Turner, Laura; (2003) The involvement of Rho GTPases in plexin mediated signal transduction. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Plexins are a family of conserved transmembrane proteins. Together with neuropilins, they act as receptors for the semaphorin family of growth cone guidance molecules. Whilst it is clear that guidance involves cytoskeletal changes, little is known of the mechanisms via which plexins regulate growth cone morphology. Hints suggesting the involvement of Rho GTPases stem from observations of localised actin rearrangements elicited by plexins and semaphorins. This feeling is consolidated by the demonstration of the Rac dependent nature of plexin induced cellular responses. To investigate plexin mediated signal transduction, a heterologous assay for semaphorin induced collapse has been developed and characterised. Studies using jasplakinolide indicate that Sema3A-Fc induced collapse requires actin dissassembly. Interestingly, a similar collapsed phenotype is observed in cells treated with latrunculin A. It is possible that Sema3A-Fc and latrunculin A may utilise comparable intracellular machinery to achieve similar morphological outcomes. Experiments using GTPase mutants demonstrated that Sema3A-Fc induced collapse required Rac and Cdc42, but not RhoA. In addition, Sema3A-Fc stimulation led to Rac activation prior to morphological collapse. Interestingly, collapse induced by constitutively active Plexin-Al did not require Rac. This suggests that Rac may act upstream of Plexin-Al, perhaps regulating the activity of Plexin-Al itself Evidence that Plexin-Al interacts directly with Rac.GTP supports this theory. A putative inter-or intramolecular interaction was identified within the cytoplasmic tails of Plexin-Al and Plexin-Bl. As the critical residues required for these interactions are also essential for Rac binding, these interactions and the association between Rac and plexin may be mutually exclusive. Finally, the cytoplasmic tail of Plexin-Al was shown to dimerise, but antibody mediated clustering of Plexin-Al was not sufficient to induce morphological collapse. In the light of these results, Rac may regulate plexin activity by modulating either conformational changes or receptor aggregation states in response to semaphorin stimulation.

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