Porchetta, C; (2012) α2-Chimaerin and Interacting Proteins in Axonal Guidance Signalling. Doctoral thesis , UCL (University College London).

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Abstract

Developing neurones require a dynamic cytoskeleton for newly forming processes to respond to guidance cues. The Rho GTPases, including Rac1, are very important in cytoskeleton remodelling. A GTPase activating protein (GAP) for Rac1, α2-chimaerin, participates in Semaphorin 3A (Sema 3A) and ephrin/EphA4 growth cone collapse pathways, and the SH2 domain is essential, suggesting phosphotyrosine interactions are involved. The activation of α2-chimaerin in collapse pathways may be through diacylglycerol binding to the cysteine rich C1 domain or via phosphotyrosine signalling. This investigation explores the interactions of α2-chimaerin and the role of tyrosine phosphorylation in retraction signalling pathways. The Src family tyrosine kinase, Fyn, is involved in Sema 3A and ephrin A1/EphA4 signalling. Results showed that Fyn can bind to the GAP domain of α2-chimaerin and can phosphorylate α2-chimaerin activated by phorbol 12-myristate 13-acetate, Sema 3A or ephrin A1. Fyn was found to phosphorylate tyrosines 143 and 303 of α2-chimaerin. Overexpression of α2-chimaerin led to increased axon number in hippocampal neurones. Selected point mutations of α2-chimaerin were sufficient to affect axon branching in developing hippocampal neurones. In COS-7 cells, the receptor EphA4 interacted with α2-chimaerin GAP domain when unstimulated and with the SH2 domain when stimulated with the ligand ephrin A1. In N1E- 115 cells permanently expressing EphA4, α2-chimaerin shRNA inhibited ephrin A1- induced cell rounding. The SH2 and C1 domains of α2-chimaerin were both necessary for ephrin A1/EphA4 cell rounding. Another partner of α2-chimaerin is adaptor protein Nck1. The α2-chimaerin Nck1 binding region has been mapped and the binding may be sufficient to activate α2- chimaerin, allowing other partners to interact. A novel α2-chimaerin interacting protein Vps28, a component of the endosomal sorting complex required for transport-I, has been investigated in endocytosis using monoclonal antibodies and shRNA. These results together suggest an important regulatory role for α2-chimaerin and its partners in axonal guidance pathways.

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