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.     

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.     

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.     

Successful inhibition of tumor development by specific class-3 semaphorins is associated with expression of appropriate semaphorin receptors by tumor cells

The class-3 semaphorins (sema3s) include seven family members. Six of them bind to neuropilin-1 (np1) or neuropilin-2 (np2) receptors or to both, while the seventh, sema3E, binds to the plexin-D1 receptor. Sema3B and sema3F were previously characterized as tumor suppressors and as inhibitors of tumor angiogenesis. To determine if additional class-3 semaphorins such as sema3A, sema3D, sema3E and sema3G possess anti-angiogenic and anti-tumorigenic properties, we expressed the recombinant full length semaphorins in four different tumorigenic cell lines expressing different combinations of class-3 semaphorin receptors. We show for the first time that sema3A, sema3D, sema3E and sema3G can function as potent anti-tumorigenic agents. All the semaphorins we examined were also able to reduce the concentration of tumor associated blood vessels although the potencies of the anti-angiogenic effects varied depending on the tumor cell type. Surprisingly, there was little correlation between the ability to inhibit tumor angiogenesis and their anti-tumorigenic activity. None of the semaphorins inhibited the adhesion of the tumor cells to plastic or fibronectin nor did they modulate the proliferation of tumor cells cultured in cell culture dishes. However, various semaphorins were able to inhibit the formation of soft agar colonies from tumor cells expressing appropriate semaphorin receptors, although in this case too the inhibitory effect was not always correlated with the anti-tumorigenic effect. In contrast, the anti-tumorigenic effect of each of the semaphorins correlated very well with tumor cell expression of specific signal transducing receptors for particular semaphorins. This correlation was not broken even in cases in which the tumor cells expressed significant concentrations of endogenous semaphorins. Our results suggest that combinations of different class-3 semaphorins may be more effective than single semaphorins in cases in which tumor cells express more than one type of semaphorin receptors.     

Structural basis of MET receptor dimerization by the bacterial invasion protein InlB and the HGF/SF splice variant NK1

The structural basis of ligand-induced dimerization of the receptor tyrosine kinase MET by its natural ligand hepatocyte growth factor/scatter factor (HGF/SF) is not well understood. However, interesting insight into the molecular mechanism of MET dimerization has emerged from crystal structures of MET in complex with a bacterial agonist, the invasion protein internalin B (InlB) from pathogenic Listeria monocytogenes. MET activation by InlB promotes uptake of bacteria into host cells. Structural and biophysical data suggest that InlB is monomeric on its own but dimerizes upon binding to the membrane-anchored MET receptor promoting the formation of a signaling active 2:2 complex. The dimerization interface is small and unusually located on the convex side of the curved InlB leucine-rich repeat (LRR) domain. As InlB does not dimerize in solution, the dimerization site could only be identified by studying packing contacts of InlB in various crystal forms and had to be proven by scrutinizing its biological relevance in cellular assays. InlB dimerization is thus an example of a low-affinity contact that appears irrelevant in solution but becomes physiologically significant in the context of 2-dimensional diffusion restricted to the membrane plane. The resulting 2:2 InlB:MET complex has an InlB dimer at its center with one MET molecule bound peripherally to each InlB. This model of ligand-mediated MET dimerization may serve as a blue-print to understand MET activation by NK1, a naturally occurring HGF/SF splice variant and MET agonist. Crystal structures of NK1 repeatedly show a NK1 dimer, in which residues implicated in MET-binding are located on the outside. Thus, MET dimerization by NK1 may also be ligand-mediated with a NK1 dimer at the center of the 2:2 complex with one MET molecule bound peripherally to each NK1. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases.     

Semaphorins in interactions between T cells and antigen-presenting cells

Although semaphorins were identified originally as guidance cues for developing neuronal axons, accumulating evidence indicates that several semaphorins are expressed also in the immune system. SEMA4D (CD100), which is expressed constitutively by T cells, enhances the activation of B cells and dendritic cells (DCs) through its cell-surface receptor, CD72. SEMA4A, which is expressed by DCs, is involved in the activation of T cells through interactions with TIM2. So, these semaphorins seem to function in the reciprocal stimulation of T cells and antigen-presenting cells (APCs). Emerging evidence indicates that additional semaphorins and related molecules are involved in T-cell-APC interactions also.     

Semaphorin-3D and Semaphorin-3E Inhibit the Development of Tumors from Glioblastoma Cells Implanted in the Cortex of the Brain

Class-3 semaphorins are secreted axon guidance factors. Some of these semaphorins have recently been characterized as suppressors of tumor progression. To determine if class-3 semaphorins can be used to inhibit the development of glioblastoma-multiforme tumors, we expressed recombinant sema-3A, 3B, 3D, 3E, 3F or 3G in U87MG glioblastoma cells. Sema3A and sema3B expressing cells contracted and changed shape persistently while cells expressing other semaphorins did not. Sema3A and sema3F differed from other semaphorins including sema3B as they also inhibited the proliferation of the cells and the formation of soft agar colonies. With the exception of sema3G and sema3B, expression of these semaphorins in U87MG cells inhibited significantly tumor development from subcutaneously implanted cells. Strong inhibition of tumor development was also observed following implantation of U87MG cells expressing each of the class-3 semaphorins in the cortex of mouse brains. Sema3D and sema3E displayed the strongest inhibitory effects and their expression in U373MG or in U87MG glioblastoma cells implanted in the brains of mice prolonged the survival of the mice by more then two folds. Furthermore, most of the mice that died prior to the end of the experiment did not develop detectable tumors and many of the mice survived to the end of the experiment. Most of the semaphorins that we have used here with the exception of sema3D were characterized previously as inhibitors of angiogenesis. Our results indicate that sema3D also functions as an inhibitor of angiogenesis and suggest that the anti-tumorigenic effects are due primarily to inhibition of tumor angiogenesis. These results indicate that class-3 semaphorins such as sema3D and sema3E could perhaps be used to treat glioblastoma patients.     

Genes that control the development of migrating muscle precursor cells

Skeletal muscles in vertebrates, despite their functional and biochemical similarities, are generated via diverse developmental mechanisms. A major subclass of hypaxial muscle groups is derived from long-range migrating progenitor cells that delaminate from the dermomyotome. The development of this lineage is controlled by Pax3, the c-Met tyrosine kinase receptor, its ligand SF/HGF (scatter factor/hepatocyte growth factor) and the homeobox factor Lbx1. These molecules are essential for establishment of the precursor pool, delamination, migration and target finding. Progress has been made in understanding patterning of the muscles, which requires a precise control of proliferation and differentiation of myogenic precursor cells.     

Developmental expression of sema3G, a novel zebrafish semaphorin

The semaphorins are a large, evolutionarily conserved family of signaling molecules with broad functions during development. The class 3 semaphorins are a subclass of secreted semaphorins found in vertebrates. There have been six class 3 semaphorins identified to date (sema3A to sema3F) and some have been shown to function in axon guidance and cardiovascular development. However, the functions of many class 3 semaphorins and their potential interactions in vivo are still not well understood. As a step toward understanding the actions of all class 3 semaphorins in vivo, we have cloned and analyzed the developmental expression pattern of a novel zebrafish class 3 semaphorin, sema3H [corrected] sema3H [corrected] is expressed in a dynamic pattern throughout the first 3 days of development. It is expressed in the adaxial cells of the somite during somitogenesis. In the brain, sema3H [corrected] is expressed in cell clusters in the midbrain and diencephalon, and is expressed in the telencephalon in close proximity to the olfactory epithelium. sema3H [corrected] also is expressed in the pharyngeal arches, the pectoral fin bud, and the developing pronephros. These results provide a basis for studying how expression of multiple semaphorins could be essential for aspects of early development.     

Semaphorin 3C preserves survival and induces neuritogenesis of cerebellar granule neurons in culture

Semaphorins (sema) constitute a family of molecules sharing a common extracellular domain (semaphorin domain). This family includes several types of secreted and membrane-associated molecules that are grouped into eight subclasses (subclasses 1-7 and viral semaphorins). Subclass 3 semaphorins are secreted molecules involved in axonal guidance, mainly through repulsive gradients and induction of growth cone collapse. More recently sema 3 molecules have been identified as positive factors in dependence of the type of neurons. Besides their axonal guidance function, some semaphorins have been implicated in apoptosis and survival. We investigated the effect of sema3C on survival and neurite outgrowth of rat cerebellar granule neurons (CGNs) in culture. 3T3 cells were stably transfected with sema3C. Several clonal lines were established and tested for their neuritogenic activity and one, S3C-8, was selected for the bulk of experiments. S3C-8 was co-cultured with CGNs. Sema3C enhanced CGN viability as assessed in co-cultures of CGNs with monolayers of S3C-8 in comparison with co-cultures of CGNs with control mock-transfected 3T3 cells. Moreover sema3C induced neuritogenesis of cultured CGNs, which express neuropilin-1 and -2. S3C-8 cells, overexpressing sema3C, were significantly more neuritogenic for CGN than poly l-lysine (PLL), a positive substrate for CGNs, as assessed by the measurement of the length of neurites and confirmed by Tau expression along the time of culture. CGNs co-cultured with S3C-8, showed up-regulation of the expression of axonal microtubule-associated proteins (MAPs) such as Tau, phosphorylated MAP2C and mode I-phosphorylated MAP1B compared with neurons cultured on control 3T3 cells. We also found increased expression of a specific marker of neuronal cell bodies and dendrites, high molecular weight MAP2 (HMW-MAP2). Interestingly, there was no accompanying up-regulation of a marker enriched within the neuronal somatodendritic domain, mode II-phosphorylated MAP1B. These data support the idea that secreted sema3C favors survival and neuritogenesis of cultured CGNs.     

Semaphorin 2a secreted by oenocytes signals through plexin B and plexin A to guide sensory axons in the Drosophila embryo

The semaphorin gene family has been shown to play important roles in axonal guidance in both vertebrates and invertebrates. Both transmembrane (Sema1a, Sema1b, Sema5c) and secreted (Sema2a, Sema2b) forms of semaphorins exist in Drosophila. Two Sema receptors, plexins (Plex) A and B, have also been identified. Many questions remain concerning the axon guidance functions of the secreted semaphorins, including the identity of their receptors. We have used the well-characterized sensory system of the Drosophila embryo to address these problems. We find novel sensory axon defects in sema2a loss-of-function mutants in which particular axons misproject and follow inappropriate pathways to the CNS. plexB loss-of-function mutants show similar phenotypes to sema2a mutants and sema2a interacts genetically with plexB, supporting the hypothesis that Sema2a signals through PlexB receptors. Sema2a protein is expressed by larval oenocytes, a cluster of secretory cells in the lateral region of the embryo and the sema2a mutant phenotype can be rescued by driving Sema2a in these cells. Ablation of oenocytes results in sensory axon defects similar to the sema2a mutant phenotype. These data support a model in which Sema2a, while being secreted from oenocytes, acts in a highly localized fashion: It represses axon extension from the sensory neuron cell body, but only in regions in direct contact with oenocytes.     

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 beta-sheet and two alpha-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.     

Semaphorin4D的作用机制与功能

Semaphorins家族是一类以结构中具有sema区域为共同特征的蛋白,Semaphorin4D(Sema4D)是其成员之一.Sema4D与受体丛状蛋白B1(PlexinB1)和分化抗原簇72(cluster of differentiation antigen72,CD72)结合,通过多种信号转导途径,在神经系统的轴突导向,免疫系统中T、B细胞的活化和免疫调节中发挥关键作用.最近发现,Sema4D在许多人体肿瘤组织中高表达,且对血管发生及肿瘤侵袭转移起重要作用.本文旨在对Sema4D的结构、作用机制及生物学功能的研究最新进展作一综述.     

Methionine acts as a "magnet" in photoaffinity crosslinking experiments

Photoaffinity crosslinking has been utilized to probe the nature of the ligand-receptor interface for a number of G protein-coupled receptor systems. Often the photoreactive benzophenone moiety incorporated in the ligand is found to react with a methionine in the receptor. We introduced methionines one-at-a-time into the region 163-176 of the parathyroid hormone receptor, and find that crosslinking occurs to the side-chain of methionine over a range of 11 amino acids. We call this the "Magnet Effect" of methionine. Hence, crosslinking contact points can be significantly shifted by the presence of methionine in a receptor domain.     

Polyubiquitin chains: polymeric protein signals

The 76-residue protein ubiquitin exists within eukaryotic cells both as a monomer and in the form of isopeptide-linked polymers called polyubiquitin chains. In two well-described cases, structurally distinct polyubiquitin chains represent functionally distinct intracellular signals. Recently, additional polymeric structures have been detected in vivo and in vitro, and several large families of proteins with polyubiquitin chain-binding activity have been discovered. Although the molecular mechanisms governing specificity in chain synthesis and recognition are still incompletely understood, the scope of signaling by polyubiquitin chains is likely to be broader than originally envisioned.     

Mechanisms of axon guidance: membrane dynamics and axonal transport in semaphorin signalling

The intricate geometry of neuronal networks poses many unique cell-biological problems regarding the way a growing axon responds to its environment. Several groups of ligand-receptor pairs have been identified to regulate such processes. In this study, we take class 3 semaphorins as an example and review what is known about the intracellular movements of semaphorins throughout neuronal cells, transport support structures and location of release sites. We discuss how their receptor trafficking may contribute to regulate membrane dynamics underlying growth cone motility and the physiological contribution made by class 3 semaphorins-induced acceleration of axoplasmic transport on neurite development.     

The orthologous human and murine semaphorin 6A-1 proteins (SEMA6A-1/Sema6A-1) bind to the enabled/vasodilator-stimulated phosphoprotein-like protein (EVL) via a novel carboxyl-terminal zyxin-like domain

Neuronal development and apoptosis critically depend on the transformation of extracellular signals to intracellular actions resulting in cytoskeletal rearrangements. Ena/VASP (enabled/vasodilator-stimulated phosphoprotein) proteins play an important role in actin and filament dynamics, whereas members of the semaphorin protein family are guidance signals in embryo- and organogenesis. Here, we report the identification of two novel transmembranous human and murine semaphorins, (HSA)SEMA6A-1 and (MMU)Sema6A-1. These semaphorin 6 variants directly link the Ena/VASP and the semaphorin protein family, since SEMA6A-1/Sema6A-1 is capable of a selective binding to the protein EVL (Ena/VASP-like protein). EVL is the third member of the Ena/VASP family of proteins that was identified sharing the same structural features as Mena (mammalian enabled) and VASP, although its functionality seems to be different from that of the other members. Here we demonstrate that SEMA6A-1/Sema6A-1 is colocalized with EVL via its zyxin-like carboxyl-terminal domain that contains a modified binding motif, which further stresses the existence of functional differences between EVL and Mena/VASP. In addition these findings suggest a completely new role for transmembranous semaphorins such as SEMA6A-1/Sema6A-1 in retrograde signaling.     

Semaphorin 3F and Neuropilin-2 Control the Migration of Human T-Cell Precursors

Neuropilins and semaphorins are known as modulators of axon guidance, angiogenesis, and organogenesis in the developing nervous system, but have been recently evidenced as also playing a role in the immune system. Here we describe the expression and role of semaphorin 3F (SEMA3F) and its receptor neuropilin-2 (NRP2) in human T cell precursors. NRP2 and SEMA3F are expressed in the human thymus, in both lymphoid and non-lymphoid compartments. SEMA3F have a repulsive effect on thymocyte migration and inhibited CXCL12- and sphingosine-1-phosphate (S1P)-induced thymocyte migration by inhibiting cytoskeleton reorganization prior to stimuli. Moreover, NRP2 and SEMA3F are expressed in human T-cell acute lymphoblastic leukemia/lymphoma primary cells. In these tumor cells, SEMA3F also blocks their migration induced by CXCL12 and S1P. Our data show that SEMA3F and NRP2 are further regulators of human thymocyte migration in physiological and pathological conditions.     

Semaphorins and cancers : an up 'dating'

Semaphorins, first described as axon guidance molecules, play an essential role in neural development, angiogenesis and immunological response. In 1996, two semaphorin genes, SEMA3B and SEMA3F, were isolated from chromosomal region 3p21.3 believed to contain a tumor suppressor gene based on frequent loss of heterozygosity in lung and breast cancer. Since these first studies, several semaphorins have been involved in tumor progression. Some semaphorins have been proposed to have pro-tumoral properties, whereas others have been shown to have tumor suppressive activity. This review summarizes the most recent data implicating semaphorins in cancers.