant ionic character in the Fe-C(alkyl) interaction. The anionic (CP(iPr)3)FeN2(-) species can be functionalized by a silyl electrophile to generate (CP(iPr)3)Fe-N2SiR3. (CP(iPr)3)FeN2(-) also functions as a modest catalyst for the reduction of N2 to NH3 when supplied with electrons and protons at -78 °C under 1 atm N2 (4.6 equiv NH3/Fe).

Allele-specific expression and eQTL analysis in mouse adipose tissue.

BackgroundThe simplest definition of cis-eQTLs versus trans, refers to genetic variants that affect expression in an allele specific manner, with implications on underlying mechanism. Yet, due to technical limitations of expression microarrays, the vast majority of eQTL studies performed in the last decade used a genomic distance based definition as a surrogate for cis, therefore exploring local rather than cis-eQTLs.ResultsIn this study we use RNAseq to explore allele specific expression (ASE) in adipose tissue of male and female F1 mice, produced from reciprocal crosses of C57BL/6J and DBA/2J strains. Comparison of the identified cis-eQTLs, to local-eQTLs, that were obtained from adipose tissue expression in two previous population based studies in our laboratory, yields poor overlap between the two mapping approaches, while both local-eQTL studies show highly concordant results. Specifically, local-eQTL studies show ~60% overlap between themselves, while only 15-20% of local-eQTLs are identified as cis by ASE, and less than 50% of ASE genes are recovered in local-eQTL studies. Utilizing recently published ENCODE data, we also find that ASE genes show significant bias for SNPs prevalence in DNase I hypersensitive sites that is ASE direction specific.ConclusionsWe suggest a new approach to analysis of allele specific expression that is more sensitive and accurate than the commonly used fisher or chi-square statistics. Our analysis indicates that technical differences between the cis and local-eQTL approaches, such as differences in genomic background or sex specificity, account for relatively small fraction of the discrepancy. Therefore, we suggest that the differences between two eQTL mapping approaches may facilitate sorting of SNP-eQTL interactions into true cis and trans, and that a considerable portion of local-eQTL may actually represent trans interactions.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-471) contains supplementary material, which is available to authorized users.

Characterization of L1 ORF1p Self-Interaction and Cellular Localization Using a Mammalian Two-Hybrid System

Long INterspersed Element-1 (LINE-1, L1) is an active retrotransposon that mobilizes using a ribonucleoprotein particle (RNP) intermediate composed of the full-length bicistronic L1 mRNA and the two proteins (ORF1p and ORF2p) encoded by that mRNA. ORF1p and ORF2p demonstrate cis-preference for their encoding mRNA. Previous studies of ORF1p, purified from bacterial and insect cells demonstrated that this protein forms trimers in vitro. While valuable for understanding ORF1p function, these in vitro approaches do not provide any information on ORF1p self-interaction in the context of mammalian cells. We used a mammalian two-hybrid (M2H) system in order to study L1 ORF1p self-interaction in human and mouse cells. We demonstrate that the M2H system successfully detects human and mouse ORF1p self-interactions in transiently transfected mammalian cells. We also generated mouse and human ORF1p-specific antibodies to characterize the expression of ORF1p fusion proteins used in the M2H system. Using these antibodies, we demonstrate that ORF1p interaction in trans leads to the formation of heterodimers that are expected to produce a positive signal in the M2H system. Although the role for L1 ORF1p cis-preference in L1 mobilization is established, the impact of ability of ORF1pto interact in trans on the L1 replication cycle is not known. Furthermore, western blot analysis of ORF1p generated by a full-length L1, wild type ORF1, or a codon-optimized ORF1 expression vector is detected in the nucleus. In contrast, the addition of a tag to the N-terminus of the mouse and human ORF1 proteins can significantly alter the subcellular localization in a tag-specific manner. These data support that nuclear localization of ORF1p may contribute to L1 (and potentially the SINE Alu) RNP nuclear access in the host cell.

Attenuation of Eph Receptor Kinase Activation in Cancer Cells by Coexpressed Ephrin Ligands

The Eph receptor tyrosine kinases mediate juxtacrine signals by interacting “in trans” with ligands anchored to the surface of neighboring cells via a GPI-anchor (ephrin-As) or a transmembrane segment (ephrin-Bs), which leads to receptor clustering and increased kinase activity. Additionally, soluble forms of the ephrin-A ligands released from the cell surface by matrix metalloproteases can also activate EphA receptor signaling. Besides these trans interactions, recent studies have revealed that Eph receptors and ephrins coexpressed in neurons can also engage in lateral “cis” associations that attenuate receptor activation by ephrins in trans with critical functional consequences. Despite the importance of the Eph/ephrin system in tumorigenesis, Eph receptor-ephrin cis interactions have not been previously investigated in cancer cells. Here we show that in cancer cells, coexpressed ephrin-A3 can inhibit the ability of EphA2 and EphA3 to bind ephrins in trans and become activated, while ephrin-B2 can inhibit not only EphB4 but also EphA3. The cis inhibition of EphA3 by ephrin-B2 implies that in some cases ephrins that cannot activate a particular Eph receptor in trans can nevertheless inhibit its signaling ability through cis association. We also found that an EphA3 mutation identified in lung cancer enhances cis interaction with ephrin-A3. These results suggest a novel mechanism that may contribute to cancer pathogenesis by attenuating the tumor suppressing effects of Eph receptor signaling pathways activated by ephrins in trans.