Insights into Eph receptor tyrosine kinase activation from crystal structures of the EphA4 ectodomain and its complex with ephrin-A5

Eph receptor tyrosine kinases and their ephrin ligands mediate cell signaling during normal and oncogenic development. Eph signaling is initiated in a multistep process leading to the assembly of higher-order Eph/ephrin clusters that set off bidirectional signaling in interacting cells. Eph and ephrins are divided in two subclasses based on their abilities to bind and activate each other and on sequence conservation. EphA4 is an exception to the general rule because it can be activated by both A- and B-class ephrin ligands. Here we present high-resolution structures of the complete EphA4 ectodomain and its complexes with ephrin-A5. The structures reveal how ligand binding promotes conformational changes in the EphA4 ligand-binding domain allowing the formation of signaling clusters at the sites of cell–cell contact. In addition, the structural data, combined with structure-based mutagenesis, reveal a previously undescribed receptor–receptor interaction between the EphA4 ligand-binding and membrane-proximal fibronectin domains, which is functionally important for efficient receptor activation.Eph receptors, the largest family of receptor tyrosine kinases, and their ephrin ligands regulate a variety of cell–cell interactions during development and in the adult organism (1–3). Because both receptors and ligands are membrane-bound, their interactions at sites of cell–cell contact initiate unique bidirectional signaling cascades (4). The signaling downstream of the Eph is referred to as “forward,” and the signaling downstream of the ephrins is referred to as “reverse.”The 16 Eph receptors and 9 ephrins are divided into two subclasses based on sequence homology and binding affinities. Receptor–ligand binding within each subclass is fairly promiscuous, whereas cross-subclass signaling happens rarely (5). The best-known exception to this general rule is EphA4, which has long been known to bind and be activated by both A- and B-class ligands (6, 7). Because of its unique properties, EphA4 is an attractive target for studying the structural details of subclass specificity (8).Eph and ephrins were originally identified as axon guidance molecules; they have now been implicated in a vast array of cell communication events. Those include bone morphogenesis and homeostasis, immunological and inflammatory host responses, stem cell plasticity, learning and memory, and Alzheimer’s disease (9). However, currently, the most intensely studied function of the Eph/ephrin system is that during development and progression of different cancers. Many A- and B-class receptors were shown to be overexpressed in various tumor types (10–13) and to regulate critical steps of blood vessel formation (vasculogenesis) and remodeling (angiogenesis) and hence tumor growth.Structural studies on the minimal binding domains of Eph receptors and ephrins revealed important details of receptor–ligand recognition (2, 14). The initial binding force comes from a penetration of a long, hydrophobic ephrin loop into a hydrophobic cavity on the surface of the receptor to form high-affinity 1:1 ligand–receptor heterodimers (15). The ligand-binding cavity of the receptor consists of loops DE, FG, and JK, which were shown to exhibit various amounts of conformational flexibility, depending on the subclass and the individual receptor. Several structures of the EphA4 ligand-binding domain (LBD) alone and in complex with its ligands have addressed its unusually high ligand promiscuity (8, 16–18).More recently, the crystal structures of the EphA2 ectodomain (ECD) bound to ephrin-A1 and ephrin-A5 suggested a structural basis for the formation of Eph/ephrin signaling clusters at the interfaces of interacting cells (19, 20). Specifically, it was proposed that these signaling clusters nucleate from high-affinity Eph/ephrin heterodimers, which assemble into heterotetramers that further aggregate into higher-order oligomers. To understand the structural basis of EphA4 ligand-induced clustering and explore underlying receptor–receptor and receptor–ligand interactions, we determined and report here the structures of the complete EphA4 ECD, alone and in complexes with its ephrin-A5 ligand.     

Fetal cardiac function after labetalol or pindolol for maternal hypertension in a sheep model of increased placental vascular resistance

Objective

We hypothesized that labetalol and pindolol have no detrimental effects on fetal cardiac function and pulmonary hemodynamics when administered for norepinephrine-induced maternal hypertension in a chronic sheep model of increased placental vascular resistance. Specifically, we investigated the effects of labetalol and pindolol on fetal cardiopulmonary responses to acute hypoxemia.

Study design

Twenty chronically instrumented near-term ewes with increased placental vascular resistance after placental embolization were anesthetized and randomized to receive labetalol or pindolol for norepinephrine-induced hypertension. Thereafter, maternal inspiratory oxygen fraction was decreased to induce fetal hypoxemia. At the end of each phase, fetal hemodynamics were assessed by Doppler ultrasonography. The data were analyzed using repeated measures ANOVA.

Results

Maternal administration of norepinephrine had no effect on fetal hemodynamics. Pindolol decreased fetal heart rate and weight-indexed left ventricular cardiac output and increased pulmonary vascular impedances, while labetalol had no effect on these parameters. During hypoxemia, fetal heart rate increased to baseline in the pindolol group and pulmonary vascular impedances increased in the labetalol group, with no changes in fetal cardiac outputs.

Conclusion

Pindolol decreased fetal left ventricular cardiac output and induced vasoconstriction in the pulmonary vasculature, but neither pindolol nor labetalol significantly modified fetal cardiopulmonary responses to acute hypoxemia.     

The hypermethylation of the O6-methylguanine-DNA methyltransferase gene promoter in gliomas--correlation with array comparative genome hybridization results and IDH1 mutation

The use of molecular markers in the diagnostics of gliomas aids histopathological diagnosis and allows their further classification into clinically significant subgroups. The aim of this study was to characterize the methylation pattern of the O(6) -methylguanine-DNA methyltransferase (MGMT) promoter, gene copy number aberrations, and isocitrate dehydrogenase I (IDH1) mutation in gliomas. We studied 51 gliomas (15 oligodendrogliomas, 18 oligoastrocytomas, 3 astrocytomas, and 15 glioblastomas) by pyrosequencing, array comparative genome hybridization (CGH), and immunohistochemistry. MGMT hypermethylation was observed in 100% of oligoastrocytomas, 93% of oligodendrogliomas, and 47% of glioblastomas. The most frequently altered chromosomal regions were deletions of 1p31.1/21.1-22.2 and 19q13.3qter in oligodendroglial tumors, and losses of 9p21.3, 10q25.3qter, and 10q26.13-26.2 in glioblastomas. Deletions on 9p and 10q, and gain of 7p were associated with the unmethylated MGMT phenotype, whereas deletion of 19q and oligodendroglial morphology was associated with MGMT hypermethylation. IDH1 mutation showed positive correlation with MGMT hypermethylation and loss of 1p/19q. Our results suggest that MGMT promoter methylation, analyzed by pyrosequencing, is a frequent event in oligodendroglial tumors, and it correlates with IDH1 mutation and 19q loss in gliomas. Pyrosequencing proved a good method for assessing the degree of MGMT methylation in formalin-fixed paraffin-embedded glioma samples. However, further studies are needed to confirm a clinically relevant cut-off point for MGMT methylation in gliomas.     

Observ-OM and Observ-TAB: Universal syntax solutions for the integration, search, and exchange of phenotype and genotype information

Genetic and epidemiological research increasingly employs large collections of phenotypic and molecular observation data from high quality human and model organism samples. Standardization efforts have produced a few simple formats for exchange of these various data, but a lightweight and convenient data representation scheme for all data modalities does not exist, hindering successful data integration, such as assignment of mouse models to orphan diseases and phenotypic clustering for pathways. We report a unified system to integrate and compare observation data across experimental projects, disease databases, and clinical biobanks. The core object model (Observ-OM) comprises only four basic concepts to represent any kind of observation: Targets, Features, Protocols (and their Applications), and Values. An easy-to-use file format (Observ-TAB) employs Excel to represent individual and aggregate data in straightforward spreadsheets. The systems have been tested successfully on human biobank, genome-wide association studies, quantitative trait loci, model organism, and patient registry data using the MOLGENIS platform to quickly setup custom data portals. Our system will dramatically lower the barrier for future data sharing and facilitate integrated search across panels and species. All models, formats, documentation, and software are available for free and open source (LGPLv3) at http://www.observ-om.org.     

Alveolar gas exchange and tissue deoxygenation during exercise in type 1 diabetes patients and healthy controls

We used near-infrared spectroscopy to investigate whether leg and arm skeletal muscle and cerebral deoxygenation differ during incremental cycling exercise in men with type 1 diabetes (T1D, n=10, mean±SD age 33±7 years) and healthy control men (matched by age, anthrometry, and self-reported physical activity, CON, n=10, 32±7 years) to seek an explanation for lower aerobic capacity (˙VO2peak) often reported in T1D. T1D had lower ˙VO2peak (35±4mlkg(-1)min(-1) vs. 43±8mlkg(-1)min(-1), P<0.01) and peak work rate (219±33W vs. 290±44W, P<0.001) than CON. Leg muscle deoxygenation (↑ [deoxyhemoglobin]; ↓ tissue saturation index) was greater in T1D than CON at a given absolute submaximal work rate, but not at peak exercise, while arm muscle and cerebral deoxygenation were similar. Thus, in T1D compared with CON, faster leg muscle deoxygenation suggests limited circulatory ability to increase O(2) delivery as a plausible explanation for lower ˙VO2peak and earlier fatigue in T1D.     

Measurement of aminoterminal propeptide of type I procollagen (PINP) in serum

The aminoterminal propeptide of type I procollagen (PINP) in serum is a sensitive indicator of the synthesis of type I collagen. Four assays are available for PINP, two of them (intact PINP assays) measure the intact propeptide and the other two (total PINP assays) also detect a smaller antigen in serum. In many clinical situations, these assays give similar information, but renal insufficiency increases the concentration of the smaller antigen, influencing both the apparent concentration of PINP and assay calibration. Serum PINP is mostly affected by changes in bone metabolism. In infants and children, the concentration is much higher than in adults. Serum PINP (s-PINP) is a useful indicator of disease activity in Paget's disease of bone, in bone metastases of osteoblastic nature, and in the follow-up of treatment of osteoporosis. The IFCC and IOF recently recommended the use of s-PINP as a reference marker for bone formation in studies concerning fracture risk assessment and treatment response.     

Flotillin-1 (Reggie-2) contributes to Chlamydia pneumoniae growth and is associated with bacterial inclusion

Chlamydiae are obligate intracellular pathogens replicating only inside the eukaryotic host. Here, we studied the effect of human flotillin-1 protein on Chlamydia pneumoniae growth in human line (HL) and A549 epithelial cell lines. RNA interference was applied to disrupt flotillin-1-mediated endocytosis. Host-associated bacteria were detected by quantitative PCR, and C. pneumoniae growth was evaluated by inclusion counts. C. pneumoniae attachment to host cells was unaffected, but bacterial intracellular growth was attenuated in the flotillin-1-silenced cells. By using confocal microscopy, we detected flotillin-1 colocalized with the inclusion membrane protein A (IncA) in the C. pneumoniae inclusion membranes. In addition, flotillin-1 was associated with IncA in detergent-resistant membrane microdomains (DRMs) in biochemical fractioning. These results suggest that flotillin-1 localizes to the C. pneumoniae inclusion membrane and plays an important role for intracellular growth of C. pneumoniae.