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ACTIN-RELATED PROTEIN 2; ACTR2

ACTIN-RELATED PROTEIN 2; ACTR2

Alternative titles; symbolsARP2Other entities represented in this entry:ARP2/3 COMPLEX, INCLUDEDHGNC Approved Gene Symbol: ACTR2Cytogenetic location: 2p14 Ge...

Alternative titles; symbols

  • ARP2

Other entities represented in this entry:

  • ARP2/3 COMPLEX, INCLUDED

HGNC Approved Gene Symbol: ACTR2

Cytogenetic location: 2p14 Genomic coordinates (GRCh38): 2:65,227,830-65,271,252 (from NCBI)

▼ Cloning and Expression
The protrusion of the cell membrane is fundamental to cell shape change and locomotion. Actin polymerization (see 102560) plays a critical role in this process. The leading edge of motile cells is dominated by thin actin-rich structures called lamellipodia, which exhibit highly dynamic behavior characterized by rapid extension and retraction. Many aspects of the mechanism of lamellipodial protrusion are echoed in the intracellular motility of certain bacterial and viral pathogens, such as the bacterium Listeria monocytogenes. Welch et al. (1997) purified an approximately 220-kD multiprotein complex from human platelets that induces actin polymerization at the L. monocytogenes cell surface and mediates bacterial motility. This complex contains actin-related proteins (Arps) in the Arp2 and Arp3 families and therefore was named the Arp2/3 complex. In addition to 43-kD ARP2 and 50-kD ARP3 (ACTR3; 604222), the human complex consists of 41/40- (ARPC1B; 604223), 34- (ARPC2; 604224), 21- (ARPC3; 604225), 20- (ARPC4; 604226), and 16-kD (ARPC5; 604227) subunits, all present in approximately equal stoichiometry. By searching an EST database with peptide sequences from the 7 subunits of the human ARP2/3 complex, Welch et al. (1997) identified full-length human cDNAs encoding each subunit. The ARP2 cDNA encodes a deduced 394-amino acid protein that is 99% identical to chicken Arp2 and 67% identical to S. cerevisiae Arp2. Welch et al. (1997) localized several subunits of the ARP2/3 complex to the lamellipodia of stationary and locomoting fibroblasts, as well as to the actin tails assembled by L. monocytogenes. They suggested that the ARP2/3 complex promotes actin assembly in lamellipodia and may participate in lamellipodial protrusion.

Machesky et al. (1997) purified the ARP2/3 complex from human neutrophils and sequenced peptides from each of the subunits.

▼ Gene Function
Loisel et al. (1999) used pure components of the actin cytoskeleton to reconstitute sustained movement in Listeria and Shigella in vitro. Actin-based propulsion was driven by the free energy released by ATP hydrolysis linked to actin polymerization and did not require myosin (see 601478). In addition to actin and activated Arp2/3 complex, actin depolymerizing factor and capping protein (see 601571) were also required for motility as they maintained a high steady-state level of G-actin (see 102610), which controls the rate of unidirectional growth of actin filaments at the surface of the bacterium. The movement was more effective when profilin (see 176610), alpha-actinin (see 102575), and, in the case of Listeria, VASP (601703) were also included.

The protein N-WASP (WASL; 605056) regulates actin polymerization by stimulating the actin-nucleating activity of the Arp2/3 complex. N-WASP is tightly regulated by multiple signals; only costimulation by CDC42 (116952) and phosphatidylinositol (4,5)-bisphosphate (PIP2) yields potent polymerization. Prehoda et al. (2000) found that regulation requires N-WASP's constitutively active output domain (verprolin/cofilin/acidic (VCA) domain) and 2 regulatory domains, a CDC42-binding domain and a PIP2-binding domain. In the absence of stimuli, the regulatory modules together hold the VCA-Arp2/3 complex in an inactive 'closed' conformation. In this state, both the CDC42- and PIP2-binding sites are masked. Binding of either input destabilizes the closed state and enhances binding of the other input. This cooperative activation mechanism shows how combinations of simple binding domains can be used to integrate and amplify coincident signals.

Using fluorescence anisotropy analysis, Marchand et al. (2001) showed that efficient actin nucleation requires both recruitment of an actin monomer to the ARP2/3 complex and a subsequent activation step. The initial steps in this pathway involve binding by the WA domain of WASP/SCAR (605035) proteins, which consists of a WH2 motif (W) that binds to the actin monomers and an acidic tail (A) that binds to the ARP2/3 complex. Actin filaments seem to stimulate nucleation by enhancing binding of WA to the ARP2/3 complex and favoring the formation of a productive nucleus.

Weisswange et al. (2009) analyzed the dynamics of N-WASP, WASP-interacting protein (WIP; 602357), GRB2 (108355), and NCK (600508), which are required to stimulate ARP2/3 complex-dependent actin-based motility of vaccinia virus, using fluorescence recovery after photobleaching. Weisswange et al. (2009) showed that all 4 proteins are rapidly exchanging, albeit at different rates, and that the turnover of N-WASP depends on its ability to stimulate ARP2/3 complex-mediated actin polymerization. Conversely, disruption of the interaction of N-WASP with GRB2 and/or the barbed ends of actin filaments increases its exchange rate and results in a faster rate of virus movement. Weisswange et al. (2009) suggested that the exchange rate of N-WASP controls the rate of ARP2/3 complex-dependent actin-based motility by regulating the extent of actin polymerization by antagonizing filament capping.

Nolen et al. (2009) described 2 classes of small molecules that bind to different sites on the Arp2/3 complex and inhibit its ability to nucleate actin filaments. CK-0944636 binds between Arp2 and Arp3, where it appears to block movement of Arp2 and Arp3 into their active conformation. CK-0993548 inserts into the hydrophobic core of Arp3 and alters its conformation. Both classes of compounds inhibit formation of actin filament comet tails by Listeria and podosomes by monocytes.

Using immunofluorescence microscopy, Western blot analysis, and knockdown strategies with human lung fibroblasts, Hanisch et al. (2011) showed that Salmonella entered nonphagocytic cells by manipulating 2 machineries of actin-based motility in the host: actin polymerization through the ARP2/3 complex, and actomyosin-mediated contractility in a myosin IIA (MYH9; 160775)- and myosin IIB (MYH10; 160776)-dependent manner. Hanisch et al. (2011) concluded that Salmonella entry can be effected independently of membrane ruffling.

Li et al. (2012) showed that interactions between diverse synthetic, multivalent macromolecules (including multidomain proteins and RNA) produce sharp liquid-liquid-demixing phase separations, generating micrometer-sized liquid droplets in aqueous solution. This macroscopic transition corresponds to a molecular transition between small complexes and large, dynamic supramolecular polymers. The concentrations needed for phase transition are directly related to the valency of the interacting species. In the case of the actin-regulatory protein N-WASP (605056) interacting with its established biologic partners NCK (600508) and phosphorylated nephrin (602716), the phase transition corresponds to a sharp increase in activity towards an actin nucleation factor, the ARP2/3 complex. The transition is governed by the degree of phosphorylation of nephrin, explaining how this property of the system can be controlled to regulatory effect by kinases. Li et al. (2012) concluded that the widespread occurrence of multivalent systems suggests that phase transitions may be used to spatially organize and biochemically regulate information throughout biology.

In Drosophila, Caridi et al. (2018) showed that relocalization of DNA repair sites to the nuclear periphery occurs by directed motion along nuclear actin (see 102610) filaments assembled at repair sites by the Arp2/3 complex. Relocalization requires nuclear myosins associated with the heterochromatin repair complex Smc5/6 (609386/609387) and the myosin activator Unc45 (611219), which is recruited to repair sites by Smc5/6. Arp2/3, actin nucleation, and myosins also relocalize heterochromatic double-strand breaks in mouse cells. Defects in this pathway result in impaired heterochromatin repair and chromosome rearrangements. Caridi et al. (2018) concluded that their findings identified de novo nuclear actin filaments and myosins as effectors of chromatin dynamics for heterochromatin repair and stability in multicellular eukaryotes.

Using Xenopus laevis cell-free extracts and mammalian cells, Schrank et al. (2018) established that nuclear actin, WASP, and the actin-nucleating ARP2/3 complex are recruited to damaged chromatin undergoing homology-directed repair. They demonstrated that nuclear actin polymerization is required for the migration of a subset of double-strand breaks into discrete subnuclear clusters. Actin-driven movements specifically affect double-strand breaks repaired by homology-directed repair in G2 cell cycle phase; inhibition of actin nucleation impairs DNA end-processing and homology-directed repair. By contrast, ARP2/3 is not enriched at double-strand breaks repaired by nonhomologous end joining and does not regulate nonhomologous end joining. Schrank et al. (2018) concluded that nuclear actin-based mobility shapes chromatin organization by generating repair domains that are essential for homology-directed repair in eukaryotic cells.

▼ Biochemical Features
Volkmann et al. (2001) performed electron cryomicroscopy and 3-dimensional reconstruction of Acanthamoeba castellanii and S. cerevisiae Arp2/3 complexes bound to the WASP (301000) carboxy-terminal domain. Asymmetric, oblate ellipsoids were revealed. Image analysis of actin branches indicated that the complex binds the side of the mother filament, and ARP2 and ARP3 are the first 2 subunits of the daughter filament. Comparison to the actin-free WASP-activated complexes suggests that branch initiation involves large-scale structural rearrangements within ARP2/3.

Robinson et al. (2001) determined the crystal structure of bovine ARP2/3 complex at 2.0-angstrom resolution. ARP2 and ARP3 are folded like actin, with distinctive surface features. Subunits ARPC2 and ARPC4 in the core of the complex associate through long carboxy-terminal alpha helices and have similarly folded amino-terminal alpha/beta domains. ARPC1 is a 7-blade beta propeller with an insertion that may associate with the side of an actin filament. ARPC3 and ARPC5 are globular alpha-helical subunits. Robinson et al. (2001) predicted that WASP/SCAR proteins activate ARP2/3 complex by bringing ARP2 into proximity with ARP3 for nucleation of a branch on the side of a preexisting actin filament.

Tags: 2p14