InteractiveFly: GeneBrief

plexin B: Biological Overview | References

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 (PlexinA and PlexinB), have also been identified. Many questions remain concerning the axon guidance functions of the secreted semaphorins, including the identity of their receptors. The well-characterized sensory system of the Drosophila embryo was used to address these problems. Novel sensory axon defects were found 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 (Bates, 2007).

The detailed knowledge of the cellular basis for axon pathfinding in the sensory system of the Drosophila embryo has been exploited to shed further light on the mechanisms by which semaphorins mediate axon guidance. Analysis of sensory axon trajectories in semaphorin mutants reveals that secreted semaphorins play roles in early pathfinding decisions by these axons. In sema2a loss-of-function mutants, the v'ch1 axon often projects to a nearby, inappropriate pathway, the ISN, instead of the SN. Two other defects are seen at low frequencies in sema2a mutants: one or more of the lateral cluster axons projects aberrantly to the SN and axons of dorsal sensory neurons project anteriorly or posteriorly, instead of growing ventrally towards the lateral sensory cluster (Bates, 2007).

The modest penetrance levels of sema/plex LOF phenotypes suggest that in the sensory system, as in many other situations, axon pathfinding events are likely to involve the simultaneous action of multiple guidance factors that act cooperatively and/or redundantly. Indeed, it has been shown that PlexA and PlexB have partially redundant roles in motor axon pathfinding (Ayoob, 2006) while motor axon branching over muscles is governed by the relative balance of Sema2a, Netrins and FasII, rather than the level of any one of these molecules. Sema2a alone apparently makes only a moderate contribution to sensory axon guidance. Several other molecules have been identified that play a role in the guidance of the same sensory axons that are affected by sema and plex mutations. A future goal of this research is to elucidate how these various guidance factors interact to mediate sensory axon guidance (Bates, 2007).

Overexpression of Sema2a in oenocytes, epidermis or trachea, or ectopic expression in neurons did not result in defective sensory axon morphologies. This finding contrasts with the reported disruption of muscle innervation following overexpression of Sema2a on muscles. Using anti-Sema2a staining, it was cofirmed that the GAL4 driver lines used in these overexpression experiments led to expression of high levels of Sema2a protein in the relevant tissues. Moreover, the partial rescue of the sema2a LOF mutant phenotype observed when the sal-GAL4 line was used to drive UAS-sema2a shows that at least this line can effectively drive expression of functional Sema2a protein. Thus, the absence of sensory axon defects following Sema2a overexpression suggests that sensory axons are less sensitive to semaphorin levels than are motor axons (Bates, 2007).

In contrast to the secreted semaphorins, no evidence was found from analysis of the sema1a loss-of-function mutant and overexpression of sema1a that this transmembrane semaphorin is involved in sensory axon guidance in the periphery (Bates, 2007).

The v'ch1 pathway is a potentially attractive route for lateral cluster axons, as shown by the misprojection of lateral axons along this route in a variety of mutants, including robo, slit and trachealess. However, in wild-type embryos, lateral cluster axons very rarely follow the v'ch1 axon and vice versa. At the onset of sensory axon growth, a cluster of 4 to 7 oenocytes lies between the v'ch1 and lateral cluster neurons. Filopodia extend from the v'ch1 cell body in a variety of directions: those projecting dorsally and anteriorly, towards the oenocytes, are generally short and do not develop into axon branches. These observations suggest that oenocytes form a repulsive zone, preventing axon growth between the v'ch1 and lateral cluster cell bodies. The finding that ablating some of the oenocytes in a hemisegment can result in misprojection of the v'ch1 axon to the lateral cluster neurons and vice versa supports this hypothesis (Bates, 2007).

Oenocytes strongly express Sema2a, and driving expression of Sema2a in these cells rescues the v'ch1 axon misprojection defect seen in sema2a mutants. These findings suggest that Sema2a secreted by oenocytes normally represses axon growth by v'ch1 towards the lateral cluster neurons, and vice versa. Sema2a may act by inhibiting filopodial extension and/or by repressing filopodial dilation and subsequent axon formation. Loss of Sema2a function in sema2a mutants removes that repression, allowing the v'ch1 and lateral cluster axons to misproject in a reciprocal fashion to each other (Bates, 2007).

This model of Sema2a function differs from prevailing views of the action of secreted semaphorins as diffusible chemo-repellents. Isbister (2003) has shown that sensory axons in the grasshopper limb bud are guided by two orthogonal gradients of Sema2a protein expression which span the entire width and length of the limb bud. The directional growth of the axons is apparently dictated by the fractional change in Sema2a concentration across the limb bud epithelium. Similarly, the secreted vertebrate semaphorin Sema III is proposed to have a long-range guidance function in the spinal cord (Messersmith, 1995). This study concluded that Sema III secreted by ventral spinal cord cells diffuses dorsally, repelling the axons of small diameter sensory afferents and thereby forcing them to terminate in the dorsal horn (Bates, 2007).

In contrast, the current results suggest that the secreted Drosophila semaphorin Sema2a acts in a very local fashion. Sema2a produced by oenocytes in contact with a portion of the surface of the v'ch1 cell body suppresses axon extension specifically from that region of the cell. In this way, Sema2a determines, at least in part, the initial polarity of axon extension from the v'ch1 neuron (Bates, 2007).

Sema2a is also expressed in stripes of epidermal cells at the segment border. This source of Sema2a could conceivably contribute to guidance of the v'ch1 and lateral cluster axons by inhibiting their growth in a posterior direction. However, the absence of aberrant, posteriorly projecting sensory axons on either v'ch1 or lateral cluster neurons in sema2a mutants speaks against this function (Bates, 2007).

Genetic and biochemical evidence points to PlexA being the receptor for the transmembrane class I semaphorins during motor axon guidance in the Drosophila embryo. However, until recently, the receptor(s) for the secreted class II semaphorins and the ligand(s) for PlexB had not been identified. Ayoob (2006) has now provided evidence for a physical and genetic interaction between PlexB and Sema2a during motor axon guidance (Bates, 2007).

The current study provides a number of lines of evidence, suggesting that PlexB acts as a receptor for Sema2a in a different context, regulating the growth of the v'ch1 sensory axon. (1) plexB is expressed in lateral cluster sensory neurons, including v'ch1 at the time of axon outgrowth. (2) plexB loss-of-function mutants show the same v'ch1 axon misprojection phenotypes as sema2a loss-of-function mutants. (3) The v'ch1 axon defects in plexB mutants can be rescued by driving plexB expression in sensory neurons. One caveat with this conclusion is that the P0163-GAL4 line used in these experiments drives GAL4 in the oenocytes as well as the sensory neurons. (4) Halving the gene dose of both sema2a and plexB results in the same v'ch1 axon defects as seen in single sema2a or plexB homozygous mutant embryos (Bates, 2007).

A parallel set of data leads to the tentative suggestion that PlexA may act as a receptor for Sema2a during lateral cluster axon guidance: plexA is expressed in lateral cluster neurons; plexA and sema2a LOF mutants show defects in lateral cluster axon growth, albeit at low penetrance levels; these defects can be rescued by driving PlexA in the sensory neurons; and sema2a^−/+; plexA^−/+ embryos show the same lateral cluster axon defects (Bates, 2007).

While the above results provide support for the idea that sensory axon guidance is mediated by Sema2a signaling through both PlexA and PlexB receptors, the mechanism is likely to be more complex than binding of Sema2a to PlexA and PlexB and consequent independent activation of these two receptors. The increased penetrance of the v'ch1 defect in double homozygous sema2a; plexA and sema2a; plexB mutants, compared to single sema2a, plexA or plexB mutants, suggests that additional ligands are involved. Direct interactions between the PlexA and PlexB receptors, as demonstrated by the co-immuno-precipitation experiments of (Ayoob, 2006), may also contribute to the increased frequency of v'ch1 axon defects observed in double sema2a; plexA and sema2a; plexB mutants compared to single mutants. It is believed that the sensory system provides a valuable platform in which to further investigate this and other issues related to semaphorin function in axon guidance (Bates, 2007).

Drosophila Plexin B is a Sema-2a receptor required for axon guidance

Plexin receptors play a crucial role in the transduction of axonal guidance events elicited by semaphorin proteins. In Drosophila, Plexin A (PlexA) is a receptor for the transmembrane semaphorin semaphorin-1a (Sema-1a) and is required for motor and central nervous system (CNS) axon guidance in the developing embryonic nervous system. However, it remains unknown how PlexB functions during neural development and which ligands serve to activate this receptor. This study shows that plexB, like plexA, is robustly expressed in the developing CNS and is required for motor and CNS axon pathfinding. PlexB and PlexA serve both distinct and shared neuronal guidance functions. A physical association is observed between these two plexin receptors in vivo, and they can utilize common downstream signaling mechanisms. PlexB does not directly bind to the cytosolic semaphorin signaling component MICAL (molecule that interacts with CasL), but requires MICAL for certain axonal guidance functions. Ligand binding and genetic analyses demonstrate that PlexB is a receptor for the secreted semaphorin Sema-2a, suggesting that secreted and transmembrane semaphorins in Drosophila use PlexB and PlexA, respectively, for axon pathfinding during neural development. These results establish roles for PlexB in central and peripheral axon pathfinding, define a functional ligand for PlexB, and implicate common signaling events in plexin-mediated axonal guidance (Ayoob, 2006).

Semaphorins, along with other families of guidance cues, play key roles in neural development. Through both repulsion and attraction, semaphorins guide neuronal growth cones and thereby promote the establishment of neuronal connectivity and circuit formation. By relaying guidance information to the growth cone cytoskeleton of responding neurons, plexin proteins serve as central signaling components of many semaphorin receptor complexes. Understanding how neurons integrate a complex palette of guidance cue information through the action of related guidance cue receptors is necessary to reveal the molecular mechanisms underlying the steering of neuronal processes during development and also following nerve injury (Ayoob, 2006).

In vertebrates, nine different plexin proteins are known and they are organized into four distinct classes based upon their degree of evolutionary conservation (Plexin A-D); seven of these plexins belong to classes A and B. In the fruit fly this complexity is not as great as the Drosophila melanogaster genome includes only two plexins, one belonging to class A and one to class B (PlexA and PlexB). PlexA functions as a receptor for the transmembrane semaphorins Sema-1a and Sema-1b (Winberg, 1998). In vivo analyses demonstrate that, through the action of PlexA, Sema-1a regulates the defasciculation of motor axon bundles during embryogenesis. Although gain-of-function (GOF) studies strongly suggest that Drosophila PlexB mediates repulsive guidance events in vivo (Hu, 2001), and in vitro studies demonstrate that vertebrate plexin-B proteins mediate growth cone and COS cell collapse (Oinuma, 2003; Swiercz, 2002), the consequences of removing PlexB function in Drosophila, or in vertebrates, have not been determined. It is unclear, therefore, how Plexin B proteins function during neural development (Ayoob, 2006).

It is also unclear whether the different classes of plexins play distinct or redundant roles in the establishment of neuronal connectivity. In Drosophila, plexA and plexB are both expressed throughout the nervous system during development, indicating that they are likely to function within the same neuronal classes (Winberg, 1998). When overexpressed in all neurons, both plexA and plexB can produce similar phenotypes, suggesting that these receptors participate in related signaling events (Hu, 2001; Winberg, 1998). Interestingly, vertebrate plexin A1 and plexin B1 both modulate R-Ras activation through their intrinsic GTPase activating protein (GAP) domains, and this is essential for semaphorin-mediated repulsion in vitro (Oinuma, 2004; Toyofuku, 2005). These data point towards common, or perhaps redundant, signaling mechanisms that may underlie the in vivo functions of A and B class plexin receptors (Ayoob, 2006).

By contrast, although A and B class plexins are highly conserved, many differences exist among proteins belonging to these two plexin classes. Plexin A proteins are functional receptors for transmembrane class 1 semaphorins in Drosophila and class 6 transmembrane semaphorins in vertebrates. Secreted class 3 semaphorins also signal through class A plexins; however, this requires the assembly of a distinct holo-receptor complex that includes either neuropilin 1 or neuropilin 2, obligate co-receptors that serve to facilitate class 3 semaphorin binding and plexin A activation. Plexin B proteins in vertebrates bind to different transmembrane semaphorin ligands, including those from classes 4 and 5; however, no ligand has been identified for Drosophila PlexB (Kruger, 2005). Differences also exist between the downstream signaling events mediated by A and B class plexins (Negishi, 2005). The cytoplasmic domains of Drosophila PlexA and PlexB share a high degree of amino acid sequence identity (Winberg, 1998), yet they appear to differ with respect to the signaling molecules with which they directly associate. For example, although PlexB directly interacts with the small GTPase Rac, PlexA does not (Driessens, 2001; Hu, 2001). Likewise MICAL, a large cytosolic oxidoreductase that is crucial for semaphorin-mediated repulsion, associates with PlexA but not PlexB (Terman, 2002). Therefore, PlexA and PlexB may also serve non-overlapping roles during neural development (Ayoob, 2006).

This study examined the consequences of disrupting PlexB function for Drosophila neural development, allowing for a direct comparison between PlexB and PlexA axon guidance functions. Using genetic and biochemical analyses, it has been shown that PlexB and PlexA serve distinct and overlapping roles in motor and CNS axon guidance. The similarities observed in PlexA and PlexB functions may be explained by the findings that these receptors can assemble into a heteromultimeric complex, and also that they employ common downstream signaling components to guide axons during development. Finally, plexin interactions observed with different semaphorin ligands are likely to contribute the distinct roles PlexA and PlexB serve in establishing neuronal connectivity (Ayoob, 2006).

Plexin receptors expressed at the leading edge of navigating axonal growth cones receive and transduce instructive signals encoded by semaphorins. Deciphering how plexins translate external stimuli into intracellular responses is paramount for understanding how the nervous system is wired. Analysis of neural development in Drosophila allows for direct functional comparisons between plexins from distinct classes, as Drosophila contains only two plexin proteins. This study has analyzed plexB mutants and compared the phenotypes observed to those found in plexA mutants, detecting both similarities and differences in the LOF phenotypes of these two genes. A direct physical interaction was detected between PlexA and PlexB, and a convergence of these signaling pathways upon the effector molecule MICAL was demonstrated. These two receptors bind to semaphorin ligands from different classes. These results demonstrate that plexins from different classes can respond in vivo to distinct semaphorin ligands, but have the capacity to work cooperatively using common downstream signaling molecules (Ayoob, 2006).

In Drosophila, PlexA is required for the proper defasciculation of motor and CNS axon bundles (Winberg, 1998). This axon-axon repulsion enables individual axons to overcome the adhesive forces holding them together, to separate from each other, and to innervate their appropriate targets. This study examined the role played by PlexB in motor and CNS axon pathfinding during Drosophila embryogenesis and found that plexB mutants display defects in axon fasciculation that dramatically affect pathfinding. Similar to what has been observed in plexA mutants, plexB mutants display a failure of ISNb motor axons to initially separate from the main ISN bundle or, at later stages of ISNb pathway formation, to separate from other ISNb axons. Defasciculation errors similar to those observed in plexA mutants are also observed for the dorsal branch of the SNa in plexB mutants. However, other plexB mutant SNa axon bundles display navigation phenotypes not seen in plexA mutants. These dorsal SNa axons follow an aberrant trajectory to their target, muscle 24, and as a consequence are often unable to reach this post-synaptic partner. In the CNS, however, PlexB and PlexA play distinct roles. Loss of plexA disrupts the contiguity of the outermost bundle of axons, whereas losing plexB causes excessive defasciculation of the medial tract. This differential requirement for plexins in medial and lateral FasII-postive CNS axon bundles is strikingly reminiscent of the specific requirements for differential expression of roundabout (Robo) proteins to regulate the formation of the inner, medial and lateral FasII-positive axon tracts. Determining whether the positioning and consolidation of CNS longitudinal tracts by Robos and plexins are separate or integrated processes will lend insight into how axons respond simultaneously to distinct guidance influences that serve to regulate neuropil organization (Ayoob, 2006).

Although unique axonal fasciculation and pathfinding defects are observed in plexA and plexB mutants, ISNb motor axon phenotypes in these mutants are remarkably similar. This suggests that plexins from different classes may function collaboratively to pattern certain neuronal trajectories. Drosophila provides a robust experimental model with which to examine this issue. As there are only two Drosophila plexins, cross-rescue experiments were performed with plexA and plexB. Expression of plexA in a plexB mutant background significantly reduces the severity of plexA ISNb defects, although it does not fully rescue these defects. plexA expression is, however, unable to rescue the SNa and CNS phenotypes that were observed in plexB, but not plexA, mutants. In the reciprocal experiment, PlexB cannot replace any PlexA function, either in motor axon pathways or in the CNS. Immunoprecipitation and genetic interaction experiments provide an explanation for why PlexB cannot substitute for PlexA. When epitope-tagged versions of PlexA and PlexB were expressed in vivo, immunoprecipitating PlexA brings down PlexB, indicating that these two receptors can associate in a complex in vivo. Furthermore, for ISNb pathway phenotypes, genetic interactions were observed between plexB and MICAL heterozygotes, strongly supporting a requirement for MICAL in PlexB signaling, although these two proteins do not interact directly. It is proposed that PlexB gains access to MICAL through its association with PlexA. Because MICAL is a crucial downstream signaling component for plexin-mediated axonal repulsion, PlexA may be able to substitute in a limited fashion for PlexB through its ability to recruit MICAL and mediate repulsion of ISNb axons. However, the inability of PlexB to substitute at all for PlexA may stem from its inability to directly recruit MICAL (Ayoob, 2006).

Two other transmembrane proteins play important roles in PlexA-mediated axon guidance events and may facilitate the formation of complexes that contain PlexB and PlexA. The catalytically inactive receptor tyrosine kinase Off-track (Otk), which binds to and functions with PlexA in Sema-1a signaling, is also able to associate with two vertebrate plexins from classes A and B. It is unknown whether Otk binds to Drosophila PlexB. However, in the Drosophila CNS, Otk may function separately with PlexA and PlexB. Otk mutants display a disrupted outer Fas-II-positive fascicle, a phenotype specific to plexA, and also a defasciculated middle Fas-II-positive axon bundle, a phenotype specific to plexB. Overexpression of another PlexA signaling component produces phenotypes also seen in plexB mutants. Increasing in all neurons the levels of Gyc76C, a receptor guanylyl cyclase involved in PlexA signaling, produces an SNa pathfinding defect very similar to the 'double turn' SNa phenotype seen in plexB mutants (Ayoob, 2004). Future work will reveal whether either of these transmembrane proteins involved in PlexA signaling serve as co-receptors for PlexB ligands and participate in the PlexB signaling cascade (Ayoob, 2006).

There are five semaphorins in Drosophila. Sema-1a and Sema-1b, two class 1 transmembrane semaphorins, bind to PlexA. This study found that AP-tagged versions of the extracellular domains of these transmembrane semaphorins do not bind to PlexB in vitro. However, robust binding of AP-tagged Sema-2a, a secreted semaphorin, was observed to insect cells expressing PlexB. Genetic analysis shows that this interaction is indeed functional, since plexB LOF suppresses a Sema-2a GOF phenotype. These data also suggest that there are additional PlexB ligands. plexB mutants show more severe and complex phenotypes than do the low-penetrance phenotypes reported for Sema-2a mutants. Sema-2b, the other Drosophila secreted semaphorin, is a likely candidate PlexB ligand. Sema-2b resides at cytolocation 53C4 on chromosome 2 and is only separated from Sema-2a by a few genes. Sema-2a and Sema-2b share 70% amino acid identity (84% similarity), and it seems likely this semaphorin duo is a product of a genetic duplication and that these two secreted semaphorins share certain neuronal signaling functions. Sema-2b is expressed in a small subset of neurons within the CNS suggesting that, alone, or in combination with Sema-2a, it is responsible for maintaining the medial bundle of longitudinally projecting CNS axons as a tight fascicle (Kolodkin, 1993; Rajagopalan, 2000). Consistent with findings for class A and B plexins in vertebrates, it was found that PlexA and PlexB in Drosophila serve as receptors for different classes of semaphorins. This specificity provides a basis for postulating distinct functions for the two Drosophila plexins in motor and CNS axon guidance. Because secreted semaphorins are not tethered to their substrate, as are transmembrane semaphorins, the range over which these cues might act is greater, enabling PlexB to mediate not only axonal defasciculation, but also growth cone steering and surround repulsion (Ayoob, 2006).

In addition to being repulsive axon guidance receptors, plexins also interact homophilically. Therefore, it is possible that in some instances PlexB might function in a semaphorin ligand-independent manner, perhaps even as an adhesive molecule. Proteolytic processing may also regulate PlexB function. The extracellular domains of B-class plexins contain a protease site, located close to the plasma membrane, that is cleaved by a subtilisin-like proprotein convertase (Artigiani, 2003). In western blots of Myc-PlexB extracts, in addition to full-length PlexB at 250 kDa, a smaller protein is detected at 150 kDa. The size of this protein is equal to that of the PlexB ectodomain and correlates well with a predicted PlexB protease cleavage product. These bands were also observed in the lysates of S2R+ cells transfected with Myc-PlexB. Conditioned media from these transfected cells contains only the smaller (150 kDa) form of PlexB, presumably the ectodomain released from the membrane and into the media. This proteolytic processing of the PlexB receptor may play a role in the modulation of its activity (Ayoob, 2006).

In conclusion, evidence is presented that plexin B receptors, like plexin A receptors, are crucial for the generation of neuronal connectivity in vivo. The results show that A and B class plexins can regulate similar axon guidance events collaboratively, whereas interactions with distinct classes of semaphorin ligands are likely to mediate receptor-specific functions. Further analysis of how these guidance receptors function in Drosophila will allow for a better understanding of the complex roles played by plexins during neural development, and will define plexin-mediated convergent and divergent signaling events (Ayoob, 2006).

Plexin B mediates axon guidance in Drosophila by simultaneously inhibiting active Rac and enhancing RhoA signaling

Plexins are neuronal receptors for the repulsive axon guidance molecule Semaphorins. Plexin B (PlexB) binds directly to the active, GTP-bound form of the Rac GTPase. A seven amino acid sequence in PlexB is required for Rac^GTP binding. The interaction of PlexB with Rac^GTP is necessary for Plexin-mediated axon guidance in vivo. A different region of PlexB binds to RhoA. Dosage-sensitive genetic interactions suggest that PlexB suppresses Rac activity and enhances RhoA activity. Biochemical evidence indicates that PlexB sequesters Rac^GTP from its downstream effector PAK. These results suggest a model whereby PlexB mediates repulsion by coordinately regulating two small GTPases in opposite directions: PlexB binds to Rac^GTP and downregulates its output by blocking its access to PAK and, at the same time, binds to and increases the output of RhoA (Hu, 2001).

Plexin B binds to the active form of Rac (Rac^GTP); the binding maps to a 147 amino acid region, PlexBDelta3 (amino acids 1617 through 1765). To identify the critical binding sequence in PlexBDelta3, small deletions and point mutations were introduced and a seven amino acid sequence NTLAHYG (1722 through 1728) toward the C terminus of PlexBDelta3 has been identified that, when deleted, abolishes Rac binding (PlexBDelta3d7). Deletions in neighboring regions 1743 through 1759 (PlexBDelta3d17) and 1707 through 1714 do not affect Rac binding (Hu, 2001).

The NTLAHYG sequence is highly conserved among Plexin family members. In particular, the tyrosine residue within the sequence is invariable. In human Plexin B1, a putative Cdc42/Rac interactive binding (CRIB)-like motif right after this conserved sequence has been described. Although the CRIB-like motif is not found in Drosophila PlexB, this may reflect a conservation of the binding mechanism at a higher structural level. The Psi blast program predicts two blocks of sequences in the Plexin cytoplasmic domain that share similarity with R-ras family GAP proteins. The sequence needed for Rac^GTP binding is located between these two GAP-like regions (Hu, 2001).

In the Drosophila genome, there are six Rho family small GTPases: Rac1 (referred to here as Rac), Rac2, Cdc42, RhoA, Mtl, and RhoL. To gain some insight into the specificity of the interaction, the binding of PlexBDelta3 with all six Drosophila Rho-like GTPases was examined. Only Rac and Rac2, which share the highest degree of sequence similarity (93% identity), show strong interactions with the BDelta3 region of PlexB (Hu, 2001).

Several lines of evidence suggest that RhoA is also involved in PlexiB signaling. Clustering of the vertebrate PlexB in Swiss 3T3 cells leads to stress fiber formation, indicative of Rho activation. The response can be blocked by inhibitors of Rho or of its downstream effector Rho kinase. Genetic data also indicate that RhoA mediates part of Plexin B signaling in embryonic axon guidance. It was of interest, then, to enquire whether RhoA may also directly associate with PlexB (Hu, 2001).

PlexBDelta, a larger piece of the PlexB cytoplasmic domain (1617 through 1827) binds to RhoA. In contrast to a preferential binding to GTPgammaS-bound Rac, PlexBDelta binds to the GTPgammaS and GDP-bound forms of RhoA equally well. The binding requires the last 40 amino acids of PlexBDelta. The seven amino acid internal deletion that eliminates PlexBDelta binding to Rac does not affect its binding to RhoA. Thus, two independent regions in PlexB cytoplasmic domain have been defined that are important for PlexB association with Rac and RhoA, respectively. Cdc42, another Rho family GTPase, does not bind to PlexBDelta (Hu, 2001).

Dosage-sensitive genetic interactions suggest Rac antagonizes Plexin B signaling. Since there is no mutant available for PlexB with which to examine genetic interactions, whether the gain of function phenotype of PlexB is sensitive to the level of expression of Rac or RhoA was examined. PlexB is endogenously expressed by CNS neurons. Overexpression of PlexB in all embryonic CNS neurons can be achieved with the UAS-GAL4 binary expression system. Flies containing the UAS-PlexB transgene reporter are crossed to flies carrying a neuron-specific transcriptional control driver, elav-GAL4. With two independent UAS-PlexB transgenic lines, a consistent, GAL4-dependent phenotype was observed in specific motor nerve branches. In particular, a striking defect was observed in the ability of the ISNb (intersegmental nerve b) motor axons to innervate the ventral longitudinal muscles 7, 6, 13, and 12. In wild-type embryos, the ISNb projects into the ventral longitudinal muscles. Particular motor axons innervate specific muscles; for example, the RP3 motor axon innervates muscles 7 and 6, while other ISNb axons innervate muscles 13 and 12. When PlexB is overexpressed in these neurons, two types of phenotypes are observed that are consistent with PlexB being a repulsive guidance receptor for muscle-expressed Semaphorins: (1) the RP3 axon frequently fails to defasciculate from the ISNb motor nerve branch, and as a result muscles 7 and 6 are uninnervated; (2) ISNb axons often fail to reach their distal-most target muscle 12 (scored as 'stall'). The copy number of UAS-PlexB transgene and elav-GAL4 driver was varied to generate embryos with a range of levels of expression of PlexB; 'RP3 missing' and 'stall' phenotypes are dose dependent. This dosage sensitivity suggests that the PlexB gain-of-function phenotype may provide a sensitive background for revealing genetic interactions with genes encoding downstream components involved in Plexin B signaling (Hu, 2001).

Does the binding of PlexB to Rac increase or decrease the output of Rac? It was reasoned that if increasing PlexB expression produces its effect by activating Rac, then genetically limiting Rac gene dose might suppress the PlexB overexpression phenotype. Alternatively, if PlexB signals by turning down Rac activity, then an enhancement of the plexB overexpression phenotypes might result when Rac is reduced. Indeed, the results support the second alternative: PlexB inactivates Rac. Rac protein level was reduced by 50% using a small deficiency line, Df(3L)Ar14-8 (61C04-62A08), in a moderate PlexB overexpression background (one copy transgene, one copy driver). This resulted in a distinct increase in the penetrance of PlexB gain of function phenotypes. A complementary effect results when Rac dosage is increased in the same neurons where PlexB is overexpressed with a UAS-Rac transgene. Under such conditions, a suppression on the PlexB gain of function phenotypes is observed (Hu, 2001).

Consistent with the idea that PlexB signals by downregulating Rac activity, the Plexin gain-of-function stall phenotype is reminiscent of the loss-of-function phenotype of a positive regulator of Rac, the Trio GEF. Trio has been shown to play a role in axon guidance in Drosophila and nematode and has provided additional evidence that, in this capacity, Trio interacts with Rac and regulates PAK activity. Similar to reducing Rac, reducing Trio enhances PlexB gain-of-function phenotype. However, the enhancement caused by reducing Trio is not as great as that caused by reducing Rac. This probably reflects that Trio is not directly coupled to PlexB and is not the only positive regulator (GEF) for Rac in motor axons. Rather, Trio is likely to be one of many positive regulators of Rac in these axons (Hu, 2001).

The role of RhoA in PlexB signaling was examined by reducing RhoA gene dosage with two different RhoA mutant alleles, Rho^rev220 and RhoA^l(2)k07236. Instead of enhancing the PlexB gain-of-function phenotypes as the Rac deficiency does, partially removing RhoA suppresses the PlexB gain-of-function phenotypes. This result suggests that RhoA acts antagonistically to Rac and, moreover, that RhoA partially mediates Plexin B signaling (Hu, 2001).

To further test the model that PlexB downregulates Rac output, the effect of increasing Plexin was examined in Rac dominant-negative embryos. No mutant for Drosophila Rac has yet been published, but a loss-of-function analysis for Rac, achieved by overexpressing a dominant-negative form of Drac (N17Rac) in neurons, has revealed dramatic defects in motor axon guidance. The same ISNb nerve branch that is affected by PlexB overexpression is also sensitive to overexpression of dominant-negative Rac (N17Rac). The predominant ISNb defect in N17Rac embryos occurs at an earlier target entry point, where the whole ISNb branch normally branches off from ISN nerve. In N17Rac embryos, the ISNb fails to enter the ventral muscles and instead follows the ISN distally toward dorsal muscles (scored as 'bypass'). The difference in the quality of the ISNb phenotype of N17Rac and Plexin B gain of function embryos may likely reflect the fact that Rac is downstream of multiple guidance receptors (Hu, 2001).

The penetrance of the N17Rac bypass phenotype is very sensitive to gene dosage. When the N17Rac transgene is expressed using drivers of different strengths, different frequencies of defects result. This suggests that N17Rac only partially knocks out the wild-type gene function and that expressing N17Rac with a driver of medium strength may provide a sensitized background for testing genes that regulate the remaining Rac activity. It was reasoned that if the Plexin and Rac interaction regulates Rac activity, then it might be possible to alter the penetrance of the N17Rac bypass phenotype by simultaneously increasing PlexB gene dose in the same neurons. Indeed, coexpressing PlexB and RacN17 results in a distinct enhancement of the ISNb bypass phenotypes. N17Rac embryos also show bypass defects in the SNa motor axons that project to lateral muscle targets. This SNa bypass phenotype has never been observed in any other mutant background, and it also turns out to be enhanced by simultaneously overexpressing PlexB in these neurons (Hu, 2001).

In a reciprocal experiment PlexB was reduced in Rac dominant-negative embryos to see if this had an opposite effect. This was done by injecting double-strand RNA of PlexB into N17Rac embryos. N17Rac embryos injected with Plexin B dsRNA show distinct reduction in bypass defects compared with N17Rac embryos injected with buffer. Thus, reducing PlexB and increasing PlexB in Rac dominant embryos produces opposite modulations, consistent with the model that PlexB downregulates Rac activity (Hu, 2001).

To test whether the PlexB gain-of-function and the genetic interactions depend on the direct association between PlexB and Rac, a mutant PlexB transgene, UAS-Plex Bd7, was constructed containing the seven amino acid NTLAHYG deletion in the Rac binding region of an otherwise wild-type PlexB. The same enhancement test on N17Rac embryos was performed with this mutant transgene, and no enhancement was observed. The PlexB gain-of-function phenotypes also seem to be dependent on this Rac binding region. In contrast to wild-type PlexB, when the d7 mutant transgene is overexpressed under the control of the same neuronal GAL4 driver elav, the frequency of ISNb phenotypes is significantly lower. This low-penetrance phenotype is not enhanced by removing one copy of Rac (Hu, 2001).

In vivo expression and targeting of PlexBd7 transgene could not be examined due to the lack of PlexB antibody. Nevertheless, the seven amino acid deletion does not affect protein expression and stability of PlexBDelta3 in in vitro experiments. Three independent lines of PlexBd7 transgenes show consistent behavior when tested for their phenotypes and interactions with Rac, arguing that the negative result is not caused by the insertion site (Hu, 2001).

In light of the genetic interactions between PlexB and Rac, the biochemical nature of this negative regulation was investigated. PlexB was found to compete with the Rac downstream effector PAK (p21-activated kinase) for binding to Rac^GTP. PAK is a serine/threonine kinase that mediates a major part of Rac signaling output to actin polymerization. Upon binding to Rac^GTP, PAK undergoes a conformational change that releases an autoinhibition on the kinase domain and becomes active. Since Rac binding is critical for PAK activation and also because PlexB and PAK bind to Rac in the same GTP-dependent manner, it was asked whether PlexB and PAK may bind to the same region of Rac and whether their binding to Rac^GTP is mutually exclusive (Hu, 2001).

An in vitro pull-down competition assay was used in which in vitro translated L61Rac was incubated with bead-bound GST-PAK^1-141 in the presence or absence of soluble PlexB protein fragment: PlexBDelta2(1619-1753). (PlexBDelta2 is a 135 amino acid fragment of PlexB. It binds to Rac^GTP equally well as PlexBDelta3, but it can be expressed at a higher level.) When Plexin BDelta2 is present in the binding solution, the amount of RacL61 pulled down by PAK^1-141 is greatly reduced. The extent of reduction is dependent on the amount of PlexBDelta2 used. At the 48:1 molar ratio of PlexBDelta2 to PAK^1-141, the reduction is close to complete. PlexBDelta2d7, a deletion PlexB fragment that is incapable of binding to Rac, does not compete with PAK for the Rac binding. Conversely, the presence of PAK protein fragment PAK^78-151 also reduces RacL61 binding to PlexBDelta3. This shows that the binding of the two proteins to Rac^GTP is indeed mutually exclusive (Hu, 2001).

Does this competition exist in vivo? If it does, then it may be expected that overexpressing PAK together with PlexB in embryos will cancel out PlexB gain-of-function effect. Indeed, overexpressing PAK in a PlexB gain-of-function background suppresses the phenotypes of the latter, demonstrating that PlexB signaling can be antagonized by the Rac effector PAK in vivo (Hu, 2001).

It is concluded that PlexB mediates repulsion in vivo in part by binding to active Rac (Rac^GTP) and downregulating its effector output and in part by binding to and activating RhoA. Biochemical analysis shows that PlexB binds to Rac^GTP. A seven amino acid sequence in the cytoplasmic domain of PlexB is required for this binding. Genetic analysis shows that PlexB downregulates the output of Rac^GTP. Removal of one copy of Rac enhances a PlexB gain-of-function phenotype, while overexpression of PlexB enhances a Rac dominant-negative phenotype in motor axon guidance. Overexpression of a mutant form of PlexB that lacks the seven amino acid sequence required for Rac binding does not generate its own gain-of-function phenotype, and it does not enhance a Rac dominant-negative phenotype. It is also shown that PlexB binds to RhoA through a different region of its cytoplasmic domain. Although the biochemical mechanism is not known, genetic analysis suggests that PlexB increases the output of RhoA (Hu, 2001).

The results presented here allow a confirmation and extension of a current model concerning the role of GTPases in axon guidance. This model suggests that attractive guidance cues locally activate Rac or Cdc42 in the growth cone while repulsive guidance cues locally activate RhoA. It is argued that what is important is the relative balance in the output of Rac versus RhoA. An example is provided in which the PlexiB receptor mediates repulsive axon guidance by downregulating Rac^GTP output and simultaneously upregulating RhoA output. A coordinate regulation of these two small GTPases may allow the receptor to have a finer control over actin regulatory machinery. Semaphorin signaling can be converted from repulsion to attraction by changes in cGMP level. It would be interesting to test whether and how the cGMP signaling can affect this Rac/Rho balance (Hu, 2001).

Drosophila has two Plexins: A and B. Both Plexin A and B are highly expressed in the central nervous system. The two proteins share high sequence similarity in their cytoplasmic domain, indicating a similar mode of signaling shared by the two. A direct physical association of Rac^GTP with PlexB but not with PlexA has been demonstrated. However, genetic interactions have been found between Rac and both Plexins. For example, increasing PlexA also enhances the Rac dominant-negative phenotype as does PlexB. In COS cell and DRG neurons, Rac shows coclustering with PlexA upon Sema3A ligand treatment. It is likely that ligand binding to PlexA causes Rac binding (and subsequent inactivation of Rac) just as with PlexB, but it may be that PlexA requires an unknown third protein to help mediate or facilitate this physical interaction. From a genetic perspective, they both appear to function in the same way, mediating repulsion at least in part by inactivating Rac (Hu, 2001).

The transmembrane protein OTK associates with Plexin A and contributes to the Sema 1a/Plexin A signaling pathway. Mammalian Plexin B1 also coimmunoprecipitates with OTK. In the future, it will be interesting to test whether PlexB also interacts with OTK in vivo and to what degree the Rac/Rho GTPases and OTK signaling pathways function together or in parallel downstream of Plexins (Hu, 2001).

Rac interacts with Plexin B

Semaphorins and their receptors, plexins, are widely expressed in embryonic and adult tissues. In general, their functions are poorly characterized, but in neurons they provide essential attractive and repulsive cues that are necessary for axon guidance. The Rho family GTPases Rho, Rac, and Cdc42 control signal transduction pathways that link plasma membrane receptors to the actin cytoskeleton and thus regulate many actin-driven processes, including cell migration and axon guidance. Using yeast two-hybrid screening and in vitro interaction assays, it has been shown that Rac in its active, GTP bound state interacts directly with the cytoplasmic domain of mammalian and Drosophila B plexins. Plexin-B1 clustering in fibroblasts does not cause the formation of lamellipodia, which suggests that Rac is not activated. Instead, it results in the assembly of actin:myosin filaments and cell contraction, which indicates Rho activation. Surprisingly, these cytoskeletal changes are both Rac and Rho dependent. Clustering of a mutant plexin, lacking the Rac binding region, induces similar cytoskeletal changes, and this finding indicates that the physical interaction of plexin-B1 with Rac is not required for Rho activation. The findings that plexin-B signaling to the cytoskeleton is both Rac and Rho dependent form a starting point for unraveling the mechanism by which semaphorins and plexins control axon guidance and cell migration (Driessens, 2001).

Many previously identified Rac targets contain a distinctive Rac binding site, the CRIB motif, but sequence analysis does not reveal any obvious CRIB-like sequence in plexin-B1. To identify the region of plexin-B1 that contains the Rac interaction site, a series of truncations were expressed as GST fusion proteins in E. coli. These were used in a dot blot assay. Rac interacts with a region encompassing 180 residues (amino acids 1724-1903) of the receptor (Driessens, 2001).

Plexin-B1 is a member of a large family of transmembrane proteins, and based on sequence alignments, four classes of plexins (A, B, C, and D) have been described. To test whether Rac could interact directly with other members of the family, cDNAs were obtained for human plexin-A2 (kiaa0463), plexin-B2 (kiaa0315), and plexin-D1 (kiaa0620). A region corresponding to amino acids 1724-1903 of plexin-B1 was cloned into the pGEX vector; GST fusion proteins were analyzed in the dot blot assay, but under these conditions only plexin-B1 was found to interact (Driessens, 2001).

In Drosophila, two plexins have been identified: Drosophila plexin-A and Drosophila plexin-B. Recombinant Drosophila plexin-B protein (C-terminal 435 amino acids, similar to plexin-B1 two-hybrid clone) interacts strongly with in vitro translated Drosophila L61Rac1 and weakly with wild-type Drosophila Rac1 in a pull-down experiment. Drosophila plexin-A does not interact with Drosophila Rac1 under the same conditions. A Drosophila plexin-B fragment corresponding to amino acids 1724-1903 of human plexin-B1 interacts similarly with Drosophila Rac1, as does a shorter, 149 amino acid region. Partial binding was observed with a 54 amino acid domain (Driessens, 2001).

Two blocks of sequence similarity, of approximately 320 and 150 amino acids each, have been identified in plexin cytoplasmic domains. These two blocks of sequence similarity are separated by a variable linker. This linker region is most divergent between the plexin subfamilies. The minimal Rac binding region in Drosophila plexin-B consists of the last 149 amino acids of the first conserved block but does not contain the linker region. Alignment of this 149 amino acid region of Drosophila plexin-B with other human plexins reveals a sequence highly conserved among all plexin subfamilies (Driessens, 2001).

A mechanism is proposed for plexin-B signaling to the actin cytoskeleton. In this mechanism, clustering of B plexins induces a Rac-dependent activation of Rho. These results provide a framework for the further exploration of the complex mechanisms by which plexins affect the actin cytoskeleton in different cell types, including neurons (Driessens, 2001).

Search PubMed for articles about Drosophila Plexin B

Artigiani, S., Barberis, D., Fazzari, P., Longati, P., Angelini, P., van de Loo, J. W., Comoglio, P. M. and Tamagnone, L. (2003). Functional regulation of semaphorin receptors by proprotein convertases. J. Biol. Chem. 278: 10094-10101. PubMed citation: 12533544

Ayoob, J. C., Yu, H.-H. Terman, J. R. and Kolodkin, A. L. (2004). The Drosophila receptor Guanylyl cyclase Gyc76C is required for Semaphorin-1a-Plexin A-mediated axonal repulsion. J. Neurosci. 24(30): 6639-6649. PubMed citation; Online text

Ayoob, J. C., Terman, J. R. and Kolodkin, A. L. (2006). Drosophila Plexin B is a Sema-2a receptor required for axon guidance. Development 133: 2125-2135. PubMed citation: 16672342

Bates, K. E. and Whitington, P. M. (2007). Semaphorin 2a secreted by oenocytes signals through plexin B and plexin A to guide sensory axons in the Drosophila embryo. Dev. Biol. 302(2): 522-35. PubMed citation: 17109838

Driessens, M. H. E., et al. (2001). Plexin-B semaphorin receptors interact directly with active Rac and regulate the actin cytoskeleton by activating Rho Curr. Biol. 11: 339-344. PubMed citation: 11267870

Hu, H., Marton, T. F. and Goodman, C. S. (2001). Plexin B mediates axon guidance in Drosophila by simultaneously inhibiting active Rac and enhancing RhoA signaling. Neuron 32: 39-51. 11604137

Isbister, C. M., et al. (2003). Gradient steepness influences the pathfinding decisions of neuronal growth cones in vivo. J. Neurosci. 23: 193-202. PubMed citation: 12514216

Kolodkin, A. L., Matthes, D. J. and Goodman, C.S. (1993). The semaphorin genes encode a family of transmembrane and secreted growth cone guidance molecules. Cell 75: 1389-1399. PubMed citation: 8269517

Kruger, R. P., Aurandt, J. and Guan, K. L. (2005). Semaphorins command cells to move. Nat. Rev. Mol. Cell. Biol. 6: 789-800. PubMed citation: 16314868

Messersmith, E. K., et al. (1995). Semaphorin III can function as a selective chemorepellent to pattern sensory projections in the spinal cord. Neuron 14: 949-959. PubMed citation: 7748562

Negishi, M., Oinuma, I. and Katoh, H. (2005). Plexins: axon guidance and signal transduction. Cell. Mol. Life Sci. 62: 1363-1371. PubMed citation: 15818466

Oinuma, I., Katoh, H., Harada, A. and Negishi, M. (2003). Direct interaction of Rnd1 with Plexin-B1 regulates PDZ-RhoGEF-mediated Rho activation by Plexin-B1 and induces cell contraction in COS-7 cells. J. Biol. Chem. 278: 25671-25677. PubMed citation: 12730235

Oinuma, I., Ishikawa, Y., Katoh, H. and Negishi, M. (2004). The Semaphorin 4D receptor Plexin-B1 is a GTPase activating protein for R-Ras. Science 305: 862-865. PubMed citation: 15297673

Rajagopalan, S., Vivancos, V., Nicolas, E. and Dickson, B. J. (2000). Selecting a longitudinal pathway: Robo receptors specify the lateral position of axons in the Drosophila CNS. Cell 103: 1033-1045. PubMed citation: 11163180

Swiercz, J. M., et al. (2002). Plexin-B1 directly interacts with PDZ-RhoGEF/LARG to regulate RhoA and growth cone morphology. Neuron 35: 51-63. PubMed citation: 12123608

Terman, J. R., et al. (2002). MICALs, a family of conserved flavoprotein oxidoreductases, function in Plexin-mediated axonal repulsion. Cell 109: 887-900. PubMed citation: 12110185

Toyofuku, T., Yoshida, J., Sugimoto, T., Zhang, H., Kumanogoh, A., Hori, M. and Kikutani, H. (2005). FARP2 triggers signals for Sema3A-mediated axonal repulsion. Nat. Neurosci. 8: 1712-1719. PubMed citation: 16286926

Winberg, M. L., et al. (1998). Plexin A is a neuronal semaphorin receptor that controls axon guidance. Cell 95(7): 903-16. PubMed citation: 9875845

date revised: 20 March 2008

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