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Semaphorins, Plexins and
Neuropilins and Their Role in
Vasculogenesis and
Angiogenesis
by Gera Neufeld, Niva Shraga-Heled, Tali Lange and Ofra Kessler
1. Introduction and Historical Perspective
In the late 1980s and early 1990s, the angiogenic factors belonging to the VEGF family were discovered and characterized. It was obvious from the very earliest days that multiple splice forms of VEGF existed. However, these splice forms exhibited relatively similar activities in standard in vitro assays although their potencies varied. Nevertheless, it seemed possible that receptors able to differentiate between specific splice forms
Note: The names of all semaphorins are abbreviated according to the following rules: s always appears as the first letter denoting a semaphorin. The number after the s designates the semaphorin to a specific class, and the final letter designates its place within the class. Thus, s3f means semaphorin-3F while s4d means semaphorin-4D.
VEGF:Vascular endothelial growth factor, VEGFR-1: VEGF receptor-1, VEGFR-2: VEGF receptor-2, np1: neuropilin-1, np2: neuropilin-2, and HGF: hepatocyte growth factor.
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of VEGF may exist, and that these receptors should differ from the known tyrosine-kinase VEGF receptors, VEGFR-1 and VEGFR-2, which do not differentiate between VEGF splice forms. An early indication that such splice form-specific VEGF receptors exist was obtained in experiments in which receptors that bind the VEGF splice form VEGF165 but not the VEGF121 splice form were first identified.1 However, the molecular identity of these receptors was only revealed two years later when they were found to be the products of the neuropilin-1 (np1) gene.2 Subsequently, it was realized that the related neuropilin-2 (np2) gene product also functions as a receptor for VEGF165 and for VEGF145, a VEGF splice form that does not bind to np1.3 Furthermore, additional VEGF family members such as PlGF-2, VEGF-B, and VEGF-C were also found to interact with np1 or with np2 or with both receptors.4−6
Np1 had been identified almost a decade ago as the A5 antigen, a cell surface protein involved in neuronal recognition.7,8 It was subsequently realized that np1 functions as a receptor for the axon guidance factor semaphorin-3A (s3a).9,10 S3a belongs to a semaphorin subfamily that includes the seven class-3 semaphorins. S3a was previously known as collapsin-1 because it induces an np1-mediated collapse of the actin cytoskeleton in axon growth cones of responsive nerve cells.11 At the same time, Np2 was also found to function as a receptor for class-3 semaphorins such as semaphorin-3F (s3f ). Following these findings it was realized that np1 is unlikely to be able to transduce semaphorin signals on its own because its intracellular domain is very short. It was postulated therefore that class-3 semaphorin receptors had to contain an independent signal transducing component other than neuropilins. These signaling components turned out to be members of the plexin receptor family.12,13 Interestingly, plexins serve as direct signal transducing receptors for most of the semaphorins except for some members of the class-3 semaphorin subfamily (S3a–d, and s3f ) which do not bind directly to plexins and require neuropilins as obligate semaphorin binding components in semaphorin holo-receptors.
These developments raise several questions with regard to the regulation of angiogenesis. The first is concerned with the elucidation of the mechanisms by which neuropilins modulate the angiogenesis promoting activities of the members of the VEGF family. The second concerns the
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potential role of the alternative neuropilin ligands, the semaphorins, as potential modulators of angiogenesis. Another, more general question relates to the role of the nervous system in the regulation of developmental angiogenesis. These questions have been addressed to some extent in recent years although our knowledge is still far from complete.
2. The Semaphorins
The semaphorin family consists of more than 30 genes divided into eight classes, of which the first two classes are derived from invertebrates, classes 3–7 are the products of vertebrate semaphorins, and the eighth class contains viral semaphorins (Fig. 1). In the literature, the semaphorins are often referred to by an array of confusing designations.
Fig. 1. The semaphorin family. The different semaphorin subclasses are shown. Classes 3–7 contain vertebrate semaphorins. The two main semaphorin subclasses containing members reported to function as angiogenesis regulators are the class-3 and class-4 semaphorins. Class-3 semaphorins are the only secreted vertebrate semaphorins. The subfamily contains seven known members. They are distinguished by a small basic domain and by an Ig-like domain in addition to the sema domain which is present in all semaphorins. Class-4 semaphorins are membrane-anchored semaphorins containing an Ig loop-like domain.
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This situation was clarified several years ago by the adoption of a unified nomenclature for the semaphorins.14 The semaphorins are characterized by the presence of a 500 amino acid-long sema domain that is located close to their N-termini. The sema domain is essential for semaphorin signaling and determines receptor binding specificity.15 The sema domains of two different semaphorins were recently characterized by X-ray crystallography revealing a beta propeller topology.16−19 The different semaphorin classes are characterized by class-specific structural motifs. Thus, semaphorins belonging to classes 2–4 and 7 contain immunoglobulin-like domains (Fig. 1), class-5 semaphorins contain thrombospondin repeats and class-3 semaphorins contain a basic domain. Class-3 semaphorins are produced as secreted proteins but other classes of vertebrate semaphorins are produced as membrane anchored or transmembrane proteins that can be further processed into soluble proteins. The active forms of the class-3 semaphorin s3a and of the class-4 semaphorins s4a and s4d are homodimers linked by disulfide bridges.20 As a result of these observations it is assumed that the active forms of the other class-3 and class-4 semaphorins, and possibly of all the other semaphorins as well, are homodimeric. However, this assumption still requires proof.
Semaphorins have been originally characterized as axon guidance factors that participate in the regulation of the complex process by which growth cones of axons are directed to their proper targets during the formation of the nervous system.21 In recent years it was realized that semaphorins play a role in many developmental processes outside of the nervous system, in particular as regulators of cell migration,22 immune responses23 and organogenesis.24 It is therefore not surprising that s3b and s3f have been initially characterized as modulators of tumor progression,25,26 and that these semaphorins as well as other semaphorins have been recently found to function as regulators of angiogenesis and tumor angiogenesis.
3. The Plexin Receptor Family
Most of the semaphorins were found to bind directly to cell surface receptors belonging to the plexin receptor family.12 The plexins are segregated into four sub-families. There are four A-type plexins, three
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B-type plexins, and single C- and D-type plexins.27,28 Different plexins serve as direct binding receptors for different types of semaphorins. Thus, plexin-B1 is a receptor for s4d,12 plexin-B3 is a receptor for s5a,29 plexin-A1 is a s6d receptor,30 and plexin-D1 is a receptor for s3e,31 to name but a few examples. The plexins contain a split cytoplasmic SP (sex-plexin) domain (also known as the C1 and C2 domains). The intracellular domain contains putative tyrosine phosphorylation sites but no tyrosine kinase domain. The intracellular part of plexin-B1 contains a domain located between the C1 and C2 domains that functions as a GTPase activating protein (GAP) domain. This GAP-like domain is conserved quite highly throughout the plexin family although it is unclear whether it is functional in all plexins.32 The extracellular domain of all plexins contains a sema domain which serves as an auto-inhibitory domain in the basal, non-activated state of the receptor.33 The extracellular domains of the plexins also contain sequences homologous to similar sequences found in the Met subfamily of tyrosine-kinase receptors. These are designated as Met related sequence (MRS) domains and glycine-proline (G-P) rich motifs (Fig. 2).34
The intracellular domain of the plexin-A1 receptor contains a binding site for the small GTPase Rac-1 as well as a binding site for the intracellular tyrosine-kinase Fes/Fps, which phosphorylates plexin-A1 in response to s3a.35 Likewise, it was found that the intracellular tyrosine kinase Fyn binds to the intracellular domain of the plexin-A2 receptor and phosphorylates it in response to s3a.36 The serine-threonine kinase Cdk-5 also associates with plexin-A2, is activated by s3a and phosphorylates in turn the CRMP2 protein which serves as an important downstream target of plexins in neurons.36−38 The intracellular domain of the Drosophila homologue of plexin-A1, plexin-A, also contains a binding site that enables association with the flavoprotein oxidoreductase MICAL, which was found to be essential for correct s1a-induced axon repulsion in Drosophila.39
Type-B plexins resemble class-A plexins in their primary features, but there are some peculiarities specific to this plexin subclass. PlexinB1 is the best characterized type-B plexin. It also binds Rac-1 but the purpose of the binding may be sequestration of Rac-1 so as to prevent its interaction with downstream targets.40,41 Activation of plexin-B1 by s4d sequesters Rac-1 and inhibits Rac1 signaling, while simultaneously