In eukaryotic cells, protein transport between membrane compartments is dependent on membrane carriers, mainly vesicles. The molecular mechanisms of vesicular transport have been studied extensively. A series of protein machineries have been identified to mediate each step of vesicular transport, including vesicle formation, translocation, and docking and fusion with target membrane. Specifically, the machineries for vesicle formation have been defined as coat complexes, which are usually regulated by small GTPases Sar1/ADP ribosylation factor (ARF) (Donaldson and Jackson 2011). Like all other small GTPases, the functions of Sar1/ARFs depend on associated nucleotides. When a Sar1/ARF is GTP-bound, it is in active state to recruit effectors such as coat proteins for downstream events, including vesicular transport. However, its intrinsic GTPase activity enhanced by a negative regulator, GTPase activating proteins (GAP), will hydrolyze GTP and transform Sar1/ARF to GDP-bound inactive state. To reactivate Sar1/ARF, another regulatory factor, named guanine nucleotide exchange factor (GEF), releases GDP from Sar1/ARF and facilitates GTP reloading. Single Sar1/ARF protein may regulate multiple pathways. For example, ARF6 plays roles in endocytosis, endocytic recycling, cytoskeleton rearrangement, and signaling. In contrast, ARF GAPs and GEFs may be more confined in one or a few pathways. Historically, ACAP1 is identified to function as a GAP for ARF6 specifically in endocytic recycling pathway from the recycling endosome to plasma membrane (Li and Hsu 2015).
Basis of ACAP1
Coat Function of ACAP1 as ARF6 Effector
Membrane Deformation Activity of ACAP1
ACAP1 contains a BAR domain at N-terminal. The function of BAR domain has been studied intensively. The first understanding came from the protein structure. As “head-to-head” homo-dimer, BAR domains exhibit very similar banana shape conformation (Qualmann et al. 2011). The curved underneath surface of BAR domains fits certain degree of membrane curvature. Besides, the positive-charged patches on the surface interact with negative-charged phospholipids in the membrane. Through these interactions, BAR domain-containing proteins can associate with curved membrane structures with a diameter around 50–100 nm (Frost et al. 2009).
Cargo Sorting Activity of ACAP1
One of the major roles of coat proteins is to actively sort cargoes into vesicles. Coat proteins recognize specific amino acid sequences, named sorting signals, located at the cytoplasmic domains of cargo proteins. Distinct sorting signals have been identified for most of vesicular transport pathways. In ACAP1-associated endocytic recycling, sorting signals have been identified in several model cargo proteins, including Leu-Phe (LF) and Arg-Phe (RF) sequences in transferrin receptor (TfR) cytoplasmic domain, Lys-Arg (LR) and Pro-Leu-Ser-Leu-Leu (PLSLL) sequences in glucose transporter type 4 (Glut4) cytoplasmic domains, and a single Arg-Asn-Phe-Ala-Lys-Phe (REFALF) sequence in integrin β1 cytoplasmic domain (Hsu et al. 2012). Although the sequences vary, the common character of these sorting signals is of hydrophobic and positively-charged residues. ACAP1 has been showed to directly bind to these sorting signals. When the sorting signals are mutated in cargo proteins, ACAP1 lost recognition ability and the mutant cargoes could not be able to recycle (Hsu et al. 2012).
In general, endocytic recycling could be divided into two categories, constitutive recycling, which happens constantly without regulation, and regulated recycling, which is mediated by signaling. TfR is the typical example for constitutive recycling. ACAP1 continuously sorts internalized TfR at the recycling endosome into vesicles for its return back to plasma membrane. In contrast, integrin β1 is the typical cargo for regulated recycling. Internalized integrins accumulate at the recycling endosome in the absence of signaling. No/little interaction could be detected between integrin and ACAP1 under starvation condition. Upon stimulation, extracellular signaling triggers PI3K/ Akt signaling pathway. Activated Akt phosphorylates ACAP1 at Ser544 residue, which is located at a flexible loop region linking ARFGAP and ANK domain. The phosphorylation dramatically enhances the interaction between ACAP1 and integrin β1 cytoplasmic domain, and promotes integrin recycling. Structure studies have been performed to understand the molecular mechanism underling the phosphorylation-dependent interaction. In the crystal structure of ACAP1 C-portion, which is composed of ARFGAP and ANK domains, the linker loop region containing Ser544 residue could not be visualized due to high mobility. As an alternative way, molecular simulation has been used to predict the conformational change of the loop region. The results have suggested that under nonphosphorylated condition, this loop functions as a cover to prevent integrin binding (Fig. 5b). Upon phosphorylation, the loop will lift up to allow integrin sorting signal fit into pocket (Fig. 5c). For constitutive recycling cargoes such as TfR, the relatively shorter length of sorting signal peptides (two amino acid compared to six in integrin β1) may allow it fitting into binding pocket without lifting the loop.
Interacting Proteins of ACAP1
Besides ARF6, clathrin, Akt, and cargo proteins, ACAP1 recently has been found to interact with other proteins. The first is small GTPase Rab10 which also plays an important role in endocytic recycling (Shi et al. 2012). Rab proteins mainly involves in the regulation of later stages of vesicular transport including vesicle movement, docking, and fusion with target membrane. It could be speculated that ACAP1 recruits Rab10 onto recycling vesicles for the later stages of vesicular transport.
GULP (Engulfment Adaptor PTB Domain Containing) has also been identified to interact with ACAP1 and regulate its ARFGAP activity (Ma et al. 2007). It has been reported that GULP interacts with ACAP1 to prevent it from interacting with ARF6. As consequence, the cellular level of GTP-bound active form of ARF6 increases. Then active ARF6 recruits downstream effectors other than ACAP1 to regulate cytoskeleton rearrangement and signaling.
Nucleotide-binding oligomerization domain (NOD) containing proteins have been implicated in initiating innate immune response by recognizing pathogen components in the cytoplasm and activating NFγB signaling. ACAP1 has been found to directly interact with NODs to attenuate NFγB signaling (Yamamoto-Furusho et al. 2006). ACAP1 expression in epithelial cells is strongly elevated by tumor necrosis factor α, interleukin 1β, and the NOD ligands. The role of ACAP1 here is implicated to form a negative feedback loop for inflammatory response.
Physiological Events Associated with ACAP1
The physiological functions of ACAP1 have been studied in the past decade. ACAP1 mediates TfR and Glut4 recycling, which are critical for nutrient homeostasis (Li and Hsu 2015). ACAP1 also mediates integrin recycling, which are essential for cell migration and invasion and cancer metastasis (Li and Hsu 2015). In neuron, ACAP1 has been shown to dynamically regulate the activity of ARF6, which in turn regulates axon growth. It has been reported that USP6 (ubiquitin specific protease 6) controls cytokinesis and cell proliferation as an oncogenic protein via regulating ARF6/ACAP1 (Rueckert and Haucke 2012).
A recent translational study to identify new biomarkers for Alzheimer’s disease revealed a potentially novel function of ACAP1. In the tissue and plasma samples of patients, the protein level of ACAP1 has been found to strongly correlate with Mini-Mental State Examination scores of the patients with Alzheimer’s disease (Sun et al. 2015). However, it is unclear how ACAP1 involves in Alzheimer’s disease. The expression level of ACAP1 is also found to be elevated in patients with ulcerative colitis, which may be related to the role of ACAP1 in NODs-regulated NFγB signaling (Yamamoto-Furusho et al. 2013).
It is well known that some pathogens use host machineries for their invasion into cells, such as salmonella, which remodels the actin cytoskeleton by not only activating ARF1 and ARF6 but also recruiting several ARF GAPs including ACAP1 (Davidson et al. 2015). Since the GTPase cycle of ARFs is important for cytoskeleton rearrangement, it is plausible that ACAP1 acts as GAP to promote GTPase cycle of ARF6.
ACAP1 exhibits multiple important functions, including ARF GAP activity, membrane deformation ability, and cargo sorting function in endocytic recycling. However, how these functions cooperate together in a spatial-temporal manner during vesicle formation remains obscure. Besides, the precise roles of ACAP1 in NFγB signaling and Alzheimer’s disease are intriguing. It should also be noted that the ACAP subfamily of ARF6 GAPs contains three members. Although the protein sequences are highly homologous, ACAP2 and ACAP3 don’t participate in endocytic recycling. The functions of these two ACAP1 homologs deserve further exploration. It could be possible that these ACAPs participate in distinct transport pathways dependent on ARF6.
- Davidson AC, Humphreys D, Brooks AB, Hume PJ, Koronakis V. The Arf GTPase-activating protein family is exploited by Salmonella enterica serovar Typhimurium to invade nonphagocytic host cells. mBio. 2015;6. doi: 10.1128/mBio.02253-14.