Ephrins stimulate neurite outgrowth during early cortical neurogenesis.

The Eph receptor ligands, the ephrins, are membrane-bound molecules that play important roles in establishing intercellular communication after neurogenesis by regulating cell migration, axon pathfinding, and topographic mapping. In diverse systems, such as embryonic day 17.5 (E17.5) hippocampal and cortical neurons, repulsive/inhibitory mechanisms underlie these cellular effects. However, although ligand/receptor expression occurs far earlier, during brain neurogenesis, little is known about potential ephrin functions in initial process outgrowth. We have examined ligand/receptor expression in E13.5 cortex in vivo and in culture, using alkaline phosphatase (AP)-conjugated reagents and RNase protection assay. B ephrins are highly expressed, including B1, B2, and B3, whereas A ephrins exhibit low expression levels. In contrast, the Eph receptors demonstrate an opposite pattern, exhibiting high levels of Eph A3, A4, and A5 mRNA transcripts and low levels of the B-class receptors. To examine effects on neurite outgrowth, soluble ephrins were incubated with antihuman IgG antibody, producing oligomeric agonist complexes, and dried onto culture dishes. Unexpectedly, both ephrin A and B complexes increased process outgrowth: Seventy to eighty percent of neuronal precursors exhibited long neurites on ephrins, whereas only 5-10% of cells had neurites on IgG control substrates, indicating that ephrins stimulated neuritogenesis by early cortical neurons. These observations suggest that ephrin ligand/receptor systems play ontogenetic roles not previously considered, activating mechanisms other than cellular repulsion. Ephrin systems may induce initial process elaboration by early cortical neurons that is restricted at later stages by well-characterized repulsive signaling mechanisms.

Functional analysis of the domain structure of tumor necrosis factor-alpha converting enzyme.

Many membrane-bound proteins, including cytokines, receptors, and growth factors, are proteolytically cleaved to release a soluble form of their extracellular domain. The tumor necrosis factor (TNF)-alpha converting enzyme (TACE/ADAM-17) is a transmembrane metalloproteinase responsible for the proteolytic release or "shedding" of several cell-surface proteins, including TNF and p75 TNFR. We established a TACE-reconstitution system using TACE-deficient cells co-transfected with TACE and substrate cDNAs to study TACE function and regulation. Using the TACE-reconstitution system, we identified two additional substrates of TACE, interleukin (IL)-1R-II and p55 TNFR. Using truncations and chimeric constructs of TACE and another ADAM family member, ADAM-10, we studied the function of the different domains of TACE in three shedding activities. We found that TACE must be expressed with its membrane-anchoring domain for phorbol ester-stimulated shedding of TNF, p75 TNFR, and IL-1R-II, but that the cytoplasmic domain is not required for the shedding of these substrates. The catalytic domain of ADAM-10 could not be functionally substituted for that of TACE. IL-1R-II shedding required the cysteine-rich domain of TACE as well as the catalytic domain, whereas TNF and p75 TNFR shedding required only the tethered TACE catalytic domain.

Spinal motor axons and neural crest cells use different molecular guides for segmental migration through the rostral half-somite.

The peripheral nervous system in vertebrates is composed of repeating metameric units of spinal nerves. During development, factors differentially expressed in a rostrocaudal pattern in the somites confine the movement of spinal motor axons and neural crest cells to the rostral half of the somitic sclerotome. The expression patterns of transmembrane ephrin-B ligands and interacting EphB receptors suggest that these proteins are likely candidates for coordinating the segmentation of spinal motor axons and neural crest cells. In vitro, ephrin-B1 has indeed been shown to repel axons extending from the rodent neural tube (Wang & Anderson, 1997). In avians, blocking interactions between EphB3 expressed by neural crest cells and ephrin-B1 localized to the caudal half of the somite in vivo resulted in loss of the rostrocaudal patterning of trunk neural crest migration (Krull et al., 1997). The role of ephrin-B1 in patterning spinal motor axon outgrowth in avian embryos was investigated. Ephrin-B1 protein was found to be expressed in the caudal half-sclerotome and in the dermomyotome at the appropriate time to interact with the EphB2 receptor expressed on spinal motor axons. Treatment of avian embryo explants with soluble ephrin-B1, however, did not perturb the segmental outgrowth of spinal motor axons through the rostral half-somite. In contrast, under the same treatment conditions with soluble ephrin-B1, neural crest cells migrated aberrantly through both rostral and caudal somite halves. These results indicate that the interaction between ephrin-B1 and EphB2 is not required for patterning spinal motor axon segmentation. Even though spinal motor axons traverse the same somitic pathway as neural crest cells, different molecular guidance mechanisms appear to influence their movement.

Selective inhibition of spinal cord neurite outgrowth and cell survival by the Eph family ligand ephrin-A5.

The Eph family tyrosine kinase receptors and their ligands, the ephrins, have been shown to play critical roles in cell migration, tissue morphogenesis, and axonal guidance in many different systems. However, their function in the spinal cord has not been examined carefully. We showed in this study that several Eph receptors, including EphA3, Eph A4, and Eph A5, are expressed in the ventral spinal cord in partially overlapping patterns, with EphA5 exhibiting the most widespread transcription in the entire ventral spinal cord during early development. Complementary to the receptor expression, a ligand of these receptors, ephrin-A5, is transcribed in the dorsal half of the spinal cord. Consistent with the spatial location of receptor expression, the ligand selectively inhibits neurite outgrowth and induces cell death of the ventral, but not the dorsal, spinal cord neurons. These observations suggest that interactions between the Eph family receptors and ligands exerts negative influences on ventral spinal cord neurons and thus may play important roles in regulating morphogenesis and axon guidance in the spinal cord.

The identification of seven metalloproteinase-disintegrin (ADAM) genes from genomic libraries.

Metalloproteinase-disintegrins (ADAMs) are membrane-spanning multi-domain proteins containing a zinc metalloproteinase domain and a disintegrin domain which may serve as an integrin ligand. Based on a conserved sequence within the disintegrin domain, GE(E/Q)CDCG, seven genes were isolated from a human genomic library. Two of these genes lack introns and show testis-specific expression (ADAM20 and ADAM21), while the other two genes contain introns (ADAM22 and ADAM23) and are expressed predominantly in the brain. In addition, three pseudogenes were isolated; one of which evolved from ADAM21. Human chromosomal mapping indicated that ADAM22 and ADAM23 mapped to chromosome 7q21 and 2q33, respectively, while the three pseudogenes 1-2, 3-3, and 1-32 mapped to chromosome 14q24.1, 8p23, and 14q24.1, respectively. An ancestral analysis of all known ADAMs indicates that the zinc-binding motif in the catalytic domain arose once in a common ancestor and was lost by those members lacking this motif.

Isolation of two novel metalloproteinase-disintegrin (ADAM) cDNAs that show testis-specific gene expression.

Metalloproteinase-disintegrins (ADAMs) are type 1 transmembrane proteins that contain a unique domain structure including a zinc-binding metalloproteinase domain. We have isolated cDNAs encoding two novel members of this family, ADAM29 and ADAM30 which show testis-specific expression. Three forms of ADAM29 were found that encode proteins of 820, 786 and 767 amino acids. All of the amino acid differences are located in the cytoplasmic domain. Two forms of ADAM30 were isolated that encode proteins of 790 and 781 amino acids, with the difference in the coding region occurring in the cytoplasmic domain. ADAM29 and ADAM30 map to human chromosome 4q34 and 1p11-13, respectively. An ancestral analysis of all known mammalian ADAMs indicates that the zinc-binding motif in the catalytic domain arose once in a common ancestor and was subsequently lost by those members lacking this motif.

Characterization of the cDNA and gene for mouse tumour necrosis factor alpha converting enzyme (TACE/ADAM17) and its location to mouse chromosome 12 and human chromosome 2p25.

Numerous proteins are cleaved or "shed" from their membrane-bound form. One such protein, tumour necrosis factor alpha (TNF-alpha), is synthesized as a type 2 transmembrane protein. Recently, a human protease responsible for this shedding, the TNF-alpha converting enzyme (TACE/ADAM17), was isolated. TACE/ADAM17 is a member of the adamalysin class of zinc-binding metalloproteases or ADAM (a disintegrin and metalloprotease). We report the isolation and characterization of the mouse TACE/ADAM17 cDNA and gene. Mouse TACE/ADAM17 has a 92% amino-acid identity with the human protein and was ubiquitously expressed. A recombinant form of the protease is found to cleave a peptide representing the cleavage site of precursor mouse TNF-alpha. An alternatively spliced form of mouse TACE/ADAM17 was found that would produce a soluble protein. The gene for TACE/ADAM17 is approximately 50 kb and contains 19 exons. Chromosomal mapping places TACE/ADAM17 on mouse chromosome 12 and human chromosome 2p25.

Surface densities of ephrin-B1 determine EphB1-coupled activation of cell attachment through alphavbeta3 and alpha5beta1 integrins.

Receptors of the Eph family and their ligands (ephrins) mediate developmental vascular assembly and direct axonal guidance. Migrating cell processes identify appropriate targets within migratory fields based on topographically displayed ephrin gradients. Here, EphB1 regulated cell attachment by discriminating the density at which ephrin-B1 was displayed on a reconstituted surface. EphB1-ephrin-B1 engagement did not promote cell attachment through mechanical tethering, but did activate integrin-mediated attachment. In endothelial cells, attachment to RGD peptides or fibrinogen was mediated through alphavbeta3 integrin. EphB1 transfection conferred ephrin-B1-responsive activation of alpha5beta1 integrin-mediated cell attachment in human embryonic kidney cells. Activation-competent but signaling-defective EphB1 point mutants failed to stimulate ephrin-B1 dependent attachment. These findings lead us to propose that EphB1 functions as a 'ligand density sensor' to signal integrin-mediated cell-matrix attachment.

Specification of distinct dopaminergic neural pathways: roles of the Eph family receptor EphB1 and ligand ephrin-B2.

Dopaminergic neurons in the substantia nigra and ventral tegmental area project to the caudate putamen and nucleus accumbens/olfactory tubercle, respectively, constituting mesostriatal and mesolimbic pathways. The molecular signals that confer target specificity of different dopaminergic neurons are not known. We now report that EphB1 and ephrin-B2, a receptor and ligand of the Eph family, are candidate guidance molecules for the development of these distinct pathways. EphB1 and ephrin-B2 are expressed in complementary patterns in the midbrain dopaminergic neurons and their targets, and the ligand specifically inhibits the growth of neurites and induces the cell loss of substantia nigra, but not ventral tegmental, dopaminergic neurons. These studies suggest that the ligand-receptor pair may contribute to the establishment of distinct neural pathways by selectively inhibiting the neurite outgrowth and cell survival of mistargeted neurons. In addition, we show that ephrin-B2 expression is upregulated by cocaine and amphetamine in adult mice, suggesting that ephrin-B2/EphB1 interaction may play a role in drug-induced plasticity in adults as well.

Ephrin-dependent growth and pruning of hippocampal axons.

Neuronal connections are arranged topographically such that the spatial organization of neurons is preserved by their termini in the targets. During the development of topographic projections, axons initially explore areas much wider than the final targets, and mistargeted axons are pruned later. The molecules regulating these processes are not known. We report here that the ligands of the Eph family tyrosine kinase receptors may regulate both the initial outgrowth and the subsequent pruning of axons. In the presence of ephrins, the outgrowth and branching of the receptor-positive hippocampal axons are enhanced. However, these axons are induced later to degenerate. These observations suggest that the ephrins and their receptors may regulate topographic map formation by stimulating axonal arborization and by pruning mistargeted axons.

An essential role for ectodomain shedding in mammalian development.

The ectodomains of numerous proteins are released from cells by proteolysis to yield soluble intercellular regulators. The responsible protease, tumor necrosis factor-alpha converting enzyme (TACE), has been identified only in the case when tumor necrosis factor-alpha (TNFalpha) is released. Analyses of cells lacking this metalloproteinase-disintegrin revealed an expanded role for TACE in the processing of other cell surface proteins, including a TNF receptor, the L-selectin adhesion molecule, and transforming growth factor-alpha (TGFalpha). The phenotype of mice lacking TACE suggests an essential role for soluble TGFalpha in normal development and emphasizes the importance of protein ectodomain shedding in vivo.

Evidence that tumor necrosis factor alpha converting enzyme is involved in regulated alpha-secretase cleavage of the Alzheimer amyloid protein precursor.

The amyloid protein, Abeta, which accumulates in the brains of Alzheimer patients, is derived by proteolysis of the amyloid protein precursor (APP). APP can undergo endoproteolytic processing at three sites, one at the amino terminus of the Abeta domain (beta-cleavage), one within the Abeta domain (alpha-cleavage), and one at the carboxyl terminus of the Abeta domain (gamma-cleavage). The enzymes responsible for these activities have not been unambiguously identified. By the use of gene disruption (knockout), we now demonstrate that TACE (tumor necrosis factor alpha converting enzyme), a member of the ADAM family (a disintegrin and metalloprotease-family) of proteases, plays a central role in regulated alpha-cleavage of APP. Our data suggest that TACE may be the alpha-secretase responsible for the majority of regulated alpha-cleavage in cultured cells. Furthermore, we show that inhibiting this enzyme affects both APP secretion and Abeta formation in cultured cells.

Regulation of thalamic neurite outgrowth by the Eph ligand ephrin-A5: implications in the development of thalamocortical projections.

The cerebral cortex is parcellated into different functional domains that receive distinct inputs from other cortical and subcortical regions. The molecular mechanisms underlying the specificity of connections of cortical afferents remain unclear. We report here that the Eph family tyrosine kinase receptor EphA5 and the ligand ephrin-A5 may play a key role in the exclusion of the limbic thalamic afferents from the sensorimotor cortex by mediating repulsive interactions. In situ hybridization shows that the EphA5 transcript is expressed at high levels in both cortical and subcortical limbic regions, including the frontal cortex, the subiculum, and the medial thalamic nuclei. In contrast, ephrin-A5 is transcribed abundantly in the sensorimotor cortex. Consistent with the complementary expression, the ligand inhibited dramatically the growth of neurites from neurons isolated from the medial thalamus but was permissive for the growth of neurites from lateral thalamic neurons, which is primarily nonlimbic. Similarly, the growth of neurites from Eph-A5-expressing neurons isolated from the subiculum was inhibited by ephrin-A5. Our studies suggest that the Eph family ligand ephrin-A5 serves as a general inhibitor of axonal growth from limbic neurons, which may serve to prevent innervation of inappropriate primary sensorimotor regions, thus contributing to the generation of specificity of thalamic cortical afferents.

Expression and purification of correctly processed, active human TACE catalytic domain in Saccharomyces cerevisiae.

Human tumor necrosis factor-alpha (TNF alpha) converting enzyme (TACE) releases soluble TNF alpha from cells. It is a member of the adamalysin family of metalloproteases. A truncated form of TACE cDNA was expressed in Saccharomyces cerevisiae and purified to homogeneity in order to study TACE structure and function. Recombinant TACE was expressed as a preproprotein including the pro- and catalytic (PROCAT) domains fused to the yeast alpha-factor leader. A C-terminal immunoreactive FLAG peptide was added for Western blot detection and anti-FLAG antibody column purification. We constructed two glycosylation mutant PROCAT TACE isoforms to facilitate purification. A PROCAT isoform, mutated to eliminate two N-linked glycosylation sites, was buffer exchanged and purified to homogeneity by ion exchange chromatography and an anti-FLAG antibody affinity step. N-terminal sequence analysis showed that the mutant preproprotein was processed in yeast at the furin protease cleavage site and yielded an active catalytic domain which has TNF alpha peptide-specific protease activity. Mass spectrometry of the purified catalytic domain showed that removal of both N-linked sites results in a homogeneous sized polypeptide lacking further posttranslational modifications.

Crystal structure of the catalytic domain of human tumor necrosis factor-alpha-converting enzyme.

Tumor necrosis factor-alpha (TNFalpha) is a cytokine that induces protective inflammatory reactions and kills tumor cells but also causes severe damage when produced in excess, as in rheumatoid arthritis and septic shock. Soluble TNFalpha is released from its membrane-bound precursor by a membrane-anchored proteinase, recently identified as a multidomain metalloproteinase called TNFalpha-converting enzyme or TACE. We have cocrystallized the catalytic domain of TACE with a hydroxamic acid inhibitor and have solved its 2.0 A crystal structure. This structure reveals a polypeptide fold and a catalytic zinc environment resembling that of the snake venom metalloproteinases, identifying TACE as a member of the adamalysin/ADAM family. However, a number of large insertion loops generate unique surface features. The pro-TNFalpha cleavage site fits to the active site of TACE but seems also to be determined by its position relative to the base of the compact trimeric TNFalpha cone. The active-site cleft of TACE shares properties with the matrix metalloproteinases but exhibits unique features such as a deep S3' pocket merging with the S1' specificity pocket below the surface. The structure thus opens a different approach toward the design of specific synthetic TACE inhibitors, which could act as effective therapeutic agents in vivo to modulate TNFalpha-induced pathophysiological effects, and might also help to control related shedding processes.

Eph receptors discriminate specific ligand oligomers to determine alternative signaling complexes, attachment, and assembly responses.

Eph family receptor tyrosine kinases (including EphA3, EphB4) direct pathfinding of neurons within migratory fields of cells expressing gradients of their membrane-bound ligands. Others (EphB1 and EphA2) direct vascular network assembly, affecting endothelial migration, capillary morphogenesis, and angiogenesis. To explore how ephrins could provide positional labels for cell targeting, we tested whether endogenous endothelial and P19 cell EphB1 (ELK) and EphB2 (Nuk) receptors discriminate between different oligomeric forms of an ephrin-B1/Fc fusion ligand. Receptor tyrosine phosphorylation was stimulated by both dimeric and clustered multimeric ephrin-B1, yet only ephrin-B1 multimers (tetramers) promoted endothelial capillary-like assembly, cell attachment, and the recruitment of low-molecular-weight phosphotyrosine phosphatase (LMW-PTP) to receptor complexes. Cell-cell contact among cells expressing both EphB1 and ephrin-B1 was required for EphB1 activation and recruitment of LMW-PTP to EphB1 complexes. The EphB1-binding site for LMW-PTP was mapped and shown to be required for tetrameric ephrin-B1 to recruit LMW-PTP and to promote attachment. Thus, distinct EphB1-signaling complexes are assembled and different cellular attachment responses are determined by a receptor switch mechanism responsive to distinct ephrin-B1 oligomers.

CD30 and its ligand: possible role in regulation of teratoma stem cells.

Like the oocyte, the cells of the early embryo, and primordial germ cells, human teratocarcinoma stem cells are pluripotent, capable of giving rise to a wide range of somatic and extraembryonic tissues. Growth factors which regulate the growth of multipotent stem cells in the mouse have been identified, but none of these have been shown conclusively to have similar effects on human or primate multipotent stem cells. CD30 is a member of the tumour necrosis factor receptor superfamily with a restricted pattern of tissue distribution, limited to immune cells, decidual tissue, and human embryonal carcinoma: in common with other embryonal carcinoma markers, CD30 is found in foci of cells in a sub-population of seminomas. CD30 ligand is a transmembrane protein, structurally related to tumour necrosis superfamily members TNF alpha, TNF beta, and CD40. CD30 ligand is expressed by T and B lymphocytes, macrophages, and a variety of normal haematopoietic cells and tumours derived from them, and exerts pleiotropic effects on normal and malignant lymphoid cells, including death, differentiation, or cell division. Studies on cultured cell lines derived from human embryonal carcinomas and yolk sac carcinomas confirm CD30 expression in the former but not the latter, and show that CD30 expression is down-regulated during stem cell differentiation in vitro. Transcripts for CD30 ligand are found at highest levels in yolk sac carcinoma cell lines, but are also found in embryonal carcinoma. CD30 ligand protein is detected in yolk sac carcinoma and nullipotent embryonal carcinoma cell lines. Exogenous CD30 ligand has no effect on multipotent human stem cell growth in vitro. However, the receptor-ligand pair may function in autocrine regulation of embryonal carcinoma stem cells. CD30 and its ligand are candidate stem cell identity factors, juxtacrine regulators whose sole function is to identify a cell's position in a developmental hierarchy.

Characterization of the genes for mouse LERK-3/Ephrin-A3 (Epl3), mouse LERK-4/Ephrin-A4 (Epl4), and human LERK-6/Ephrin-A2 (EPLG6): conservation of intron/exon structure.

We have isolated the genes for the eph receptor family ligands mouse LERK-3/Ephrin-A3 (Epl3), mouse LERK-4/Ephrin-A4 (Epl4), and human LERK-6/Ephrin-A2 (EPLG6). These genes show a high level of conservation in their intron/exon structures encoding the receptor-binding region. In addition, the nucleotide sequences of the genes reveal the predicted cDNA sequence of mouse LERK-3/Ephrin-A3, mouse LERK-4/Ephrin-A4, and human LERK-6/Ephrin-A2.

Nck recruitment to Eph receptor, EphB1/ELK, couples ligand activation to c-Jun kinase.

Eph family receptor tyrosine kinases signal axonal guidance, neuronal bundling, and angiogenesis; yet the signaling systems that couple these receptors to targeting and cell-cell assembly responses are incompletely defined. Functional links to regulators of cytoskeletal structure are anticipated based on receptor mediated cell-cell aggregation and migratory responses. We used two-hybrid interaction cloning to identify EphB1-interactive proteins. Six independent cDNAs encoding the SH2 domain of the adapter protein, Nck, were recovered in a screen of a murine embryonic library. We mapped the EphB1 subdomain that binds Nck and its Drosophila homologue, DOCK, to the juxtamembrane region. Within this subdomain, Tyr594 was required for Nck binding. In P19 embryonal carcinoma cells, activation of EphB1 (ELK) by its ligand, ephrin-B1/Fc, recruited Nck to native receptor complexes and activated c-Jun kinase (JNK/SAPK). Transient overexpression of mutant EphB1 receptors (Y594F) blocked Nck recruitment to EphB1, attenuated downstream JNK activation, and blocked cell attachment responses. These findings identify Nck as an important intermediary linking EphB1 signaling to JNK.