Replacing two conserved tyrosines of the EphB2 receptor with glutamic acid prevents binding of SH2 domains without abrogating kinase activity and biological responses.

Eph receptor tyrosine kinases play key roles in pattern formation during embryonic development, but little is known about the mechanisms by which they elicit specific biological responses in cells. Here, we investigate the role of tyrosines 605 and 611 in the juxtamembrane region of EphB2, because they are conserved Eph receptor autophosphorylation sites and demonstrated binding sites for the SH2 domains of multiple signaling proteins. Mutation of tyrosines 605 and 611 to phenylalanine impaired EphB2 kinase activity, complicating analysis of their function as SH2 domain binding sites and their contribution to EphB2-mediated signaling.

Multiple in vivo tyrosine phosphorylation sites in EphB receptors.

Autophosphorylation regulates the function of receptor tyrosine kinases. To dissect the mechanism by which Eph receptors transmit signals, we have developed an approach using matrix-assisted laser desorption-ionization (MALDI) mass spectrometry to map systematically their in vivo tyrosine phosphorylation sites. With this approach, phosphorylated peptides from receptors digested with various endoproteinases were selectively isolated on immobilized anti-phosphotyrosine antibodies and analyzed directly by MALDI mass spectrometry.

An Eph receptor regulates integrin activity through R-Ras.

The ability of integrins to mediate cell attachment to extracellular matrices and to blood proteins is regulated from inside the cell. Increased ligand-binding activity of integrins is critical for platelet aggregation upon blood clotting and for leukocyte extravasation to inflamed tissues. Decreased adhesion is thought to promote tumor cell invasion.

A novel signaling intermediate, SHEP1, directly couples Eph receptors to R-Ras and Rap1A.

The Eph family of receptor tyrosine kinases has been implicated in many developmental patterning processes, including cell segregation, cell migration, and axon guidance. The cellular components involved in the signaling pathways of the Eph receptors, however, are incompletely characterized. Using a yeast two-hybrid screen, we have identified a novel signaling intermediate, SHEP1 (SH2 domain-containing Eph receptor-binding protein 1), which is expressed in the embryonic and adult brain.

Signal transfer by Eph receptors.

The Eph receptors are a unique family of receptor tyrosine kinases that enforce cellular position in tissues through mainly repulsive signals generated upon cell-cell contact. Together, Eph receptors and their membrane-anchored ligands. the ephrins, are key molecules for establishing tissue organization through signaling pathways that control axonal projection, cell migration, and the maintenance of cellular boundaries.

Signal transfer by eph receptors.

The Eph receptors are a unique family of receptor tyrosine kinases that enforce cellular position in tissues through mainly repulsive signals generated upon cell-cell contact. Together, Eph receptors and their membrane-anchored ligands, the ephrins, are key molecules for establishing tissue organization through signaling pathways that control axonal projection, cell migration, and the maintenance of cellular boundaries. Through their SH2 (Src Homology 2) and PDZ (postsynaptic density protein, disks large, zona occludens) domains, several signaling molecules have been demonstrated to interact with the activated cytoplasmic domain of Eph receptors by using the yeast two-hybrid system and in vitro biochemical assays.

Eph receptors and ephrins demarcate cerebellar lobules before and during their formation.

The formation of the ten cerebellar lobules is an unsolved problem in brain development. We report a screen for the four subfamilies of Eph receptors and their ligands (ephrins) in developing mouse cerebellum, using soluble receptor-immunoglobulin and ligand-immunoglobulin fusion proteins, and antibodies against EphA and ephrin-B proteins. Our results identify Eph receptors and ephrins as the first molecules known to demarcate individual lobules during development.

[Gastric pull-up in pharyngo-esophageal reconstruction: a personal experience].

During the five-year period from 1992 to 1997 a total of 62 patients with advanced hypopharyngeal carcinoma extended to the cervical esophagus came to our attention. Of these 42 (67.7%) were deemed operable and of these 31 (23 males, 8 females; age range 48 to 74 years; mean age 58.

Substance P and central respiratory activity: a comparative in vitro study on foetal and newborn rat.

Experiments were performed in vitro on foetal (embryonic days 18 to 21, E18-21) and newborn rat (postnatal days 0 to 3, P0-3) brainstem spinal cord preparations to analyse the perinatal developmental changes in the effects induced by substance P. Superfusion of the preparations with SP-containing artificial cerebrospinal fluid (aCSF) induced significant increase in the respiratory frequency of newborn rats (10-9 M), whereas concentration up to 10-7 M induced no change in foetal preparations. A whole cell patch clamp approach was used to record intracellularly from phrenic motoneurones.

Complex formation between EphB2 and Src requires phosphorylation of tyrosine 611 in the EphB2 juxtamembrane region.

The cellular components of the neuronal signaling pathways of Eph receptor tyrosine kinases are only beginning to be elucidated. Here we show that in vivo tyrosine phosphorylation sites of the Eph receptors EphA3, EphA4, and EphB2 in embryonic retina serve as binding sites for the Src-homology 2 (SH2) domain of Src kinase. Furthermore, tyrosine-phosphorylated EphB2 was detected in Src immunoprecipitates from transfected Cos cells, indicating that EphB2 and Src can physically associate.

Expression and tyrosine phosphorylation of Eph receptors suggest multiple mechanisms in patterning of the visual system.

The EphA3 receptor tyrosine kinase has been implicated in guiding the axons of retinal ganglion cells as they extend in the optic tectum. A repulsive mechanism involving opposing gradients of the EphA3 receptor on retinal axons and its ligands, ephrin-A2 and ephrin-A5, in the tectum influences topographic mapping of the retinotectal projection. To investigate the overall role of the Eph family in patterning of the visual system, we have used in situ hybridization to localize nine Eph receptors in the chicken retina and optic tectum at Embryonic Day 8.

Immunolocalization of the receptor tyrosine kinase EphA4 in the adult rat central nervous system.

EphA4 is a receptor tyrosine kinase of the Eph family previously designated Cek8 in chicken, Tyro1 in rat, and Sek1 in mouse, which is preferentially expressed in the embryonic and adult nervous system. We have mapped the distribution of EphA4 in the adult rat brain and spinal cord using a polyclonal antibody raised against a synthetic carboxy-terminal peptide. Immunoblotting experiments revealed that EphA4 is widely distributed in various regions of the adult rat brain.

The Eph family of receptors.

Eph receptor tyrosine kinases have recently been identified as instructive molecules that guide the topographic movement of cells and growth cones. The activation of Eph receptors by their ligands, which are membrane-anchored molecules, involves a cell-cell recognition event that often causes cell repulsion. Therefore, Eph receptors mediate signals that can override cell adhesion.

The Eph family: a multitude of receptors that mediate cell recognition signals.

The Eph receptor tyrosine kinases are emerging as molecules that guide the migration of cells and growth cones during embryonic development. Based on their concentration in embryonic regions containing growing neuronal processes, the Eph receptors were suspected early on to have a role in regulating aspects of axon growth. The most distinctive role of the Eph receptors appears to be their ability to mediate cell-cell repulsion through the binding of a ligand on an adjacent cell surface.

Serotonergic inhibition of phrenic motoneuron activity: an in vitro study in neonatal rat.

In vitro experiments were conducted on neonatal rat brainstem-spinal cord preparations to test the hypothesis of an inhibitory modulation of phrenic activity by serotonin (5-HT) via non-5-HT2A receptors [Lindsay, A.D. and Feldman, J.

Shared and distinct functions of RAGS and ELF-1 in guiding retinal axons.

Two ligands for Eph-related receptor tyrosine kinases, RAGS and ELF-1, have been implicated in the control of development of the retinotectal projection. Both molecules are expressed in overlapping gradients in the tectum, the target area of retinal ganglion cell axons. In two in vitro assays ELF-1 is shown to have a repellent axon guidance function for temporal, but apparently not for nasal axons.

Tyrosine phosphorylation of L1 family adhesion molecules: implication of the Eph kinase Cek5.

The L1 family comprises transmembrane cell adhesion molecules of the immunoglobulin superfamily that play an important role in neuronal migration and axon outgrowth, fasciculation, and myelination. Consistent with a crucial role in developmental processes, mutations in L1 cause severe brain malformations. Although L1 activates intracellular signaling pathways, little is known about the membrane proximal events of L1 signaling.

Tyrosine phosphorylation of transmembrane ligands for Eph receptors.

Axonal pathfinding in the nervous system is mediated in part by cell-to-cell signaling events involving members of the Eph receptor tyrosine kinase (RTK) family and their membrane-bound ligands. Genetic evidence suggests that transmembrane ligands may transduce signals in the developing embryo. The cytoplasmic domain of the transmembrane ligand Lerk2 became phosphorylated on tyrosine residues after contact with the Nuk/Cek5 receptor ectodomain, which suggests that Lerk2 has receptorlike intrinsic signaling potential.

Reciprocal expression of the Eph receptor Cek5 and its ligand(s) in the early retina.

Recent evidence suggests that Eph receptor tyrosine kinases and their ligands provide positional information in the developing visual system. We previously found that the Eph receptor Cek5 is more highly expressed in the ventral than dorsal chicken embryonic retina. We now report the identification of a chicken ligand for Cek5 (cCek5-L) that is 75% identical to the ligand LERK2.

Increased excitability and inward rectification in layer V cortical pyramidal neurons in the epileptic mutant mouse Stargazer.

The excitability of layer V cortical pyramidal neurons was studied in vitro in the single-locus mutant mouse stargazer (stg), a genetic model of spike wave epilepsy. Field recordings in neocortical slices from mutant mice bathed in artificial cerebrospinal fluid revealed spontaneous synchronous network discharges that were never present in wild-type slices. Intracellular and whole cell recordings from stg/stg neurons in deep layers showed spontaneous giant depolarizing excitatory post-synaptic potentials generating bursts of action potentials, and a 78% reduction in the afterburst hyperpolarization.

Developmental expression and distinctive tyrosine phosphorylation of the Eph-related receptor tyrosine kinase Cek9.

Cek9 is a receptor tyrosine kinase of the Eph subfamily for which only a partial cDNA sequence was known (Sajjadi, F.G., and E.

B61, a ligand for the Eck receptor protein-tyrosine kinase, exhibits neurotrophic activity in cultures of rat spinal cord neurons.

Although the Eph subfamily represents the largest group of receptor protein-tyrosine kinases, the biological roles of the Eph-related receptors and their ligands are not well understood. B61 has been identified recently by receptor affinity chromatography as a ligand for the Eph-related receptor Eck (Bartley et al.: Nature 368:558-560, 1994).

Perinatal developmental changes in respiratory activity of medullary and spinal neurons: an in vitro study on fetal and newborn rats.

Experiments were performed in vitro on fetal and newborn rat brainstem-spinal cord preparations to analyse the perinatal developmental changes in inspiratory motor output. The amplitude of the inspiratory bursts of the whole C4 ventral root (global extracellular recording), the firing patterns of 80 medullary inspiratory neurons (unitary extracellular recording) and the firing and membrane properties of 71 respiratory neurons in the C4 ventral horn (whole-cell recording) were analysed at embryonic day 18 (E18), 21 (E21) and post natal days 0 to 3 (P0-3). At E18, the amplitude of the C4 bursts was weak and variable from one respiratory cycle to the next, as well as the discharge pattern of most of the medullary inspiratory neurons.

Genomic organization and alternatively processed forms of Cek5, a receptor protein-tyrosine kinase of the Eph subfamily.

The genomic organization of Cek5, a receptor tyrosine kinase of the Eph subfamily, was elucidated utilizing a strategy involving PCR amplification of Cek5 genomic DNA. Cek5 is the first Eph-related kinase for which the exon-intron structure of the entire coding region has been determined. The Cek5 gene spans over 35 kb and comprises at least 16 exons.

Polarized expression of the receptor protein tyrosine kinase Cek5 in the developing avian visual system.

Receptor protein tyrosine kinases of the Eph subfamily have been proposed to play roles in pattern formation based on their distribution during embryonic development. Cek5 (chicken embryo kinase 5) and Cek8 (chicken embryo kinase 8) are Eph-related kinases highly expressed in the chicken embryonic retina. To assess their potential roles in the development of the visual pathway, we examined their distribution by immunoperoxidase labeling.