The ADP-ribosylation factor (ARF) family of small GTP-binding proteins are ubiquitously expressed and involved in many cellular events such as cell adhesion, cell migration, neurite outgrowth, cell secretion, and endocytosis (D’Souza-Schorey and Chavrier 2006). In mammals, the ARF family consists of six members (ARFs 1–6), and ARFs 1–5 function at the Golgi whereas ARF6 regulates cellular events at the plasma membrane (Donaldson and Jackson 2011). Since ARFs belong to the Ras superfamily of GTPases, they act as molecular switches by cycling between inactive GDP-bound and active GTP-bound forms. They depend on guanine exchange factors (GEFs) for activation and GTPase-activating proteins (GAPs) for inactivation (Donaldson and Honda 2005). Mammalian cells express more than 30 ARF GAPs. Many of them are multidomain proteins that are proposed to function as scaffolds for cellular events. The 31 ARF GAPs predicted in the human genome have been classified into 11 subfamilies based on their sequence similarities and phylogenetic analysis. The AGAP subfamily is predicted to contain 11 members, with AGAP1 and AGAP2 being the most extensively studied (Kahn et al. 2008). Most of the other predicted human AGAP proteins result from gene duplications on the chromosome 10q region.
Human and mouse AGAP1 genes have two alternate transcripts that encode proteins with calculated molecular masses of 94 kDa (857 amino acids [aa]) and 89 kDa (804 aa), respectively. AGAP1 is a multidomain protein, which contains an N-terminal GTP-binding protein-like domain (GLD) followed by split pleckstrin homology (PH), a ARF GAP, and a region of ankyrin (ANK) repeat domains. The GAP activity of recombinant AGAP1 with ARF1 as substrate is approximately two-fold higher compared to that with ARF5, and it is inactive as a GAP against ARF6. Moreover, the ARF-GAP activity of recombinant AGAP1 is dependent on phosphatidic acid (PA) and phosphatidylinositol(4,5)-bisphosphate (PtdIns(4,5)P2) (Nie et al. 2002). Although PtdIns(4,5)P2 and PA can activate the ARF-GAP activity of AGAP1, they can be substituted by other phospholipids without losing any activity, indicating that they may not be required specifically for the GAP activity of AGAP1. Recently Kif2A – a kinesin motor protein – has been shown to cooperate with PtdIns(4,5)P2 to regulate the ARF-GAP activity (Luo et al. 2016).
Sites of Expression and Splice Variants of AGAP1
Sites of Expression
Although AGAP1 is widely expressed in human tissues, its expression levels fluctuate. High levels of AGAP1 mRNA were found in skeletal muscle, kidney, placenta, brain, heart, colon, and lung tissues. Lower levels of AGAP1 mRNA were present in the spleen, liver, and small intestine, while the thymus and peripheral leukocytes did not appear to have detectable levels of AGAP1 mRNA (Meurer et al. 2004).
When AGAP1 is expressed exogenously in U87, NIH 3T3, HeLa, and COS-1 cells, it shows punctate localization in the cell periphery at low-expression levels and diffused cytosolic localization at high levels of expression (Nie et al. 2002, 2003, 2005). Exogenously expressed AGAP1 associates with Rab4-containing endosomes and it is reliant on the ARF-GAP domain for this localization. The colocalization with Rab4 on endosomes is unique to AGAP1 (and AGAP2) and hence this subcellular localization distinguishes it (and AGAP2) from other known ARF GAPs (Nie et al. 2002, 2005). However, it must be noted that the exact subcellular distribution of endogenous AGAP1 has not yet been established. The endogenous AGAP1 localization may not necessarily be the same as that of the exogenously expressed AGAP1, which is located in endocytic compartments containing Rab4 and AP-1 (Nie et al. 2002, 2005).
The AGAP1 split PH domain possesses nine of the ten amino acids identified in the PtdIns(3,4,5)P3 binding consensus sequence, including a tyrosine residue that is present in all known PtdIns(3,4,5)P3 binding PH domains. However, PtdIns(3,4,5)P3 has not yet been shown to either bind to AGAP1 or regulate AGAP1 function (Nie et al. 2002). The split PH domain of AGAP1 is thought to be important in endosome targeting. Since it colocalizes with Rab4 on endosomes, AGAP1 is implicated in transport between early and recycling endosomes. Similarly, its colocalization with AP-1 implies a role for AGAP1 in endosome transport from the trans-Golgi network (TGN) (Nie et al. 2002).
AGAP1 Interacting Proteins Role in Regulating Its Functions
AGAP1 Interaction with AP-3
AGAP1 directly interacts with AP-3 to regulate clathrin-coated vesicle formation and their subsequent transport between intracellular compartments. This association is mediated by the split PH domain of AGAP1 and the δ and σ3 subunits of AP-3 (Nie et al. 2003). Adaptor protein complexes are involved in the formation of intracellular transport vesicles and in the selection of cargo for incorporation into the vesicles (Robinson and Bonifacino 2001). The AGAP1/AP-3 complex regulates endosomal trafficking via ARF1 (Nie et al. 2003). Overexpressed AGAP1 colocalizes with endogenous AP-3 and affects its functions by inhibiting ARF1 activation. Overexpression of AGAP1 also changes the subcellular localization of AP-3, resulting in a reduction in AP-3 association with large punctate structures, suggesting a possible role for it in AP-3-containing endosomes (Nie et al. 2003). Furthermore, overexpression of AGAP1 in HeLa cells increases LAMP1 antibody internalization, thereby suggesting that AGAP1 disrupts AP-3-dependent LAMP1 trafficking to lysosomes (Nie et al. 2003).
AGAP1 Interaction with Muscarinic Acetylcholine Receptor (M5R)
AGAP1 also binds to M5R and affects its trafficking. This G-protein coupled receptor (GPCR) is located in dopaminergic neurons in the midbrain, where it potentiates dopamine release. AGAP1 has been shown to bind to the M5R 3rd intracellular (i3) loop region through residues 552–609 of its split PH domain, thereby allowing AP-3 to bind with M5R. Interaction of AGAP1 and AP-3 is essential for endocytic recycling of M5R in neurons. Inhibition or elimination of AGAP1-M5R binding reduced the cell surface receptor density following sustained receptor stimulation. It has been hypothesized that AP-3-dependent trafficking targets presynaptic M5R for recycling through an intrinsic cargo-recognition function of AGAP1 (Bendor et al. 2010).
AGAP1 Regulation of Rab4 and AP-1 Localization
Overexpression of AGAP1 results in the redistribution of Rab4 and AP-1 to AGAP1-containing punctate structures. The ARF-GAP activity of AGAP1 is essential for the redistribution of Rab4 and AP-1. However, this effect was not dependent on the GLD domain. As both Rab4 and AP-1 are involved in endocytic traffic, the punctate structures are probably an endocytic intermediate affected by AGAP1 (Nie et al. 2002). AGAP1 overexpression in NIH 3T3 cells inhibits platelet-derived growth factor (PDGF)-induced membrane ruffling, in an ARF-GAP activity-dependent and GLD-independent manner, and also affects the organization of the actin cytoskeleton. The AGAP1-overexpressing cells show reduced numbers of stress fibres and a thickened cortical actin around the cell periphery. This effect of AGAP1 requires both the GLD and ARF-GAP domains (Nie et al. 2002).
AGAP1 Role in NO/cGMP Signaling Pathway
AGAP1 may also play a role in the nitric oxide (NO)/cGMP signaling pathway. AGAP1 binds with the α1 and β1 subunits of NO receptor and soluble guanylyl cyclase (sGC) via its C-terminus. Tyrosine (Tyr) phosphorylation of AGAP1 by Src increases its interaction with sGC, indicating that AGAP1 binding to this protein is regulated by its C-terminus Tyr phosphorylation (Meurer et al. 2004) AGAP1 has been shown to homodimerize via its N-terminal region (1–238 aa), which contains a truncated GLD domain. Since sGC binds to the C-terminus of AGAP1, it is thought that homodimerization of AGAP1 allows the binding of sGC to AGAP1 (Meurer et al. 2004).
AGAP1 Interaction with RhoA
Several AGAP1-binding proteins have been identified by yeast two-hybrid screening. These include Rock1, Calcoco2, RhoA, Cdc42, and Rac1. RhoA, which is a member of the Rho family of small GTPases, regulates actin cytoskeleton and cellular adhesion with possible roles in endocytic pathway (Jaffe and Hall 2005). RhoA has been shown to directly bind the GLD domain of AGAP1 and AGAP2 via its C-terminus and subsequently increase their GAP activity towards ARF1 (Luo et al. 2012). It has been suggested that the association of AGAP1 with RhoA contributes to lysosomal maturation given that both AGAP1 and Rho have been implicated in intracellular trafficking (Nie et al. 2002; Steffan et al. 2009).
AGAP1 Interaction with Kif2A
Recently, a novel set of AGAP1-binding partners, which play diverse roles in intracellular trafficking and transport of vesicles and organelles along microtubules, have been identified (Luo et al. 2016). These include dynactin subunit 1, Kif5, Kif2A, myosin 1c, Rab11, and actin. In eukaryotic cells, Kif5 and Kif2A kinesin motor proteins support the movement of cargo along microtubules throughout the cell. They are powered by the hydrolysis of ATP that allows them to control cytoskeleton dynamics (Hirokawa et al. 2009). By using immunoprecipitation studies, KiF2A has shown to associate specifically to AGAP1 but not with the other ARF GAPs. Exogenously expressed AGAP1 and Kif2A have shown to colocalize in Hela cells at the cell periphery. This association is mediated by both the GLD and PH domains of AGAP1 and the motor domain of Kif2A (Luo et al. 2016).
Luo and colleagues have also investigated the effect of point mutations within the GLD domain of AGAP1 on its GAP activity (Luo et al. 2016). In the presence of Kif2A, the mutants E100Q, E110Q, E125Q of AGAP1 shown to have less ARF-GAP activity whereas the D124N has exhibited higher ARF-GAP activity when compared to that of wild-type AGAP1. Furthermore, Kif2A stimulated ARF-GAP activity of AGAP1 has also been shown to be dependent on two pairs of residues within the AGAP1-PH domain (K474/K475 and K479/K480). These results confirm previous findings that both the PH and GLD domains contribute to the ARF-GAP activity of AGAP1. Interestingly, Kif2A has failed to regulate the ARF-GAP activity of AGAP1 in the absence of PtdIns(4,5)P2. Moreover, the GLD and PH domains of AGAP1 have shown to increase Kif2A ATPase activity. These results suggest that the ARF-GAP activity of AGAP1 is cooperatively regulated by both Kif2A and PtdIns(4,5)P2. It has also been shown that cell spreading increases while cell migration decreases as a result of siRNA-mediated downregulation of AGAP1 and Kif2A. The effect of knockdown of Kif2A on cell spreading can be rescued by expression of AGAP1 but the effect of knockdown of AGAP1 can not be rescued by Kif2A overexpression, suggesting that Kif2A functions upstream of AGAP1 (Luo et al. 2016).
The Emerging Role of AGAP1 in Disease
AGAP1 has been identified as a novel therapeutic target in several diseases. A link between chromosome 2q37.3 terminal deletions and autism susceptibility has been found (Wassink et al. 2005). Autism is a disorder of neural development characterized by impaired social interaction and communication and by restricted and repetitive behavior. The AGAP1 gene is located on chromosome 2q37, indicating a possible link between AGAP1 and autism (Wassink et al. 2005). In a cerebral palsy (a neurological condition that affect movement and coordination) patient, whole-exome sequencing has identified a single de nova mutation in AGAP1 affecting its alternate splicing and is also predicted to affect its role as a regulator of AP-3-mediated trafficking events (McMichael et al. 2015). In patients with acute lymphoblastic leukemia (ALL), a cluster of outlier genes – among which is AGAP1 – has been shown to be overexpressed, the functional relevance of which is yet to be found (Harvey et al. 2010). Finally, AGAP1 has also been shown to be a target for the microRNA miR-1269, which is upregulated in hepatocellular carcinoma tissues (Gan et al. 2015). Overall, very few studies have been undertaken to investigate the functional role of AGAP1 in disease pathogenesis.
A rabbit polyclonal antibody raised against residues 784–804 of human AGAP1 has been used for detecting the protein by Western blotting (WB) (Nie et al. 2002). There are several commercially available polyclonal antibodies to AGAP1. A rabbit polyclonal antibody raised against residues 795–857 of human AGAP1 is available from Abcam (#ab96827) and Novus Biologicals (#NBP1-31261). This antibody can be used for detection of endogenous AGAP1 by WB and immunocytochemistry (ICC) or immunofluorescence (IF). Santa Cruz Biotechnology supplies a goat polyclonal anti-AGAP1 antibody (N-15; #sc-47786) raised against a peptide near the N-terminus of human AGAP1, and this antibody can be used for WB, IF, or immunoprecipitation. A rabbit polyclonal antibody raised against a synthetic peptide (corresponding to the middle region of human AGAP1) is available from Origen (#TA343187) and Thermo Fisher Scientific (#PA5-21741). Although it is worth noting that no publications currently cite any of these antibodies, their data sheets contain evidence of their efficiency in the methods outlined above.
The AGAP1 activation is allosterically regulated by various protein and inositol lipid interactions. AGAP1 binds the β3A subunit of AP-3 via its PH domain to regulate clathrin vesicle formation and intracellular trafficking. Its ARF-GAP activity has been shown to depend on its association with PtdIns(4,5)P2 through the split PH domain and PA via the GLD domain. Recently, it has been shown that Kif2A also cooperates with PtdIns(4,5)P2 to regulate AGAP1-GAP activity. The amino acid residues within the GLD (D124) and PH (K474/K475 and K479/K480) critical for Kif2A-stimulated ARF-GAP activity of AGAP1 have been identified. Further, the functional roles for AGAP1/Kif2A complex include regulation of cell migration and cell invasion, which cellular events depend on cytoskeleton remodeling. The siRNA-mediated knockdown of either AGAP1 or Kif2A reduces the rate of cell migration while accelerates the rate of cell spreading. AGAP1 may also bind to phospholipids anchoring sGC to the plasma membrane, thereby enabling sGC to increase cyclic GMP concentrations at specific cellular locations. In addition to the AGAP1-AP3 interaction in regulating clathrin-coated vesicle formation, the AGAP1/AP3 complex also interacts with the M5R to regulate its recycling. Further, AGAP1 interacts with RhoA, via its GLD domain, to potentially regulate the actin cytoskeleton and membrane trafficking. In summary, AGAP1 has been implicated in intracellular trafficking events and its role in disease pathogenesis is mostly unknown.
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