As a group, semaphorins are expressed in most tissues and this expression varies considerably with age. The expression patterns of the individual semaphorins are best characterized in the nervous system, particularly during development, where most, or perhaps all, semaphorins are widely expressed in the nervous system by neuronal and non-neuronal cells (reviewed in [17
]; see Table for details of the expression and functions of all members of the family and associated references). Semaphorins are also widely expressed in many organ systems and their derivatives, including the cardiovascular, endocrine, gastrointestinal, hepatic, immune, musculoskeletal, renal, reproductive, and respiratory systems.
Expression and function of semaphorins
No particular pattern of expression appears to define each of the different classes of semaphorins, but many are dynamically expressed in particular areas during development, and this expression often decreases with maturity. In the nervous system, for example, semaphorin expression is often associated with growing axons as they form axonal tracts, but this expression often decreases following the formation of the tracts. Interestingly, changes in the adult expression levels of semaphorins have been described following injury in neuronal and non-neuronal tissues, during tumorigenesis, and in association with other pathological conditions.
The diverse expression patterns of the different semaphorins suggest that they are important in a variety of functions during development and into adulthood. Indeed, genetic analyses in both invertebrates and vertebrates indicate that semaphorins are often required for viability and reveal, in combination with additional functional assays, distinct roles in various physiological and pathological processes in most or perhaps all tissues. These studies reveal that semaphorins on cellular processes such as adhesion, aggregation, fusion, migration, patterning, process formation, proliferation, viability, and cytoskeletal organization.
Semaphorins are best known for their roles in nervous system development, and a number of approaches in vivo
and in vitro
indicate that semaphorins can enable axons to find and connect with one another and their other targets (reviewed in [18
]). An important way in which semaphorins guide these growing axons is by repelling them or preventing them from entering certain regions. For example, characterization of their normal expression patterns, the defects observed in particular semaphorin mutants, and assays in vivo
and in vitro
have revealed that at least some semaphorins form molecular boundaries to prevent axons and cells from entering inappropriate areas. Semaphorins also have roles in physiological and pathological processes in the adult. In the nervous system, altered semaphorin function has been linked to epilepsy, retinal degeneration, Alzheimer's disease, motor neuron degeneration, schizophrenia, and Parkinson's disease [19
Semaphorins may also limit the ability of axons to regrow after injury and prevent abnormal sprouting of axons involved in pain or autonomic function [23
]. In the immune system, semaphorins are critical for various phases of the immune response (Table ; reviewed in [27
]). Semaphorins are also involved in cancer progression, by affecting chemotaxis, viability, tumorigenesis, metastasis, and angiogenesis (reviewed in [28
]). More recently, semaphorins have also been implicated in vascular health and heart disease (reviewed in [29
Receptors and signaling proteins associated with semaphorins
The molecular mechanisms by which semaphorins mediate their functional effects are far from clear. Semaphorin-mediated axon repulsion is a result of the modification of the axonal cytoskeleton at the growing tips or growth cones of axons. The control of axon outgrowth or growth-cone motility depends critically upon the dynamics of F-actin polymerization and depolymerization, coupled with the regulation of F-actin translocation and microtubule dynamics. Following exposure to secreted Sema3A, growth cones undergo a rapid collapse that is accompanied by the depolymerization of F-actin, a decreased ability to polymerize new F-actin, attenuated microtubule dynamics, and collapsed microtubule arrays (reviewed in [30
]). The molecular mechanisms underlying these phenomena are poorly understood but may also be responsible for many of the functional effects that semaphorins have in non-neuronal tissues. For example, the cytoskeleton is required for cells to move, polarize, change shape, engulf particles, and interact with other cells; even the most divergent family member, the viral semaphorin SemaVA, induces actin cytoskeletal rearrangement in dendritic cells of the immune system and alters the ability of these cells to adhere and migrate [31
Post-translational processing underlies at least some of the functional effects of semaphorins. Several secreted and transmembrane semaphorins undergo proteolytic processing, and this is important in semaphorin-mediated repulsive axon guidance, growth-cone collapse, cell migration, invasive growth, and metastasis (for example, see [32
]). For example, mouse Sema3A, Sema3B, and Sema3C are synthesized as inactive precursors and become repulsive for axons upon proteolytic cleavage [32
Oligomerization is another modification that is important for semaphorin function. The secreted vertebrate semaphorin Sema3A is a dimer [9
], and dimerization is important for its activity in repulsive axon guidance and growth-cone collapse [36
]. Cysteine residues in the carboxy terminus are important for this dimerization, although weak dimerization also occurs between sema domains [8
]. Transmembrane semaphorins also form disulfide-linked dimers and depend on oligomerization for at least some of their functional effects [5
Semaphorin receptors and signaling
Semaphorins exert the majority of their effects by serving as ligands and binding to other proteins through their extracellular domains. All classes of semaphorins except class 2 have been found to bind directly to members of the plexin (Plex) family of transmembrane receptors (reviewed in [41
]; see Table for a summary of the receptors and signaling proteins associated with semaphorins and Figure for the primary structure of known semaphorin receptors). Interestingly, plexins also contain sema domains, albeit highly divergent, that are important for binding to semaphorins [8
]. Several other proteins have also been identified that bind to the extracellular portions of semaphorins (Figure ). In particular, members of the neuropilin (Npn) family of transmembrane proteins are receptors for class 3 semaphorins [30
]. Both the basic tail and the sema domain of Sema3A are important for binding to Npn-1, although binding to the sema domain is weaker. Neuropilins, however, only have short cytoplasmic tails that are not required for the effects of semaphorins on axon guidance [30
]. Interestingly, neuropilins also bind plexins, such that class 3 semaphorins, which bind to neuropilins, signal their effects through the cytoplasmic domain of plexins.
Figure 3 Semaphorin receptors. Members of the plexin protein family are organized into four classes (A, B, C, and D); plexins are known to bind to semaphorins from all classes except class 2, whose receptors are unknown. Class 3 semaphorins bind both members of (more ...)
The signal transduction cascades used by semaphorins are poorly understood. No canonical signal transduction pathways seem to mediate the effects of semaphorins, making the identification of semaphorin signaling intermediates difficult. Over the past few years, however, a number of proteins have been identified and linked with semaphorin signaling, including G proteins, kinases, regulators of cyclic nucleotide levels, oxidation-reduction enzymes, and regulators of the actin cytoskeleton (Table ). These intermediates suggest that novel signaling cascades implement semaphorin function (reviewed in [21
]), although a complete signaling pathway through which these proteins direct semaphorin function has not yet been characterized. Furthermore, semaphorin signaling intermediates have been identified using several different functional assays, complicating a precise determination of the roles of these proteins in the different semaphorin functions.
At the moment, the best characterized semaphorin signaling cascades are those used for axon guidance and cell migration. Semaphorin-mediated repulsive axon-guidance signaling depends on the large cytoplasmic domains of plexins, at least some of which have GTPase-activating protein (GAP) activity: these domains show sequence similarity to a group of Ras-family-specific GAPs, and mammalian PlexA1 and PlexB1 have GAP activity towards R-Ras [45
]. The cytoplasmic domains of plexins also bind other small GTPases as well as binding regulators of GTPase activity, including guanine-nucleotide exchange factors (GEFs) and GAPs [44
]. The functional implications of these interactions are best understood for mammalian Sema4D and mammalian PlexB1: activation of PlexB1 by Sema4D enhances the activity of RhoGEFs, activating the small GTPase RhoA, and leads to cytoskeletal rearrangement and repulsive axon guidance. There may be variation, however, in the signaling cascades activated by the different semaphorins. Repulsive axon guidance signaling by invertebrate Sema-1a or vertebrate Sema3A through class A plexins, for example, uses many proteins not currently characterized as important for repulsive axon guidance by Sema4D and PlexB1 [18
Specific signaling proteins may also be required for the distinct functions of semaphorins. For example, Sema4D, together with PlexB1, limits cell migration or axon outgrowth by signaling through signaling proteins including the epidermal growth factor receptor ErbB2, Rho kinase, 12-15 lipoxygenase, and PlexC1; whereas Sema4D signaling through PlexB2 and the hepatocyte growth factor receptor Met, the receptor tyrosine kinase Ron, p190RhoGap, the tyrosine kinases Pyk2, Src, and Akt, and phosphatidylinositol 3-kinase enables cell migration or axon outgrowth (reviewed in [41
Importantly, recent work has also begun to identify mechanisms by which semaphorin signaling and its functional effects can be modulated. Neurotrophins, growth factors, chemokines, cell adhesion molecules, and integrins have all been shown to modulate semaphorin signaling, and some of these effects seem to occur through cyclic nucleotides, nitric oxide, and semaphorin receptor endocytosis [21
]. Interestingly, semaphorins can also serve as cell-surface receptors for plexins and perhaps other proteins, and mediate some of their functional effects through 'reverse signaling' [48
] (Table ). In particular, transmembrane semaphorins can function as receptors essential for generating proper neuronal connectivity [49
] and cardiac development [48
], and these effects have been linked to the association of their cytoplasmic portions with signaling and anchoring proteins (Table ).