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Guanylyl cyclase at 76C: Biological Overview | References

Cyclic nucleotide levels within extending growth cones influence how navigating axons respond to guidance cues. Pharmacological alteration of cAMP or cGMP signaling in vitro dramatically modulates how growth cones respond to attractants and repellents, although how these second messengers function in the context of guidance cue signaling cascades in vivo is poorly understood. Using a novel Sema-1a-dependent forward genetic screening approach, it was found that Drosophila receptor-type guanylyl cyclase: Gyc76C, a protein possessing a single transmembrane domain, is required for semaphorin-1a (Sema-1a)-plexin A repulsive axon guidance of motor axons in vivo. Genetic analyses define a neuronal requirement for Gyc76C in axonal repulsion. Additionally, it was found that the integrity of the Gyc76C catalytic cyclase domain is critical for Gyc76C function in Sema-1a axon repulsion. These results support a model in which cGMP production by Gyc76C facilitates Sema-1a-plexin A-mediated defasciculation of motor axons, allowing for the generation of neuromuscular connectivity in the developing Drosophila embryo (Ayoob, 2004).

During neural development, axons extend along complex, but precisely defined, routes to contact their appropriate targets and establish the connectivity of the adult nervous system. Guidance cues belonging to several families have been identified that direct axons along these pathways through attractive and repulsive mechanisms. For many of these extracellular cues, including ephrins, netrins, slits, and semaphorins, cell surface receptors have been identified that are required for the establishment of these neuronal trajectories. The signal transduction pathways by which these guidance cue receptors direct the cytoskeletal alterations critical for attractive or repulsive steering events, however, are only now beginning to be understood (Ayoob, 2004).

Two well characterized intracellular effectors that can dictate how an axon responds to extracellular signals are the second messengers cAMP and cGMP. Experiments with cultured Xenopus spinal neurons, in an in vitro growth cone turning assay, showed that changing the intracellular levels of cAMP or cGMP alters how an axon responds to extracellular guidance cues. For example, the attractive response of an axon to the guidance cue netrin-1 can be converted to repulsion by decreasing the effective levels of cAMP within the responding neuron. Conversely, the axonal response to the potent chemorepellent semaphorin 3A (Sema3A) can be converted from repulsion to attraction by increasing cGMP levels within the neuron. cAMP and cGMP have been shown to function together to influence how an axon responds to a particular attractant or repellent. The ratio of cAMP to cGMP determines how extending axons respond to netrin-1 in the growth cone turning assay: high cAMP to cGMP ratios produce an attractive response, whereas low ratios lead to repulsion (Nishiyama, 2003). Related observations have been made for Sema3A. When cAMP levels are raised in cultured neurons, the potent growth cone collapsing effect of Sema3A is neutralized (Dontchev, 2002; Chalasani, 2003). However, in these same cultures, raising the levels of cGMP potentiates the growth cone collapsing effect of Sema3A, suggesting that both cyclic nucleotides can modulate the response to a single axon guidance cue (Ayoob, 2004 and references therein).

The molecular mechanisms underlying axon guidance effects caused by pharmacologically altering cyclic nucleotide levels are still unclear. Insight into how cAMP dictates axonal steering responses has been gained from the identification of Nervy, a protein that couples plexin A (PlexA), the receptor for the invertebrate transmembrane semaphorin-1a (Sema-1a), with the cAMP-dependent protein kinase A (PKA). Sema-1a is present on motor axons in the developing Drosophila nervous system and through its receptor PlexA acts as a repellent and directs individual axons away from the tightly fasciculated bundles in which they travel. Nervy tethers PKA to the PlexA, positioning PKA to antagonize Sema-1a-mediated repulsion in response to local increases in cAMP (Ayoob, 2004).

Several studies provide hints as to which proteins involved in cGMP signaling may be involved in modulating or supporting axon guidance events (Gibbs, 1998; Polleux, 2000; Seidel, 2000; Schmidt, 2002; Nishiyama, 2003). How specific proteins function in particular axon guidance signaling pathways to alter cGMP levels is, however, unknown. Using a novel Sema-1a-dependent forward genetic screening approach, it was found that the Drosophila receptor guanylyl cyclase (rGC) Gyc76C, a member of the phylogenetically conserved family of single transmembrane domain guanylyl cyclases (Wedel, 2001), is necessary for Sema-1a-mediated repulsive signaling in the developing Drosophila embryonic nervous system. Furthermore, the data strongly suggest that cGMP production by Gyc76C is essential for its function in vivo. Together, these findings provide a functional link between local production of cGMP within the growth cone and Sema-1a repulsive axon guidance signaling (Ayoob, 2004).

These experiments provide an important molecular link between semaphorin-mediated repulsion and cGMP signaling in vivo. Gyc76C is critical for Sema-1a-Plexin A-mediated selective defasciculation of axon bundles in the developing Drosophila neuromuscular system. A conserved amino acid residue within the Gyc76C cyclase domain, a residue required for receptor guanylyl cyclase (rGC) catalytic activity, is also required in Gyc76C for correct motor axon pathfinding. The identification of Gyc76C as an essential component of the Sema-1a-PlexA repulsive axon guidance signaling pathway provides insight into how cyclic nucleotide production is linked to the cascade of events downstream of semaphorin-mediated repulsion. These observations also provide a potential target for modulating repulsive semaphorin signaling by alterations of cGMP levels directly through rGCs (Ayoob, 2004).

These analyses demonstrate a role for the rGC Gyc76C in Sema-1a-mediated axon-axon repulsion. LOF mutations were generated in the Gyc76C gene and highly penetrant phenotypes were observed similar to the motor axon guidance defects observed in sema1a, plexA, and mical mutants. Micals are a family of conserved flavoprotein oxidoreductases that function in Plexin-mediated axonal repulsion (Terman, 2002). Neuronal expression of a Gyc76C cDNA restores the wild-type innervation pattern in gyc76C mutant embryos and also restores viability to the lethal gyc76C mutant line, demonstrating a requirement for Gyc76C in neurons for correct axonal pathfinding. Neuronal overexpression of wild-type Gyc76C also results in phenotypes resembling PlexA GOF phenotypes. The genetic interaction analyses confirm a role for Gyc76C in Sema-1a-PlexA repulsive signaling. Embryos heterozygous for both Gyc76C and other members of this signaling cascade, including Sema-1a, PlexA, and MICAL, display motor axon pathway disruptions. These phenotypes are qualitatively similar to LOF mutant phenotypes observed in sema1a, plexA, and mical LOF mutants and are seen at comparable frequencies. In addition to suppressing the Sema-1a- dependent midline phenotype, loss of Gyc76C function also suppresses a PlexA- dependent phenotype. However, increasing the levels of Gyc76C enhances this PlexA GOF phenotype. Finally, a Gyc76C transgene lacking a key conserved aspartate residue required for cyclase catalytic activity does not rescue either the gyc76C embryonic motor axon guidance defects or the lethality associated with gyc76C mutants and appears to function in a dominant-negative manner. Taken together, these results link Gyc76C to the proper generation of neuromuscular connectivity in Drosophila through its role in mediating semaphorin-plexin signaling events associated with axonal repulsion. In addition, these results strongly suggest that cGMP production is critical for Gyc76C participation in Sema1a neuronal signaling events (Ayoob, 2004).

Initial in vitro observations demonstrating the importance of cGMP levels in semaphorin-mediated repulsion shows that increasing cGMP signaling reverses the repulsive signal from the secreted vertebrate semaphorin Sema3A, resulting in Sema3A acting as an attractant in the single growth cone steering assay. Recent studies show that Sema3A growth cone collapse requires increased cGMP signaling and also that cAMP signaling acts in opposition to cGMP signaling in the modulation of Sema3A-mediated growth cone collapse. Support for cAMP signaling cascades modulating semaphorin-mediated repulsion in vivo is provided by a demonstration that the A-kinase anchoring protein Nervy serves to antagonize Sema-1a-mediated axonal repulsion in Drosophila motor axons. Presumably, Nervy acts by localizing cAMP activation of PKA to the Plexin receptor and decreases Sema-1a repulsive signaling (Terman, 2004). The identification of Gyc76C as a positive effector in vivo of Sema-1a-PlexA-mediated repulsion is consistent with these Sema3A growth cone collapse studies. A model recently proposed for cyclic nucleotide modulation of netrin-1-mediated attraction and repulsion provides insight into how cGMP might effect semaphorin-mediated steering, collapse, and in vivo axonal repulsion. Using the in vitro growth cone steering assay, Nishiyama (2004) has shown that the [cAMP]/[cGMP] ratio determines whether netrin-1 acts in an attractive or a repulsive manner: high ratios promote attraction, whereas lower ratios promote repulsion. Importantly, a basal level of cGMP signaling is required for both netrin-mediated attractive and repulsive responses in this system. Although it remains to be determined, it is tempting to speculate that, like the observations for netrin-1-mediated guidance, the [cAMP]/[cGMP] ratio also serves to modulate semaphorin signaling events. In Drosophila motor axons, Gyc76C and Nervy could function antagonistically to regulate Sema-1a signaling in this manner. Gyc76C production of cGMP would lower a [cAMP]/[cGMP] ratio and thus promote repulsion, whereas increases in cAMP levels would decrease repulsion through PKA tethered to PlexA by Nervy. A loss of Gyc76C altogether would result in abolition of Sema-1a repulsion because of a cGMP requirement for any guidance response, and this is what was observed in the gyc76C mutants. Future experiments will determine how raising or lowering Gyc76C activity affects the guidance response to Sema-1a in vivo (Ayoob, 2004).

This study describes a role for a receptor-type guanylyl cyclase in axon guidance as an effector of transmembrane Sema1a axonal repulsion. Soluble guanylyl cyclases in both vertebrates and invertebrates have been implicated in axonal and dendritic guidance. However, in a GOF genetic screen for Sema-1a signaling components, genomic regions containing genes encoding all of the identified Drosophila soluble guanylyl cyclase subunits were assayed, including one known to be expressed in the nervous system, yet heterozygosity at these loci did not suppress or enhance the Sema-1a GOF phenotype. This may reflect a requirement for cGMP production at or near the PlexA receptor to provide a local increase in cGMP levels essential for semaphorin-mediated axonal repulsion and suggests that basal cGMP signaling provided by soluble gyanylyl cyclases is not essential for semaphorin-mediated repulsion. The initial genetic screen covered an additional two of the seven Drosophila rGCs, however, neither of the deficiencies that remove these rGCs genetically interacted with the Sema-1a GOF phenotype. Taken together, these results from the genetic screen suggest that Gyc76C is an integral component of the semaphorin signaling cascade and that cGMP production by other sources may not contribute to this repulsion. These results also motivate future experiments to investigate specific interactions between Gyc76C and PlexA (Ayoob, 2004).

Vertebrate receptor guanylyl cyclases that have a single transmembrane domain like Gyc76C are best known for their roles as receptors for natriuretic peptides that regulate blood pressure and volume and also for their role in the visual phototransduction cascade. The other vertebrate rGCs, however, have no known ligands or functions. In addition, very little is known about what roles, if any, these vertebrate rGCs play during neural development. It will be of great interest to investigate whether any vertebrate rGCs participate in semaphorin repulsive signaling (Ayoob, 2004).

Because Gyc76C is a multidomain protein, it is likely that regions other than the cyclase domain are important for its function. Interestingly, like the transmembrane protein Off-track, which is also required for Sema1a-mediated motor axon repulsion in Drosophila, Gyc76C contains a catalytically inactive kinase homology domain (KHD). In the vertebrate receptor guanylyl cyclase GC-A, this region has been shown to play a regulatory role by inhibiting the catalytic cyclase domain. The KHD of Gyc76C, or possibly Off-track, may function as an important modulator of cyclase activity (Ayoob, 2004).

The portion of Gyc76C that is C terminal to the conserved cyclase domain is unique among rGC family members; it is much longer than the same region in other rGCs and shares no amino acid similarity with these regions or with sequences of any known proteins. However, the last four amino acids of Gyc76C fit the consensus for a PDZ (PSD-95, Discs-large, zona occludens-1) domain binding motif. A similar motif is also found in MICAL, another component of the Sema-1a signaling cascade (Terman, 2002), raising the possibility that, as has been observed for other assemblages of signaling components, PDZ domain-containing scaffolding proteins may serve an important role in semaphorin signaling (Ayoob, 2004).

Gyc76C may provide a direct physical link between the leading edge of the growth cone and the motile machinery of the actin cytoskeleton. Vertebrate rGCs in photoreceptors are able to bind actin filaments, and the C-terminal domains of intestinal rGCs have also been implicated in interactions with the actin cytoskeleton. Perhaps the large C-terminal extension of Gyc76C functions in a similar manner to bridge the regions of signal reception and output. Whether or not Gyc76C cyclase activity is ligand gated remains unknown, and like all other Drosophila rGCs and the majority of vertebrate rGCs, Gyc76C is an orphan receptor. Future experiments will address whether Sema-1a triggers Gyc76C catalytic activity and also whether Gyc76C is indeed part of the receptor complex for Sema-1a (Ayoob, 2004).

In conclusion, using a novel genetic screening paradigm for identifying semaphorin signaling cascade components, an in vivo link was found between Sema-1a-mediated repulsive guidance and cGMP signaling pathways. Characterization of other candidates from this screen will likely provide additional insight into the mechanisms of repulsive axon guidance signaling (Ayoob, 2004).

Search PubMed for articles about Drosophila Gyc76c

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. Medline abstract: 15282266

Chalasani, S. H., et al. (2003). A chemokine, SDF-1, reduces the effectiveness of multiple axonal repellents and is required for normal axon pathfinding. J. Neurosci. 23: 1360-1371. Medline abstract: 12598624

Dontchev, V. D. and Letourneau, P. C. (2002). Nerve growth factor and semaphorin 3A signaling pathways interact in regulating sensory neuronal growth cone motility. J. Neurosci. 22: 6659-6669. Medline abstract: 12151545

Gibbs, S. M. and Truman. J. W. (1998). Nitric oxide and cyclic GMP regulate retinal patterning in the optic lobe of Drosophila. Neuron 20: 83-93. Medline abstract: 9459444

Gibbs, S. M., Becker, A., Hardy, R. W. and, Truman, J. W. (2001). Soluble guanylate cyclase is required during development for visual system function in Drosophila. J. Neurosci. 21: 7705-7714. Medline abstract: 11567060

Nishiyama, M., et al. (2003). Cyclic AMP/GMP-dependent modulation of Ca2+ channels sets the polarity of nerve growth-cone turning. Nature 424: 990-995. Medline abstract: 12827203

Polleux, F., Morrow, T. and Ghosh, A. (2000). Semaphorin 3A is a chemoattractant for cortical apical dendrites. Nature 404: 567-573. Medline abstract: 10766232

Schmidt, H., et al. (2002). cGMP-mediated signaling via cGKIalpha is required for the guidance and connectivity of sensory axons. J. Cell. Biol. 159: 489-498. Medline abstract: 12417579

Seidel, C. and Bicker, G. (2000). Nitric oxide and cGMP influence axonogenesis of antennal pioneer neurons. Development 127: 4541-4549. Medline abstract: 11023858

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

Terman, J. R. and Kolodkin, A. L. (2004). Nervy links protein kinase a to plexin-mediated semaphorin repulsion. Science 303: 1204-1207. 14976319

Wedel, B. and Garbers, D. (2001). The guanylyl cyclase family at Y2K. Annu. Rev. Physiol. 63: 215-233. Medline abstract: 11181955

date revised: 2 December 2007

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