|Ph.D Student||Smolkin Tatyana|
|Subject||Characterization of Functional Receptor Complexes for|
|Department||Department of Medicine||Supervisor||PROFESSOR EMERITUS Gera Neufeld|
|Full Thesis text|
Semaphorins are a large family of secreted and membrane-bound proteins that were first described as axon guidance factors in the nervous system, but are known today to participate in a variety of biological processes. They are divided into 8 subclasses, distinguished by class-specific structural motifs. Signals of the secreted class-3 semaphorins are mediated by binding to the neuropilin-1 or neuropilin-2 co-receptors which are not able to transduce semaphorin signals independently, due to their short intracellular domains. To transduce class-3 semaphorin signals, the neuropilins associate with type-A plexin receptors or with PlexinD1, which serve as the signal transducers, after binding a class-3 semaphorin. Class-3 semaphorins were found to function as anti-angiogenic and anti-tumorigenic guidance factors. To study the role of the PlexinA2 receptor in semaphorin signaling, we silenced its expression in endothelial cells and in glioblastoma cells. The silencing did not affect Sema3A signaling, which requires neuropilin-1, PlexinA1 and PlexinA4, but completely abolished Sema3B signaling, which required also PlexinA4 and one of the two neuropilins. Interestingly, overexpression of PlexinA2 in PlexinA1- or PlexinA4-silenced cells restored responses to both semaphorins, thereby nullifying the ability of the cells to distinguish between them. These results also suggest that overexpressed PlexinA2 may perhaps functionally replace the other class-A and class-D plexins, and that this may be true for other plexins too. However, although PlexinA4 overexpression restored Sema3A signaling in PlexinA1-silenced cells, it failed to restore Sema3B signaling in PlexinA2-silenced cells suggesting that not every plexin can compensate for the loss of every other plexin in cells. Our results suggest that differential changes in the expression levels of plexins induced by microenvironmental cues can change the responses of different populations of migrating cells to encountered gradients of semaphorins.
Sema3E is the only known class-3 semaphorin, capable of neuropilin independent signaling through PlexinD1. To study the role of neuropilins in functional class-3 semaphorin receptor complexes we expressed high levels of PlexinD1 in glioblastoma cells, which express insignificant levels of this plexin. PlexinD1 expression enabled signal transduction induced by Sema3C, Sema3D, Sema3E and Sema3G, suggesting that PlexinD1 is an essential component of the receptor complexes of these semaphorins. Interestingly, CRISPR/Cas9 induced knock-out of both neuropilin-1 and neuropilin-2 in cells expressing recombinant-PlexinD1 did-not abolish Sema3C signaling, indicating that Sema3C too can signal independently of neuropilins. However, when present, the neuropilins increased the sensitivity to Sema3C by about five-fold. Interestingly, when the expression of PlexinA4 was silenced by shRNA in cells lacking neuropilins and expressing PlexinD1, Sema3C signaling was abolished. This observation suggests that in the absence of neuropilins, PlexinA4 and PlexinD1 are required for successful Sema3C signaling. However, silencing PlexinA4 in cells expressing neuropilins and PlexinD1, did-not inhibit Sema3C signaling, suggesting that PlexinA4 can partially compensate for loss of neuropilins in PlexinD1 expressing cells
Unlike Sema3C, Sema3D and Sema3G could not signal in PlexinD1 expressing cells, silenced for both neuropilins. These results suggest that neuropilins and PlexinD1 are strictly required to enable signaling induced by Sema3D and Sema3G. Silencing each neuropilin in turn revealed that Sema3D utilizes either neuropilin-1 or neuropilin-2, while Sema3G is neuropilin-2 specific.