Sema Domain Research Articles

Semaphorins and their receptors in lamprey CNS: Cloning, phylogenetic analysis, and developmental changes during metamorphosis

The large, conserved semaphorin gene family encodes axon guidance molecules in both invertebrates and vertebrates. The primitive vertebrate lamprey diverged near the time of vertebrate origins and is useful for understanding the gene duplication events that led to the increased complexity of the vertebrate genome. We characterized the sequence and expression pattern of semaphorins and their receptors genes in the sea lamprey, Petromyzon marinus. We uncovered two members of the semaphorin family in sea lamprey. The first encodes a diffusible class 3 type semaphorin protein that is most similar to the human and mouse Sema3F (71% amino acid identity). The second encodes a transmembrane class 4 type semaphorin that is most similar to mouse Sema4D and human Sema4G, with 38% amino acid identity within the Sema domain. We also identified in lamprey two members of the semaphorin receptor family, lamprey Plexin A1 and Plexin A2. Phylogenetic analysis indicates that lamprey Sema3 and Sema4 represent precursor genes existing prior to the origin of the vertebrate Sema3A-G and Sema4A-G subfamilies. Therefore, the gene duplication event that gave rise to those subfamilies must have occurred after the divergence of jawed vertebrates from jawless fish. These semaphorins and plexins are expressed in unique and dynamic patterns in lamprey spinal cord and brain during development.

Structure determination of human semaphorin 4D as an example of the use of MAD in non-optimal cases

Semaphorins are an important class of signalling molecules involved in axon guidance, immune function and angiogenesis. They are characterized by having an extracellular sema domain of about 500 residues. The steps involved in the determination of the structure of human semaphorin 4D are described here as a case study of selenium MAD phasing in a difficult case with low symmetry, moderate diffraction and low selenium content. A particular feature of this study was the large number of diffraction images required to give data of sufficient quality for structure determination and these data are re-analyzed here to investigate the effects of radiation damage on eventual data quality and to suggest strategies for successful MAD phasing in similar difficult cases.

Plexin B3 promotes neurite outgrowth, interacts homophilically, and interacts with Rin

BackgroundPlexins, known to date as receptors of semaphorins, are implicated in semaphorin-mediated axon repulsion and growth cone collapse. However, subtype-specific functions of the majority of the nine members of the mammalian plexin family are largely unknown. In order to investigate functional properties of B-plexins, we analyzed the expression of human and murine plexin B3 and expressed full-length human plexins B2 (B2) and B3 (B3) in NIH-3T3 cells.ResultsUnexpectedly, B3 strongly and B2 moderately stimulate neurite outgrowth of primary murine cerebellar neurons. Both plexins mediate Ca2+/Mg2+-dependent cell aggregation due to homophilic trans-interaction, which is strong in the case of B3 and moderate for B2. Using different deletion constructs we show that the sema domain of B3 is essential for homophilic interaction. Using yeast two-hybrid analysis, we identified the neuron-specific and calmodulin-binding Ras-related GTPase Rin as an interaction partner of the intracellular part of B3, but not of B2. Rin, also known for its neurite outgrowth-inducing characteristics, co-localizes and co-immunoprecipitates with B3 in co-transfected COS-7 cells.ConclusionOur data suggest an involvement of homophilic interaction of B3 in semaphorin-independent signaling mechanisms positively influencing neuronal morphogenesis or function. Furthermore the neuron-specific small GTPase Rin is involved in downstream signaling of plexin B3.

Crystal structure of the HGF β-chain in complex with the sema domain of the Met receptor

Crystal structure of the HGF β-chain in complex with the sema domain of the Met receptor

Requirement of the transmembrane semaphorin Sema4C for myogenic differentiation

Semaphorins constitute a large family of signaling proteins that contribute to axonal guidance. Here we demonstrate that the transmembrane semaphorin Sema4C is up-regulated both in the early stage of differentiation of C2C12 mouse skeletal myoblasts into myotubes and during injury-induced muscle regeneration in vivo. Depletion of Sema4C in C2C12 cells resulted in marked attenuation of myotube formation. A fusion protein containing the extracellular Sema domain and a peptide corresponding to the intracellular COOH-terminal region of Sema4C each inhibited the differentiation of C2C12 cells. These findings indicate that Sema4C-mediated interaction among myoblasts plays an important role in terminal myogenic differentiation.

Class IV semaphorins promote angiogenesis by stimulating Rho-initiated pathways through plexin-B.

The semaphorins are a large family of secreted and cell surface proteins that provide attractive and repulsive cues for axon guidance during neuronal development. Semaphorins share a conserved NH(2)-terminal Sema domain with their receptors, the plexins, which mediate neuronal cell adhesion, axon guidance, and maintenance of established neuronal pathways in the adult. Both semaphorins and plexins share structural homology with the extracellular domain of c-Met, a member of the scatter factor family of receptors. However, the highly conserved cytoplasmic region of plexins has no homology with the c-Met tyrosine kinase or with any other known protein. Using a recently developed antibody and RNA analysis, we found that high levels of plexin-B1 are expressed in endothelial cells. Whereas c-Met, with which plexin-B1 can interact, is known to be a potent promoter of angiogenesis, the effects of semaphorin-mediated plexin activation in endothelial cells are still poorly understood. Here, we examined the role of plexin-B1 activation in angiogenesis using a purified, secreted form of its ligand, Semaphorin 4D (Sema4D). Sema4D potently induced chemotaxis and tubulogenesis in endothelial cells and enhanced blood vessel formation in an in vivo mouse model. Interestingly, responses to Sema4D did not require c-Met activation. Instead, the use of chimeric plexin-B1 receptors, Rho inhibitors, and lentiviral gene delivery of interfering molecules revealed that these proangiogenic effects are dependent on a COOH-terminal PDZ-binding motif of plexin-B1, which binds two guanine nucleotide exchange factors for the small GTPase Rho, PDZ-RhoGEF and LARG, and are mediated by the activation of Rho-initiated pathways.

Insights into function of PSI domains from structure of the Met receptor PSI domain

PSI domains are cysteine-rich modules found in extracellular fragments of hundreds of signaling proteins, including plexins, semaphorins, integrins, and attractins. Here, we report the solution structure of the PSI domain from the human Met receptor, a receptor tyrosine kinase critical for proliferation, motility, and differentiation. The structure represents a cysteine knot with short regions of secondary structure including a three-stranded antiparallel β-sheet and two α-helices. All eight cysteines are involved in disulfide bonds with the pattern consistent with that for the PSI domain from Sema4D. Comparison with the Sema4D structure identifies a structurally conserved core comprising the N-terminal half of the PSI domain. Interestingly, this part links adjacent SEMA and immunoglobulin domains in the Sema4D structure, suggesting that the PSI domain serves as a wedge between propeller and immunoglobulin domains and is responsible for the correct positioning of the ligand-binding site of the receptor.

The Sema domain of Met is necessary for receptor dimerization and activation

Hepatocyte growth factor (HGF) binds the extracellular domain and activates the Met receptor to induce mitogenesis, morphogenesis, and motility. The extracellular domain of Met is comprised of Sema, PSI, and four IPT subdomains. We investigated the contribution of these subdomains to Met receptor dimerization. Our observations indicate that the Sema domain is necessary for dimerization in addition to HGF binding. Treatment of Met-overexpressing tumor cells with recombinant Sema in the presence or absence of HGF results in decreased Met-mediated signal transduction, cell motility, and migration, behaving in a manner similar to an antagonistic anti-Met Fab. These data suggest that the Sema domain of Met may not only represent a novel anticancer therapeutic target but also acts as a biotherapeutic itself.

Crystal structure of the HGF beta-chain in complex with the Sema domain of the Met receptor.

The Met tyrosine kinase receptor and its ligand, hepatocyte growth factor (HGF), play important roles in normal development and in tumor growth and metastasis. HGF-dependent signaling requires proteolysis from an inactive single-chain precursor into an active alpha/beta-heterodimer. We show that the serine protease-like HGF beta-chain alone binds Met, and report its crystal structure in complex with the Sema and PSI domain of the Met receptor. The Met Sema domain folds into a seven-bladed beta-propeller, where the bottom face of blades 2 and 3 binds to the HGF beta-chain 'active site region'. Mutation of HGF residues in the area that constitutes the active site region in related serine proteases significantly impairs HGF beta binding to Met. Key binding loops in this interface undergo conformational rearrangements upon maturation and explain the necessity of proteolytic cleavage for proper HGF signaling. A crystallographic dimer interface between two HGF beta-chains brings two HGF beta:Met complexes together, suggesting a possible mechanism of Met receptor dimerization and activation by HGF.

The Soluble Sema Domain of the RON Receptor Inhibits Macrophage-stimulating Protein-induced Receptor Activation

RON is a receptor tyrosine kinase of the MET family that is involved in cell proliferation, cell survival, and cell motility in both normal and disease states. Macrophage-stimulating protein (MSP) is the RON ligand whose binding to RON causes receptor activation. RON is a trans-membrane heterodimer comprised of one alpha- and one beta-chain originating from a single-chain precursor and held together by several disulfide bonds. The intracellular part of RON contains the kinase domain and regulatory elements. The extracellular region is characterized by the presence of a sema domain (a stretch of approximately 500 amino acids with several highly conserved cysteine residues), a PSI (plexin, semaphorins, integrins) domain, and four immunoglobulin-like folds. Here we show that a soluble, secreted molecule representing the sema domain of RON (referred to as ron-sema) has a dominant negative effect on the ligand-induced receptor activation and is capable of inhibiting RON-dependent signaling pathways and cellular responses. Results suggest that the sema domain of RON participates in ligand binding by the full-length receptor. The ability of ron-sema to suppress growth of MSP-responsive cells in culture, including cancer cells, points to a potential therapeutic use of this molecule, and forced expression of it could potentially be used as a gene therapy tool for treating MSP-dependent types of cancer.

The ligand-binding face of the semaphorins revealed by the high-resolution crystal structure of SEMA4D.

Semaphorins, proteins characterized by an extracellular sema domain, regulate axon guidance, immune function and angiogenesis. The crystal structure of SEMA4D (residues 1-657) shows the sema topology to be a seven-bladed beta-propeller, revealing an unexpected homology with integrins. The sema beta-propeller contains a distinctive 77-residue insertion between beta-strands C and D of blade 5. Blade 7 is followed by a domain common to plexins, semaphorins and integrins (PSI domain), which forms a compact cysteine knot abutting the side of the propeller, and an Ig-like domain. The top face of the beta-propeller presents prominent loops characteristic of semaphorins. In addition to limited contact between the Ig-like domains, the homodimer is stabilized through extensive interactions between the top faces in a sector of the beta-propeller used for heterodimerization in integrins. This face of the propeller also mediates ligand binding in integrins, and functional data for semaphorin-receptor interactions map to the equivalent surface.

Redundant functions but temporal and regional regulation of two alternatively spliced isoforms of Semaphorin 3F in the nervous system

SEMA3F is a secreted semaphorin that affects axon and cell guidance in the developing nervous system, and is also thought to have anti-tumor activity. Two spliced forms of SEMA3F have been identified that differ by the insertion of 31 amino acids in the sema domain. Here, we investigated the bioactivity of these isoforms and show, using coculture and binding assays, that they share common axonal chemorepulsive properties and binding to neuropilin receptors. SEMA3F isoforms were also found to regulate endothelial cell morphology by remodeling lamellipodial protrusions. Although Sema3F expression globally decreased during mouse development, we noted an enrichment of the longest isoform at postnatal stages in some territories such as the brainstem and spinal cord. These results indicate that although functionally redundant in cell culture assays, Sema3F spliced forms are characterized in vivo by a temporal and regional specific regulation during maturation of the nervous system.

Transmembrane Sema4E Guides Branchiomotor Axons to Their Targets in Zebrafish

Class 4 semaphorins are a large class of transmembrane proteins that contain a sema domain and that are expressed in the CNS, but their in vivo neural function is unknown. In zebrafish, the epithelial cells that line the pharyngeal arches express Sema4E. Extension of branchiomotor axons along the mesenchymal cells bounded by these epithelial cells suggests that Sema4E may act as a repulsive guidance molecule to restrict the branchiomotor axons to the mesenchymal cells. To test this hypothesis, Sema4E was misexpressed in hsp70 promoter-regulated transgenic zebrafish in which sema4E was heat-inducible, and Sema4E was knocked down by injection of antisense morpholino oligonucleotides that acted specifically against Sema4E. Ubiquitous induction of Sema4E retarded outgrowth by the facial and gill branchiomotor axons significantly. Furthermore, outgrowth by gill motor axons was specifically inhibited when Sema4E-expressing transgenic cells were transplanted to their pathway in nontransgenic host embryos. Morpholino knockdown of Sema4E caused facial motor axons to defasciculate and follow aberrant pathways. These results show that Sema4E is repulsive for facial and gill motor axons and functions as a barrier for these axons within the pharyngeal arches.

Identification, Characterization, and Functional Study of the Two Novel Human Members of the Semaphorin Gene Family

We cloned two novel human transmembrane semaphorins, (HSA)SEMA6C and (HSA)SEMA6D, that belong to the class VI subgroup of the semaphorin family. The genes for SEMA6C and SEMA6D are mapped on chromosome 1q12-21.1 and 15q21.1, respectively. Among the adult tissues, SEMA6C is expressed only in skeletal muscle, whereas SEMA6D is expressed abundantly in kidney, brain, and placenta and moderately in the heart and skeletal muscles. During murine development, neither SEMA6C nor SEMA6D was expressed in embryonic day 10.5 (E10.5) embryos, but both were highly expressed in the areas of the lateral ventricle, the striatum, the wall of the midbrain, the pons/midbrain junction, and the choroid plexus of E13 embryos. Were neurons, neither axons nor astrocytes, highly expressed both semaphorins. Three isoforms of SEMA6C and five isoforms of SEMA6D derived from alternative splicing were identified, and their expression was regulated in a tissue- and development-dependent manner. Deletion analysis indicated that a sema domain and a PSI domain are integrally necessary for correct post-translation modification and subcellular localization. The extracellular domain of SEMA6C inhibited axonal extension of nerve growth factor-differentiated PC12 cells and induced the growth cone collapse of chicken dorsal root ganglion, rat hippocampal neurons, and rat cortical neurons in a dose-responsive manner. SEMA6D acted like SEMA6C except it had no significant effect on the growth cones of rat cortical neurons.

PlexinA1 Autoinhibition by the Plexin Sema Domain

Semaphorin 3A (Sema3A) binds to neuropilin-1 (NP1) and activates the transmembrane Plexin to transduce a repulsive axon guidance signal. Here, we show that Sema3 signals are transduced equally effectively by PlexinA1 or PlexinA2, but not by PlexinA3. Deletion analysis of the PlexinA1 ectodomain demonstrates that the sema domain prevents PlexinA1 activation in the basal state. Sema-deleted PlexinA1 is constitutively active, producing cell contraction, growth cone collapse, and inhibition of neurite outgrowth. The sema domain of PlexinA1 physically associates with the remainder of the PlexinA1 ectodomain and can reverse constitutive activation. Both the sema portion and the remainder of the ectodomain of PlexinA1 associate with NP1 in a Sema3A-independent fashion. Plexin A1 is autoinhibited by its sema domain, and Sema3A/NP1 releases this inhibition.

Cloning and Characterization of a Novel Class VI Semaphorin, Semaphorin Y

Semaphorins comprise a large family of proteins implicated in axonal guidance. We cloned a novel transmembrane semaphorin, semaphorin Y (Sema Y), which has a class VI sema domain. Sema Y shows growth cone collapsing activity on DRG neuronsin vitro,and the target regions of the DRG neurons expresssema YmRNA during development. Sema Y may be a stop signal for these neurons in their target areas. Interestingly,sema YmRNA was also detected in other neurons and their targets. Two isoforms of Sema Y derived from alternative splicing were identified and their expression was found to be regulated in a tissue- and age-dependent manner. Distribution ofsema YmRNA suggests that Sema Y might also be important during maintenance of axonal connections and/or differentiation and migration of cells. Sequence comparison among class VI semaphorins revealed two short conserved sequence stretches in their cytoplasmic domains, suggesting interaction of these semaphorins with a common intracellular component(s).

Neuropilin-2 Is a Receptor for Semaphorin IV Insight into the Structural Basis of Receptor Function and Specificity

Neuropilins bind secreted members of the semaphorin family of proteins. Neuropilin-1 is a receptor for Sema III. Here, we show that neuropilin-2 is a receptor for the secreted semaphorin Sema IV and acts selectively to mediate repulsive guidance events in discrete populations of neurons. neuropilin-2 and semaIV are expressed in strikingly complementary patterns during neurodevelopment. The extracellular complement-binding (CUB) and coagulation factor domains of neuropilin-2 confer specificity to the Sema IV repulsive response, and these domains of neuropilin-1 are necessary and sufficient for binding of the Sema III semaphorin (sema) domain. The coagulation factor domains alone are necessary and sufficient for binding of the Sema III immunoglobulin- (Ig-) basic domain and the unrelated ligand, vascular endothelial growth factor (VEGF). Lastly, neuropilin-1 can homomultimerize and form heteromultimers with neuropilin-2. These results provide insight into how interactions between neuropilins and secreted semaphorins function to coordinate repulsive axon guidance during neurodevelopment.

Neuropilin-1 Extracellular Domains Mediate Semaphorin D/III-Induced Growth Cone Collapse

Somatosensory axon outgrowth is repulsed when soluble semaphorin D (semD) binds to growth cone neuropilin-1 (Npn-1). Here, semD ligand binding studies of Npn-1 mutants demonstrate that the sema domain binds to the amino-terminal quarter, or complement-binding (CUB) domain, of Npn-1. By herpes simplex virus– (HSV-) mediated expression of Npn-1 mutants in chick retinal ganglion cells, we show that semD-induced growth cone collapse requires two segments of the ectodomain of Npn-1, the CUB domain and the juxtamembrane portion, or MAM (meprin, A5, μ) domain. In contrast, the transmembrane segment and cytoplasmic tail of Npn-1 are not required for biologic activity. These data imply that the CUB and MAM ectodomains of Npn-1 interact with another transmembrane growth cone protein that in turn transduces a semD signal into axon repulsion.

Molecular cloning of a novel member of semaphorin family genes, semaphorin Z

Semaphorins/collapsins (semaphorins) comprise a large family of proteins implicated in axonal guidance during development. We cloned a novel member ( semaZ) of the semaphorin gene family from a rat brain cDNA library. Sema Z was thought to be an integral membrane glycoprotein of 887 amino acids including a sema domain composed of 532 amino acids. The amino acid sequence of Sema Z showed 28–35% identity with other semaphorins in its sema domain, including 15 conserved cysteine residues. The cytoplasmic domain of Sema Z was found to be rich in prolines. Our phylogenetic analysis based on the amino acid sequence of the sema domains and the location of conserved N-glycosylation sites suggested that the sema domain of Sema Z belongs to a new class, class VI. We detected the semaZ mRNA in the first branchial arch of embryonic day 11 (E11) rat embryo, and subsequently in the myotomes and the dorsal root ganglia in developing somites from E11.5 through E13.5, but not in the brain. However, at E15, 18, 21 and P0, semaZ was highly expressed in the brain. Sema Z might play a role in both peripheral and central nervous system development.