Importance of Arp3 residue arginine 161 (R161) for neurite outgrowth and the interaction between WAVE1 and the Arp2/3 complex
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Axon extension results from branched actin nucleation and polymerization leading to lamellipodial expansion. This process is regulated by interaction of the Wiskott-Aldrich syndrome protein (WASP), WASP family verprolin-homologous 1 (WAVE1), with actin binding proteins of the actin related protein (Arp) 2/3 complex. The Arp2/3 complex is composed of seven proteins (Arp2, Arp3 and accessory proteins ARPC1-5) and binds to existing actin filaments to initiate (nucleate) new side chains. Both Arp2 and Arp3 bind to actin filaments; however, the amino acid residues in Arp3 that contribute to this association and interaction of the Arp2/3 complex with WAVE1 are not yet identified. Using computational structure simulations, we predicted that Arp3 arginine 161 (R161) contributes the most to Arp3 interacting with Arp2. To test whether this prediction is true, we have expressed N-terminal green fluorescent protein (GFP)-tagged wild-type and mutant Arp3 (with R161 mutated to alanine) in B35 neuroblastoma cells and determined the effects on Arp2/3 complexing with WAVE1, actin filament content and neurite outgrowth using immunocytochemistry, co-immunoprecitation and image analysis. Expression of Arp3-R161A in B35 neuroblastoma cells decreased neurite outgrowth (79.36% decrease in the number of neurite bearing cells) and increased cortical phalloidin staining by 25.74%. Arp2 and WAVE1 co-immunoprecipitated with expressed wild-type Arp3 and Arp3-R161A, providing evidence that the mutant Arp3 participated in the Arp2/3 complex formation, which could still interact with WAVE1. In vitro, actin polymerization was increased by extracts from cells expressing the Arp3-R161A gene. We interpret these data to indicate that residue R161 of Arp3 is important for regulating Arp2/3 complex binding to actin filaments and that mutation of this residue to alanine promotes this binding, increasing actin polymerization and decreasing neurite outgrowth. Understanding the mechanisms of Arp2/3 complex function may identify sites for therapeutic action for promoting axon regeneration following traumatic and neurodegenerative lesions.