The Chemorepulsive Activity of the Axonal Guidance Signal Semaphorin D Requires Dimerization

Abstract

The axonal guidance signal semaphorin D is a member of a large family of proteins characterized by the presence of a highly conserved semaphorin domain of about 500 amino acids. The vertebrate semaphorins can be divided into four different classes that contain both secreted and membrane-bound proteins. Here we show that class III (SemD) and class IV semaphorins (SemB) form homodimers linked by intermolecular disulfide bridges. In addition to the 95-kDa form of SemD (SemD(95k)), proteolytic processing of SemD creates a 65-kDa isoform (SemD(65k)) that lacks the 33-kDa carboxyl-terminal domain. Although SemD(95k) formed dimers, the removal of the carboxyl-terminal domain resulted in the dissociation of SemD homodimers to monomeric SemD(65k). Mutation of cysteine 723, one of four conserved cysteine residues in the 33-kDa fragment, revealed its requirement both for the dimerization of SemD and its chemorepulsive activity. We suggest that dimerization is a general feature of semaphorins which depends on class-specific sequences and is important for their function.The semaphorins are a large family of secreted and membrane-bound proteins that are involved in axonal navigation (1). To date, sequences of 15 vertebrate semaphorins have been published, and these can be divided into four classes (2-11) based on the similarity of their semaphorin domains and the presence of distinct sequence motifs in their COOH-terminal domains such as Ig homologies (classes III and IV), thrombospondin repeats (class V), and transmembrane segments (classes IV, V, and VI). The best studied vertebrate semaphorins are the murine SemD 1 and its chick ortholog collapsin-1 (3,6,(12)(13)(14)(15). When added to cultures of dorsal root ganglia, both induce a rapid and reversible collapse of sensory growth cones (3,12,15). Gradients of SemD originating from aggregates of cells transfected with an expression vector repel sensory and sympathetic axons in collagen gel co-cultures, demonstrating that semaphorins have the ability to exclude axons from regions expressing these proteins (6,13,15).The secreted class III semaphorins are synthesized as proproteins that are processed proteolytically to 95-or 65-kDa isoforms (designated 95k and 65k, respectively) at several conserved dibasic cleavage sites (15). Semaphorins SemA, SemD, and SemE act as repellents for specific populations of axons, and the potency of this repulsion is regulated by proteolysis (15). Cleavage of pro-SemD at a COOH-terminal processing site generates the 95k isoform (SemD95k)) and is required to activate its repulsive activity. Further cleavage of SemA, SemD, or SemE to a 65k form reduces their repulsive activities by at least 1 order of magnitude (15). Semaphorins display specific and highly dynamic expression patterns in the developing nervous system as well as in nonneural tissues (2-6, 8, 9, 13, 14, 16 -18). In vitro, specific subsets of spinal sensory afferents display a differential responsiveness to SemD which is regulated developmentally (5,13,14). It therefore has been propos...

The axonal chemorepellant semaphorin 3A is increased in the cerebellum in schizophrenia and may contribute to its synaptic pathology

“…The heterogeneity is because a combination of proteolytic processing of pro-sema3A and dimerization of sema3A cleavage products. 35,36 Our Western blots of unfractionated protein showed a band of 50 kDa in fetal whole brain (Figure 1b) and 40 kDa in adult cerebellum (Figure 1a), consistent with results seen using a similar carboxy-terminal antibody in HEK293 cells and mouse embryo, respectively. 35,36 The fact that we did not see any clear bands at 125, 95, or 65 kDa, as also reported in some circumstances by these authors, may reflect intrinsically low levels of these sema3A species in human cerebellum, or their susceptibility to perimortem degradation.…”

“…35,36 Our Western blots of unfractionated protein showed a band of 50 kDa in fetal whole brain (Figure 1b) and 40 kDa in adult cerebellum (Figure 1a), consistent with results seen using a similar carboxy-terminal antibody in HEK293 cells and mouse embryo, respectively. 35,36 The fact that we did not see any clear bands at 125, 95, or 65 kDa, as also reported in some circumstances by these authors, may reflect intrinsically low levels of these sema3A species in human cerebellum, or their susceptibility to perimortem degradation. The change in molecular weight between infants and adults may represent a maturational shift in sema3A processing, and be related to the corresponding alteration in sema3A immunoreactivity from being primarily in Purkinje cell bodies (Figure 2b) to the adjacent neuropil (Figure 2a).…”

Crystal structure of the plexin A3 intracellular region reveals an autoinhibited conformation through active site sequestration

“…Dimerization or higher order clustering plays an important role in regulating plexin signaling (8,11,21). The plexin intracellular regions weakly dimerize in solution (Fig.…”

“…The GAP activity of the plexin cytoplasmic domains can be induced by simultaneous binding of Rnd1 and an clusterinducing antibody, whereas no activity was detected in the absence of either (8,11). While semaphorins may contribute to plexin activation by inducing a change in its oligomerization state (8,11,21,22), the mechanism by which the Rho GTPases facilitate the activation process remains unclear. A conformational change associated with GAP activation has been suggested by studies that show interactions between C1 and C2, and dissociation of the two upon RhoGTPase binding to the RBD (11,20).…”

Transmembrane Domain Interactions Control Biological Functions of Neuropilin-1

“…We hypothesized that the biological function of Sema3A was abrogated when interfering with the TM domain because of the destabilization of the receptor complex required for signal transduction. Indeed, it has been proposed that Sema3A dimer (known to be important for binding and collapsing activity; Klostermann et al, 1998;Koppel and Raper, 1998) may undergo a dimer-to-monomer transition upon binding to monomeric NRP1 (Antipenko et al, 2003), a transition that would explain the persistence of binding after destabilization of NRP1 homodimerization. In the other hand, the interference of the TM domain dimerization may be sufficient to generate conformational changes altering optimal binding at the extracellular level.…”