Spatial Organization of EphA2 at the Cell-Cell Interface Modulates Trans-Endocytosis of EphrinA1

EphA2 is a receptor tyrosine kinase (RTK) that is sensitive to spatial and mechanical aspects of the cell’s microenvironment. Misregulation of EphA2 occurs in many aggressive cancers. Although its juxtacrine signaling geometry (EphA2’s cognate ligand ephrinA1 is expressed on the surface of an apposing cell) provides a mechanism by which the receptor may experience extracellular forces, this also renders the system challenging to decode. By depositing living cells on synthetic supported lipid membranes displaying ephrinA1, we have reconstituted key features of the juxtacrine EphA2-ephrinA1 signaling system while maintaining the ability to perturb the spatial and mechanical properties of the membrane-cell interface with precision. In addition, we developed a trans-endocytosis assay to monitor internalization of ephrinA1 from a supported membrane into the apposing cell using a quantitative three-dimensional fluorescence microscopy assay. Using this experimental platform to mimic a cell-cell junction, we found that the signaling complex is not efficiently internalized when lateral reorganization at the membrane-cell contact sites is physically hindered. This suggests that EphA2-ephrinA1 trans-endocytosis is sensitive to the mechanical properties of a cell’s microenvironment and may have implications in physical aspects of tumor biology.     

EphrinA1 repulsive response is regulated by an EphA2 tyrosine phosphatase

Ephrin kinases and their ephrin ligands transduce repulsion of cells in axon guidance, migration, invasiveness, and tumor growth, exerting a negative signaling on cell proliferation and adhesion. A key role of their kinase activity has been confirmed by mutant kinase inactive receptors that shift the cellular response from repulsion to adhesion. Our present study aimed to investigate the role of low molecular weight protein-tyrosine phosphatase (LMW-PTP) in ephrinA1/EphA2 signaling. LMW-PTP, by means of dephosphorylation of EphA2 kinase, negatively regulates the ephrinA1-mediated repulsive response, cell proliferation, cell adhesion and spreading, and the formation of retraction fibers, thereby confirming the relevance of the net level of tyrosine phosphorylation of Eph receptors. LMW-PTP interferes with ephrin-mediated mitogen-activated protein kinase signaling likely through inhibition of p120RasGAP binding to the activated EphA2 kinase, thereby confirming the key role of mitogen-activated protein kinase inhibition by ephrinA1 repulsive signaling. We conclude that LMW-PTP acts as a terminator of EphA2 signaling causing an efficient negative feedback loop on the biological response mediated by ephrinA1 and pointing on tyrosine phosphorylation as the main event orchestrating the repulsive response.     

Structural and functional characterization of monomeric EphrinA1 binding site to EphA2 receptor

The EphA2 receptor is overexpressed in glioblastoma multiforme and has been to shown to contribute to cell transformation, tumor initiation, progression, and maintenance. EphrinA1 (eA1) is a preferred ligand for the receptor. Treatment with monomeric eA1, the form of eA1 found in the extracellular environment, causes receptor phosphorylation, internalization, and down-regulation with subsequent anti-tumor effects. Here, we investigated the structure-function relationship of a monomeric eA1 focusing on its G-H loop ((108)FQRFTPFTLGKEFKE(123)G), a highly conserved region among eAs that mediates binding to their receptors. Alanine substitution mutants of the G-H loop amino acids were transfected into U-251 MG glioblastoma multiforme cells, and functional activity of each mutant in conditioned media was assessed by EphA2 down-regulation, ERK and AKT activation and cellular response assays. Alanine substitutions at positions Pro-113 Thr-115, Gly-117, Glu-122, and also Gln-109 enhanced the EphA2 receptor down-regulation and decreased p-ERK and p-AKT. Substitution mutants of eA1 at positions Phe-108, Arg-110, Phe-111, Thr-112, Phe-114, Leu-116, Lys-118, Glu-119, and Phe-120 had a deleterious effect on EphA2 down-regulation when compared with eA1-WT. Mutants at positions Phe-108, Lys-18, Lys-121, Gly-123 retained similar properties to eA1-WT. Recombinant eA1-R110A, -T115A, -G117A, and -F120A have been found to exhibit the same characteristics as the ligands contained in the conditioned media mainly due to the differences in their binding to the receptor. Thus, we have identified variants of eA1 that possess either superagonistic or antagonistic properties. These new findings will be important in the understanding of the receptor/ligand interactions and in further design of anti-cancer therapies targeting the eA/EphA system.     

姜黄素对晚期宫颈癌患者Bcl-xL、Bcl-2、EphA2及EphrinA1表达的影响

目的:探究姜黄素对宫颈癌晚期放疗患者Bcl-x L、Bcl-2、Eph A2与Ephrin A1表达的影响。方法:收集160例晚期宫颈癌放疗患者的宫颈癌石蜡标本,分为姜黄素组与对照组,均给予放射治疗,姜黄素组放疗期间加服姜黄素片剂,采用免疫组化等实验方法,观察两组病例标本Bcl-x L、Bcl-2、Eph A2与Ephrin A1的表达情况,比较两组病理标本化疗前后的凋亡细胞指数(AI)与微血管密度(MVD)。结果:姜黄素组的Bcl-x L、Bcl-2、Eph A2与Ephrin A1的表达阳性率分别为8.3%、43.33%、46.67%、15.0%,均显著低于对照组(P0.05);放疗后姜黄素组的AI显著高于对照组(P0.05),MVD显著低于对照组(P0.05)。结论:姜黄素可抑制Bcl-x L、Bcl-2的表达以促进肿瘤细胞凋亡,同时降低Eph A2与Ephrin A1的表达以减少肿瘤微血管形成,有显著的抗肿瘤与放疗增敏作用。     

EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

The obligate intracellular bacterium Chlamydia trachomatis invades into host cells to replicate inside a membrane-bound vacuole called inclusion. Multiple different host proteins are recruited to the inclusion and are functionally modulated to support chlamydial development. Invaded and replicating Chlamydia induces a long-lasting activation of the PI3 kinase signaling pathway that is required for efficient replication. We identified the cell surface tyrosine kinase EphrinA2 receptor (EphA2) as a chlamydial adherence and invasion receptor that induces PI3 kinase (PI3K) activation, promoting chlamydial replication. Interfering with binding of C. trachomatis serovar L2 (Ctr) to EphA2, downregulation of EphA2 expression or inhibition of EphA2 activity significantly reduced Ctr infection. Ctr interacts with and activates EphA2 on the cell surface resulting in Ctr and receptor internalization. During chlamydial replication, EphA2 remains active accumulating around the inclusion and interacts with the p85 regulatory subunit of PI3K to support the activation of the PI3K/Akt signaling pathway that is required for normal chlamydial development. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Despite the depletion of EphA2 from the cell surface, Ctr infection induces upregulation of EphA2 through the activation of the ERK pathway, which keeps the infected cell in an apoptosis-resistant state. The significance of EphA2 as an entry and intracellular signaling receptor was also observed with the urogenital C. trachomatis-serovar D. Our findings provide the first evidence for a host cell surface receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate chlamydial replication. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism by which Chlamydia subverts the host cell and induces apoptosis resistance.     

Localization of a Region in the Fusion Protein of Avian Metapneumovirus That Modulates Cell-Cell Fusion

The genus Metapneumovirus within the subfamily Pneumovirinae of the family Paramyxoviridae includes two members, human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), causing respiratory tract infections in humans and birds, respectively. Paramyxoviruses enter host cells by fusing the viral envelope with a host cell membrane. Membrane fusion of hMPV appears to be unique, in that fusion of some hMPV strains requires low pH. Here, we show that the fusion (F) proteins of aMPV promote fusion in the absence of the attachment protein and low pH is not required. Furthermore, there are notable differences in cell-cell fusion among aMPV subtypes. Trypsin was required for cell-cell fusion induced by subtype B but not subtypes A and C. The F protein of aMPV subtype A was highly fusogenic, whereas those from subtypes B and C were not. By construction and evaluation of chimeric F proteins composed of domains from the F proteins of subtypes A and B, we localized a region composed of amino acid residues 170 to 338 in the F protein that is responsible for the hyperfusogenic phenotype of the F from subtype A. Further mutagenesis analysis revealed that residues R295, G297, and K323 in this region collectively contributed to the hyperfusogenicity. Taken together, we have identified a region in the aMPV F protein that modulates the extent of membrane fusion. A model for fusion consistent with these data is presented.     

EphrinA/EphA signal facilitates insulin-like growth factor-I-induced myogenic differentiation through suppression of the Ras/extracellular signal-regulated kinase 1/2 cascade in myoblast cell lines

Insulin-like growth factor-I (IGF-I) activates not only the phosphatidylinositol 3-kinase (PI3K)-AKT cascade that is essential for myogenic differentiation but also the extracellular signal-regulated kinase (ERK) 1/2 cascade that inhibits myogenesis. We hypothesized that there must be a signal that inhibits ERK1/2 upon cell-cell contact required for skeletal myogenesis. Cell-cell contact-induced engagement of ephrin ligands and Eph receptors leads to downregulation of the Ras-ERK1/2 pathway through p120 Ras GTPase-activating protein (p120RasGAP). We therefore investigated the significance of the ephrin/Eph signal in IGF-I-induced myogenesis. EphrinA1-Fc suppressed IGF-I-induced activation of Ras and ERK1/2, but not that of AKT, in C2C12 myoblasts, whereas ephrinB1-Fc affected neither ERK1/2 nor AKT activated by IGF-I. IGF-I-dependent myogenic differentiation of C2C12 myoblasts was potentiated by ephrinA1-Fc. In p120RasGAP-depleted cells, ephrinA1-Fc failed to suppress the Ras-ERK1/2 cascade by IGF-I and to promote IGF-I-mediated myogenesis. EphrinA1-Fc did not promote IGF-I-dependent myogenesis when the ERK1/2 was constitutively activated. Furthermore, a dominant-negative EphA receptor blunted IGF-I-induced myogenesis in C2C12 and L6 myoblasts. However, the inhibition of IGF-I-mediated myogenesis by down-regulation of ephrinA/EphA signal was canceled by inactivation of the ERK1/2 pathway. Collectively, these findings demonstrate that the ephrinA/EphA signal facilitates IGF-I-induced myogenesis by suppressing the Ras-ERK1/2 cascade through p120RasGAP in myoblast cell lines.     

Drug-resistant cancer cell-derived exosomal EphA2 promotes breast cancer metastasis via the EphA2-Ephrin A1 reverse signaling

Tumor metastasis induced by drug resistance is a major challenge in successful cancer treatment. Nevertheless, the mechanisms underlying the pro-invasive and metastatic ability of drug resistance remain elusive. Exosome-mediated intercellular communications between cancer cells and stromal cells in tumor microenvironment are required for cancer initiation and progression. Recent reports have shown that communications between cancer cells also promote tumor aggression. However, little attention has been regarded on this aspect. Herein, we demonstrated that drug-resistant cell-derived exosomes promoted the invasion of sensitive breast cancer cells. Quantitative proteomic analysis showed that EphA2 was rich in exosomes from drug-resistant cells. Exosomal EphA2 conferred the invasive/metastatic phenotype transfer from drug-resistant cells to sensitive cells. Moreover, exosomal EphA2 activated ERK1/2 signaling through the ligand Ephrin A1-dependent reverse pathway rather than the forward pathway, thereby promoting breast cancer progression. Our findings indicate the key functional role of exosomal EphA2 in the transmission of aggressive phenotype between cancer cells that do not rely on direct cell–cell contact. Our study also suggests that the increase of EphA2 in drug-resistant cell-derived exosomes may be an important mechanism of chemotherapy/drug resistance-induced breast cancer progression.Subject terms: Breast cancer, Metastasis     

CPSF6 Defines a Conserved Capsid Interface that Modulates HIV-1 Replication

The HIV-1 genome enters cells inside a shell comprised of capsid (CA) protein. Variation in CA sequence alters HIV-1 infectivity and escape from host restriction factors. However, apart from the Cyclophilin A-binding loop, CA has no known interfaces with which to interact with cellular cofactors. Here we describe a novel protein-protein interface in the N-terminal domain of HIV-1 CA, determined by X-ray crystallography, which mediates both viral restriction and host cofactor dependence. The interface is highly conserved across lentiviruses and is accessible in the context of a hexameric lattice. Mutation of the interface prevents binding to and restriction by CPSF6-358, a truncated cytosolic form of the RNA processing factor, cleavage and polyadenylation specific factor 6 (CPSF6). Furthermore, mutations that prevent CPSF6 binding also relieve dependence on nuclear entry cofactors TNPO3 and RanBP2. These results suggest that the HIV-1 capsid mediates direct host cofactor interactions to facilitate viral infection.     

N-Glycosylation at the SynCAM (Synaptic Cell Adhesion Molecule) Immunoglobulin Interface Modulates Synaptic Adhesion

Select adhesion molecules connect pre- and postsynaptic membranes and organize developing synapses. The regulation of these trans-synaptic interactions is an important neurobiological question. We have previously shown that the synaptic cell adhesion molecules (SynCAMs) 1 and 2 engage in homo- and heterophilic interactions and bridge the synaptic cleft to induce presynaptic terminals. Here, we demonstrate that site-specific N-glycosylation impacts the structure and function of adhesive SynCAM interactions. Through crystallographic analysis of SynCAM 2, we identified within the adhesive interface of its Ig1 domain an N-glycan on residue Asn⁶⁰. Structural modeling of the corresponding SynCAM 1 Ig1 domain indicates that its glycosylation sites Asn⁷⁰/Asn¹⁰⁴ flank the binding interface of this domain. Mass spectrometric and mutational studies confirm and characterize the modification of these three sites. These site-specific N-glycans affect SynCAM adhesion yet act in a differential manner. Although glycosylation of SynCAM 2 at Asn⁶⁰ reduces adhesion, N-glycans at Asn⁷⁰/Asn¹⁰⁴ of SynCAM 1 increase its interactions. The modification of SynCAM 1 with sialic acids contributes to the glycan-dependent strengthening of its binding. Functionally, N-glycosylation promotes the trans-synaptic interactions of SynCAM 1 and is required for synapse induction. These results demonstrate that N-glycosylation of SynCAM proteins differentially affects their binding interface and implicate post-translational modification as a mechanism to regulate trans-synaptic adhesion.     

EphA2 Signaling Following Endocytosis: Role of Tiam1

Eph receptors and their membrane‐bound ligands, the ephrins, represent a complex subfamily of receptor tyrosine kinases (RTKs). Eph/ephrin binding can lead to various and opposite cellular behaviors such as adhesion versus repulsion, or cell migration versus cell‐adhesion. Recently, Eph endocytosis has been identified as one of the critical steps responsible for such diversity. Eph receptors, as many RTKs, are rapidly endocytosed following ligand‐mediated activation and traffic through endocytic compartments prior to degradation. However, it is becoming obvious that endocytosis controls signaling in many different manners. Here we showed that activated EphA2 are degraded in the lysosomes and that about 35% of internalized receptors are recycled back to the plasma membrane. Our study is also the first to demonstrate that EphA2 retains the capacity to signal in endosomes. In particular, activated EphA2 interacted with the Rho family GEF Tiam1 in endosomes. This association led to Tiam1 activation, which in turn increased Rac1 activity and facilitated Eph/ephrin endocytosis. Disrupting Tiam1 function with RNA interference impaired both ephrinA1‐dependent Rac1 activation and ephrinA1‐induced EphA2 endocytosis. In summary, our findings shed new light on the regulation of EphA2 endocytosis, intracellular trafficking and signal termination and establish Tiam1 as an important modulator of EphA2 signaling .     

Rac1 Recruits the Adapter Protein CMS/CD2AP to Cell-Cell Contacts

Rac1 is a member of the Rho family of small GTPases, which regulate cell adhesion and migration through their control of the actin cytoskeleton. Rho-GTPases are structurally very similar, with the exception of a hypervariable domain in the C terminus. Using peptide-based pulldown assays in combination with mass spectrometry, we previously showed that the hypervariable domain in Rac1 mediates specific protein-protein interactions. Most recently, we found that the Rac1 C terminus associates to the ubiquitously expressed adapter protein CMS/CD2AP. CD2AP is critical for the formation and maintenance of a specialized cell-cell contact between kidney podocyte foot processes, the slit diaphragm. Here, CD2AP links the cell adhesion protein nephrin to the actin cytoskeleton. In addition, CMS/CD2AP binds actin-regulating proteins, such as CAPZ and cortactin, and has been implicated in the internalization of growth factor receptors. We found that CD2AP specifically interacts with the C-terminal domain of Rac1 but not with that of other Rho family members. Efficient interaction between Rac1 and CD2AP requires both the proline-rich domain and the poly-basic region in the Rac1 C terminus, and at least two of the three N-terminal SH3 domains of CD2AP. CD2AP co-localizes with Rac1 to membrane ruffles, and small interfering RNA-based experiments showed that CD2AP links Rac1 to CAPZ and cortactin. Finally, expression of constitutive active Rac1 recruits CD2AP to cell-cell contacts in epithelial cells, where we found CD2AP to participate in the control of the epithelial barrier function. These data identify CD2AP as a novel Rac1-associated adapter protein that participates in the regulation of epithelial cell-cell contact.     

Spatial structure and pH-dependent conformational diversity of dimeric transmembrane domain of the receptor tyrosine kinase EphA1

Eph receptors are found in a wide variety of cells in developing and mature tissues and represent the largest family of receptor tyrosine kinases, regulating cell shape, movements, and attachment. The receptor tyrosine kinases conduct biochemical signals across plasma membrane via lateral dimerization in which their transmembrane domains play an important role. Structural-dynamic properties of the homodimeric transmembrane domain of the EphA1 receptor were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics in explicit lipid bilayer. EphA1 transmembrane segments associate in a right-handed parallel alpha-helical bundle, region (544-569)(2), through the N-terminal glycine zipper motif A(550)X(3)G(554)X(3)G(558). Under acidic conditions, the N terminus of the transmembrane helix is stabilized by an N-capping box formed by the uncharged carboxyl group of Glu(547), whereas its deprotonation results in a rearrangement of hydrogen bonds, fractional unfolding of the helix, and a realignment of the helix-helix packing with appearance of additional minor dimer conformation utilizing seemingly the C-terminal GG4-like dimerization motif A(560)X(3)G(564). This can be interpreted as the ability of the EphA1 receptor to adjust its response to ligand binding according to extracellular pH. The dependence of the pK(a) value of Glu(547) and the dimer conformational equilibrium on the lipid head charge suggests that both local environment and membrane surface potential can modulate dimerization and activation of the receptor. This makes the EphA1 receptor unique among the Eph family, implying its possible physiological role as an "extracellular pH sensor," and can have relevant physiological implications.     

Tiam1 mediates neurite outgrowth induced by ephrin-B1 and EphA2

Bidirectional signals mediated by Eph receptor tyrosine kinases and their membrane-bound ligands, ephrins, play pivotal roles in the formation of neural networks by induction of both collapse and elongation of neurites. However, the downstream molecular modules to deliver these cues are largely unknown. We report here that the interaction of a Rac1-specific guanine nucleotide-exchanging factor, Tiam1, with ephrin-B1 and EphA2 mediates neurite outgrowth. In cells coexpressing Tiam1 and ephrin-B1, Rac1 is activated by the extracellular stimulation of clustered soluble EphB2 receptors. Similarly, soluble ephrin-A1 activates Rac1 in cells coexpressing Tiam1 and EphA2. Cortical neurons from the E14 mouse embryos and neuroblastoma cells significantly extend neurites when placed on surfaces coated with the extracellular domain of EphB2 or ephrin-A1, which were abolished by the forced expression of the dominant-negative mutant of ephrin-B1 or EphA2. Furthermore, the introduction of a dominant-negative form of Tiam1 also inhibits neurite outgrowth induced by the ephrin-B1 and EphA2 signals. These results indicate that Tiam1 is required for neurite outgrowth induced by both ephrin-B1-mediated reverse signaling and EphA2-mediated forward signaling.     

EphA2 receptor activation by monomeric Ephrin-A1 on supported membranes

The receptor tyrosine kinase EphA2 interacts with its glycosylphosphatidylinositol (GPI)-linked ephrin-A1 ligand in a juxtacrine configuration. The soluble ephrin-A1 protein, without its GPI membrane linker, fails to activate EphA2. However, preclustered ephrin-A1 protein is active in solution and has been frequently used to trigger the EphA2 receptor. Although this approach has yielded insights into EphA2 signaling, preclustered ligands bypass natural receptor clustering processes and thus mask any role of clustering as a signal regulatory mechanism. Here, we present EphA2-expressing cells with a fusion protein of monomeric ephrin-A1 (mEA1) and enhanced monomeric yellow fluorescent protein that is linked to a supported lipid bilayer via a nickel-decahistidine anchor. The mEA1 is homogeneously dispersed, laterally mobile, and monomeric as measured by fluorescence imaging, correlation spectroscopy, and photon counting histogram analysis, respectively. Ephrin-A1 presented in this manner activates EphA2 on the surface of MDA-MB-231 human breast cancer cells, as measured by EphA2 phosphorylation and degradation. Spatial mutation experiments in which nanopatterns on the underlying substrate restrict mEA1 movement in the supported lipid bilayer reveal spatio-mechanical regulation of this signaling pathway, consistent with recently reported observations using a synthetically cross-linked ephrin-A1 dimer.     

Interactions between EGFR and EphA2 promote tumorigenesis through the action of Ephexin1

The cell signaling factors EGFR, EphA2, and Ephexin1 are associated with lung and colorectal cancer and play an important role in tumorigenesis. Although the respective functional roles of EGFR and EphA2 are well known, interactions between these proteins and a functional role for the complex is not understood. Here, we showed that Ephexin1, EphA2, and EGFR are each expressed at higher levels in lung and colorectal cancer patient tissues, and binding of EGFR to EphA2 was associated with both increased tumor grade and metastatic cases in both cancer types. Treatment with Epidermal Growth Factor (EGF) induced binding of the RR domain of EGFR to the kinase domain of EphA2, and this binding was promoted by Ephexin1. Additionally, the AKT-mediated phosphorylation of EphA2 (at Ser897) promoted interactions with EGFR, pointing to the importance of this pathway. Two mutations in EGFR, L858R and T790M, that are frequently observed in lung cancer patients, promoted binding to EphA2, and this binding was dependent on Ephexin1. Our results indicate that the formation of a complex between EGFR, EphA2, and Ephexin1 plays an important role in lung and colorectal cancers, and that inhibition of this complex may be an effective target for cancer therapy.Subject terms: Oncogenes, Cancer models