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Title: Inhibition of Arp2/3-mediated actin polymerisation by PICK1 drives AMPA receptor endocytosis
Author: Rocca, Daniel L.
ISNI:       0000 0001 3529 3649
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2008
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Increasing evidence suggests that the dynamic regulation of the actin cytoskeleton is the principal driving factor for endocytosis, however, the mechanistic details and proteins involved remain unclear. PICK1 is a modular PDEZ-BAR domain protein that can bind the AMPA receptor (AMPAR) subunits GluR2/3 and is required for activity-dependent AMPAR endocytosis. The intemalisatjon of AMPARs has previously been shown to be dependent on regulated actin dynamics. This study demonstrates that PICKI is able to bind to the actin polymerisation machinery, via direct interactions with both F-actin and the actin-nucleating Arp2/3 complex. PICK1 is shown to be a potent inhibitor of both VCA-stimulated and Arp2/3-mediated actin polymerisation. This function is dependent on competitive interactions between PICKI and the VCA-domain of N-WASP for the Arp2/3 complex, whereby PICKl-binding does not sufficiently activate Arp2/3-mediated actin polymerisation. The inhibition of actin polymerisation is enhanced when the GluR2 C-terminus binds the PDZ domain of PICKl, which results in relief of an auto-inhibitory intra-molecular interaction between the PDZ-BAR modules. This provides a likely mechanism by which PICKI is able to localise its inhibitory effect on actin polymerisation close to sites ofAMPAR endocytosis. Mutation of a tryptophan residue at the extreme C-terminus of PICKI abolishes Arp2/3complex binding and the inhibitory effect on actin assembly. Expression of this dominant-negative mutant in hippocampal neurons resulted in a drastic block ofNMDAinduced AMPAR intemalisation, therefore demonstrating that inhibition of Arp2/3dependent actin polymerisation by PICK1 is necessary for AMPAR endocytosis. Thus this study identifies a new regulator of Arp2/3-driven actin polymerisation and provides a novel mechanism for the role of PICK1 in vesicle trafficking and AMPAR endocytosis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available