The semaphorins are a large group of cell surface and secreted proteins implicated in axonal pathfinding. Here we show that the secreted mouse semaphorin D (SemD) is synthesized as an inactive precursor (proSemD) and becomes repulsive for sensory and sympathetic neurites upon proteolytic cleavage. ProSemD processing can be blocked completely by an inhibitor selective for furin-like endoproteases or mutagenesis of three conserved dibasic cleavage sites. Its C-terminal pro-peptide contains a processing signal that is essential for SemD to acquire its full repulsive activity. SemD processing is regulated during the embryonic development of the mouse and determines the magnitude of its repulsive activity. Similarly to SemD, the secreted semaphorins SemA and SemE display repulsive properties that are regulated by processing. Our data suggest that differential proteolytic processing determines the repulsive potency of secreted semaphorins and implicate proteolysis as an important regulatory mechanism in axonal pathfinding.
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...
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.
A family of structurally related, Na ' /CI--dependent plasma-mernbranc transporters catalyze the uptake of several neurotransmitters, osmolytes and other metabolites into cells. Four different members of this transporter family have heen cloned from mamiiialian sources which all transport 4-aminobutyric acid (GABA) but differ in their pharmacological profiles and in thcir tissue distribution. We report on the cloning, sequencing and functional expression of a transporkr for GARA and /]Lalanine from the electric lobe of Torpedo. According to similarily of amino acid sequcnce (77 % identity), pharrnacological properties, and tissue distribution (nervous-system-specific), it appcars to be the counterpart of thc [Ialanine-sensitive GARA transporter, GAT-BIGAT-3IGAT4, previously cloned from rat and mouse. The identification of another GABA transportcr isoforrn from Torpedo (after the rccent characterization of a Torpedo GAT-1 transporter) indicatcs that GABA-(ransporter isoforms arc phylogenetically ancient and arose before the divergence of vertebrates. Sequence comparison between isofunctionul transporters from evolutionarily distant species aids in the identification of amino acid residucs that are critical for functional specificity. The expression of transporters for GARA and [hlanine raises questions regarding the unidentified physiological role of thesc amino acids in Torpedo electric lobe.Ke,ywwds: 4-arninobutyric acid ; jl-alanine ; transporter; Torp& ; plasma membrane. Plasma membrane bansporters driven by a Na' gradient are responsible for the removal of most neurotransmitlers from the synaptic cleft and thus for the controlled termination of synaptic signals. The modulation of their activity by pharmacological agents or by drugs of abuse can profoundly affect synaptic transmission. Some transporters [e.g., Lhosc for glutamate, 4-aminohutyric acid (GABA) and dopamine] have been shown to function also in reverse under conditions of energy deprivation or under the influence of drugs, leading to the non-vesicular release of neurotransmitter [I -31. Transporter-nicdiatcd release of &lu-tamate, for example, is believed to be an important pathogenetic mechanism i n hrain ischemia, anoxia, or epilepsy.Since 1990, molecular cloning has led to the characterkdtion of two neuro(ratisniitter-transporter families. Members of the first family 141 transport GABA, noradrenaline, dopamine, scrotonin, glycine, prolinc, [I-alanine, taurine, betaine and creatine, whercas carriers for glutamate, aspartate and several neutral amino acids constitute a second family 15-81, Both families are driven by a Na' griidient but differ i n their dependence on additional ions and have no significant sequcncc similarity. Although the characterization of the GABA/noradrenaline transporter family originatcd froin the study of neurotransmission, it has turned out that their physiological roles are not restricted to this func- tion. Ainong its members are carricrs for taurine [9][10][11], bctaine [12, 131, /Lalanine r14-161 and crcatine r17, 1x...
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