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Identification of proteins that interact with Arc/Arg3.1 leading to endocytosis of AMPA receptors

Grant number: 11/23298-0
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): February 01, 2012
Effective date (End): December 31, 2014
Field of knowledge:Biological Sciences - Morphology
Principal Investigator:Luis Lamberti Pinto da Silva
Grantee:Yunan Costa Januário
Home Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

The Arc (actin cytoskeleton-regulated) protein plays a crucial role in the modulation of glutamatergic synaptic transmission in neurons and controls synaptic plasticity mechanisms, which are essential for memory consolidation and learning. Forms of synaptic plasticity known as LTP (Long Term Potentiation) and LTD (Long Term Depression), involve the precise regulation of AMPA-type glutamate receptors availability in postsynaptic membranes. Arc controls LTD induction in the hippocampus by modulating selective endocytosis of AMPA receptors (subunits GluA1 and GluA2). It has been proposed that Arc controls the clathrin-mediated endocytosis through direct interaction with dynamin and endophilin. However, this interaction alone does not explain how AMPA receptors are selectively recruited to budding clathrin vesicles when Arc expression is induced. The recruitment of AMPA receptors to clathrin-coated endocytic vesicles during constitutive endocytosis, requires interaction of the cytosolic tail of the receptor subunits with the AP-2 adaptor protein complex. The aim of this study is to confirm and characterize the interactions between Arc and the AP-2 complex, recently discovered by the group of Dr. Sonia Correa (University of Warwick), our collaborator. Initially, we will employ GST-pull down assays using E. coli produced recombinant Arc and AP-2 to investigate whether their interaction is direct. To identify the subunit(s) of AP-2 that mediate interaction with Arc we resort to yeast two or three hybrid assays. In these assays, interaction of Arc with each of the four subunits of AP-2 will be tested individually. The results of this work will contribute to a better understanding of the mechanisms by which Arc induces AMPA receptor downregulation during LTD in hippocampal neurons.