**1.2 Addicsin**

In many papers, human addicsin and rat addicsin are called JWA and GTRAP3-18, respectively. Addicsin, GTRAP3-18, and JWA have been independently identified by several research groups (Ikemoto et al., 2002; Lin et al., 2001; Zhou et al., GeneBank, AF070523, unpublished observations). We first identified *addicsin* as a novel mRNA encoding a 22-kDa hydrophobic protein that is highly expressed in the basomedial nucleus of the mouse amygdala following repeated morphine administration (Ikemoto et al., 2002). Meanwhile, *GTRAP3-18* cDNA was identified as encoding an EAAC1 binding protein by yeast twohybrid screening of a rat brain cDNA library using the C-terminal intracellular domain of EAAC1 as bait (Lin et al., 2001). The *JWA* gene was identified as an all-*trans* retinoic acid (RA)-responsive factor from human tracheobronchial epithelial cells (Zhou et al., GeneBank, AF070523, unpublished observations). Bioinformatic analysis demonstrates that JWA has a prenylated Rab acceptor 1 (PRA1) domain and 62% similarity with Jena-Muenchen 4 (JM4), a protein recently identified as PRA1 domain family member 2 (PRAF2) (Schweneker et al., 2005). Proteins containing a large PRA1 domain form a new family of PRA1 domain family proteins (PRAFs) that regulate intracellular protein trafficking. Thus, addicsin is a new member of the PRAF family, PRAF3.

The *addicsin* cDNA is approximately 1.4 kbp and consists of a 564-bp single open reading frame (Ikemoto et al., 2002). The *addicsin* gene contains three exons separated by two introns, and the sequence is highly conserved among vertebrates (Butchbach et al., 2002). Furthermore, *addicsin* is located on mouse chromosome 6, a location corresponding to human chromosome 3p (Butchbach et al., 2002; Ikemoto et al., 2002).

Mouse addicsin is a 22-kDa protein of 188 amino acids with putative transmembrane segments (Butchbach et al., 2002; Ikemoto et al., 2002). Mouse addicsin is 98% identical to rat GTRAP3-18 and 95% similar to human JWA (Butchbach et al., 2002; Ikemoto et al., 2002). Moreover, addicsin has two putative PKC phosphorylation motifs (amino acids 18– 20 and 138–140) as well as two putative cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase II phosphorylation motifs (amino acids 27– 31 and 35–39) (Butchbach et al., 2002; Ikemoto et al., 2002) (Fig. 1). However, there is no evidence that these phosphorylation sites are phosphorylated by protein kinases *in vitro* and *in vivo*.

Expression profiles of addicsin and *addicsin* mRNA were investigated in the developing and mature brain. In the developing rat brain, the expression levels of addicsin decrease significantly from embryonic day 17 to post-natal day 0 (Maier et al., 2009). Meanwhile, *addicsin* mRNA levels increase gradually during early maturation, peaking around postnatal day 5, and then declining by about 50% by post-natal day 14 (Inoue et al., 2005). This developmental expression pattern corresponds to periods of elevated synaptogenesis,

reticulon 2B (RTN2B) interacts with EAAC1 and addicsin/GTRAP3-18, and promotes intracellular trafficking of EAAC1 in HEK293 cells and cultured cortical neurons (Liu et al., 2008). Addicsin/GTRAP3-18 interacts with EAAC1 and inhibits EAAC1 trafficking in HEK293 cells (Ruggiero et al., 2008). Thus, multiple regulatory mechanisms control EAAC1 trafficking and membrane expression, but the molecular details are generally unclear. In this

In many papers, human addicsin and rat addicsin are called JWA and GTRAP3-18, respectively. Addicsin, GTRAP3-18, and JWA have been independently identified by several research groups (Ikemoto et al., 2002; Lin et al., 2001; Zhou et al., GeneBank, AF070523, unpublished observations). We first identified *addicsin* as a novel mRNA encoding a 22-kDa hydrophobic protein that is highly expressed in the basomedial nucleus of the mouse amygdala following repeated morphine administration (Ikemoto et al., 2002). Meanwhile, *GTRAP3-18* cDNA was identified as encoding an EAAC1 binding protein by yeast twohybrid screening of a rat brain cDNA library using the C-terminal intracellular domain of EAAC1 as bait (Lin et al., 2001). The *JWA* gene was identified as an all-*trans* retinoic acid (RA)-responsive factor from human tracheobronchial epithelial cells (Zhou et al., GeneBank, AF070523, unpublished observations). Bioinformatic analysis demonstrates that JWA has a prenylated Rab acceptor 1 (PRA1) domain and 62% similarity with Jena-Muenchen 4 (JM4), a protein recently identified as PRA1 domain family member 2 (PRAF2) (Schweneker et al., 2005). Proteins containing a large PRA1 domain form a new family of PRA1 domain family proteins (PRAFs) that regulate intracellular protein trafficking. Thus, addicsin is a new

The *addicsin* cDNA is approximately 1.4 kbp and consists of a 564-bp single open reading frame (Ikemoto et al., 2002). The *addicsin* gene contains three exons separated by two introns, and the sequence is highly conserved among vertebrates (Butchbach et al., 2002). Furthermore, *addicsin* is located on mouse chromosome 6, a location corresponding to

Mouse addicsin is a 22-kDa protein of 188 amino acids with putative transmembrane segments (Butchbach et al., 2002; Ikemoto et al., 2002). Mouse addicsin is 98% identical to rat GTRAP3-18 and 95% similar to human JWA (Butchbach et al., 2002; Ikemoto et al., 2002). Moreover, addicsin has two putative PKC phosphorylation motifs (amino acids 18– 20 and 138–140) as well as two putative cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase II phosphorylation motifs (amino acids 27– 31 and 35–39) (Butchbach et al., 2002; Ikemoto et al., 2002) (Fig. 1). However, there is no evidence that these phosphorylation sites are phosphorylated by protein kinases *in vitro*

Expression profiles of addicsin and *addicsin* mRNA were investigated in the developing and mature brain. In the developing rat brain, the expression levels of addicsin decrease significantly from embryonic day 17 to post-natal day 0 (Maier et al., 2009). Meanwhile, *addicsin* mRNA levels increase gradually during early maturation, peaking around postnatal day 5, and then declining by about 50% by post-natal day 14 (Inoue et al., 2005). This developmental expression pattern corresponds to periods of elevated synaptogenesis,

study, we focus on the regulation of EAAC1 trafficking by addicsin.

human chromosome 3p (Butchbach et al., 2002; Ikemoto et al., 2002).

**1.2 Addicsin** 

and *in vivo*.

member of the PRAF family, PRAF3.

suggesting that addicsin is involved in synapse formation. Indeed, later in this chapter, we discuss evidence that addicsin participates in intracellular protein trafficking of neurotransmitter receptors. Addicsin is widely distributed in the brain (Akiduki et al., 2007; Butchbach et al., 2002). In the mature CNS, addicsin is expressed in the cerebral cortex, amygdala, striatum, hippocampus (CA1–3 fields), dentate gyrus, and cerebellum. Addicsin is expressed in the somata of glutamatergic and GABAergic neurons and exhibits presynaptic localization in restricted regions such as CA3 stratum lucidum (Akiduki et al., 2007). *In situ* hybridization analysis reveals that *addicsin* mRNA is widely distributed in the brain, predominantly expressed in principal neurons, including glutamatergic and GABAergic neurons in the mature CNS (Inoue et al., 2005). However, the precise subcellular localization of addicsin remains controversial. Recent reports found that addicsin is an integral ER membrane protein that prevents EAAC1 maturation and function by inhibiting ER trafficking (Ruggiero et al., 2008). However, our protein fractionation analysis using mouse whole brain lysates prepared in PBS, NaCl, or Na2CO3 buffer, all indicate that addicsin is predominantly present in the S1 soluble fraction, while the ER transmembrane protein calnexin is present in the P2 pellet fraction (Ikemoto et al., 2002). Our subcellular fractionation analysis with highly purified synaptic fractions prepared from mouse forebrain also support the notion that addicsin is present in the cytoplasmic and presynaptic membrane fractions (Akiduki et al., 2007). Furthermore, immunocytochemical studies reveal that addicsin is present in both the plasma membrane and the intracellular compartments, including the ER (Ikemoto et al., 2002; Watabe et al., 2007, 2008). Consistent with these findings, bioinformatic analysis demonstrates that the α-helix is not long enough for a transmembrane domain; nevertheless, addicsin is predicted to be a hydrophobic protein composed of 62% α-helix and 8% β-sheet (Butchbach et al., 2002), suggesting that it is membrane-associated. Further investigations are needed to clarify the subcellular localization of addicsin, but it is apparent that this protein can exist in both soluble and membrane-associated forms.

Addicsin easily forms homo- and heteromultimers (Ikemoto et al., 2002; Lin et al., 2001) and many reports demonstrate that addicsin can associate with a multitude of proteins (Akiduki & Ikemoto, 2008), including Arl6ip1 (Akiduki & Ikemoto, 2008), ARL6 (Ingley et al., 1999), δ opioid receptor (Wu et al., 2011), EAAC1 (Lin et al., 2001), Rab1 (Maier et al., 2009), and RTN2B (Liu et al., 2008). Moreover, recent studies using the yeast two-hybrid system revealed many potential addicsin-binding proteins (M.J. Ikemoto et al., unpublished data), strongly suggesting that addicsin exerts multiple physiological functions by forming various molecular complexes. It is vital to catalog these interacting proteins and to determine the presence and location of these molecular complexes.

These potential functions remain largely speculative, but molecular studies have provided several intriguing candidates (Fig. 2). First, addicsin is involved in apoptosis induced by 12- *O*-tetradecanoylphorbol-13-acetate, all-*trans* RA, *N*-(4-hydroxyphenyl) retinamide, arsenic trioxide, and cadmium (Mao et al., 2006; Zhou et al., 2008). Knockdown of addicsin attenuates all-*trans* RA-induced and arsenic trioxide-induced apoptosis (Mao et al., 2006; Zhou et al., 2008). Therefore, addicsin serves as a pro-apoptotic molecule. Second, addicsin acts as an environmental stress sensor to protect cells from oxidative stress and subsequent genomic damage. Addicsin is also involved in cellular responses to environmental stresses, including oxidative stress and heat shock, and in the differentiation of leukemia cells under nonphysiological conditions (Cao et al., 2007; Huang et al., 2006a, 2006b; T. Zhu et al., 2005). Addicsin is upregulated after exposure to the pro-oxidants benzo[α]pyrene and hydrogen peroxide through activation of the nuclear transcription factor I (NFI) (R. Chen et al., 2007). Addicsin facilitates DNA repair by interacting with X-ray cross-complementing group 1 protein, a regulator of the DNA base excision repair processes that translocates to the nucleus in response to oxidative stress (R. Chen et al., 2007; Wang et al., 2009). Thus, NFImediated addicsin upregulation protects against DNA damage induced by benzo[α]pyrene and hydrogen peroxide. Third, addicsin also inhibits cancer cell migration as was observed in HeLa, B16, and HCCLM3 cancer cells. (H. Chen et al., 2007). Addicsin has an important role in maintaining the stability of F-actin and in the initiation of actin cytoskeletal rearrangements. Moreover, knockdown of addicsin results in the inactivation of the MEK– ERK signaling cascade. Thus, addicsin inhibits cell migration by activating the mitogenactivated protein kinase (MAPK) cascade and regulating the rearrangement of the F-actin cytoskeleton (H. Chen et al., 2007). Fourth, addicsin participates in the regulation of GSH synthesis; the association of addicsin with EAAC1 at the plasma membrane inhibits the uptake of cysteine for GSH synthesis and thus determines the intracellular GSH content *in vitro* and *in vivo* (Watabe et al., 2007, 2008). This suggests that addicsin is a therapeutic target for enhancing GSH levels in patients with neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases, associated with oxidative stress. Fifth, addicsin significantly inhibits neurite growth in differentiated CAD cells by inactivating Rab1, a positive regulator of ER-to-Golgi trafficking (Maier et al., 2009). Finally, addicsin participates in the regulation of EAAC1-mediated glutamate uptake (Akiduki & Ikemoto, 2008) and ER protein trafficking (Liu et al., 2008; Ruggiero et al., 2008). We discuss these latter two physiological functions in detail (Section 2).

#### **1.3 Arl6ip1**

The "ADP-ribosylation factor-like 6 interacting protein 1 (Arl6ip1)" is the new name assigned to three independently described factors: the original Arl6ip, apoptotic regulator in the membrane of the ER (ARMER), and protein KIAA0069. The Arl6ip1 protein was first identified by yeast two-hybrid screening using mouse ARL6 as bait (Ingley et al., 1999) and as a negative regulatory factor during myeloid differentiation by differential display (Pettersson et al., 2000). Moreover, a novel protein, designated ARMER, initially discovered as a false-positive clone by yeast two-hybrid screening using Bcl-xL as bait, is also Arl6ip1 (Lui et al., 2003). In addition, Arl6ip1 has more than 96% homology with the human protein KIAA0069, the product of a cDNA isolated from the human myeloblast cell line KG-1 during a systematic effort to characterize complete cDNAs (Nomura et al., 1994). Amino acid analysis of Arl6ip1 demonstrates that it is composed of 203 amino acids and encodes a 23-kDa protein with four putative transmembrane segments (Pettersson et al., 2000). Several studies indicate that Arl6ip1 is an integral membrane protein localized to the ER (Lui et al., 2003; Pettersson et al., 2000). Furthermore, computational analysis of the topology of Arl6ip1 demonstrates that the N- and C-terminal ends are both exposed to the cytoplasm (Lui et al., 2003). Consistent with these results, Arl6ip1 has two putative casein kinase II phosphorylation motifs (amino acids 18–21 and 128–131), three putative PKC phosphorylation motifs (amino acids 94–96, 115–117, and 128–130), a *N*-glycosylation motif (amino acids 6–9), a prenyl group-binding motif (amino acids 72–75), and an ER retention signal in the C-terminal cytoplasmic region (amino acids 200–203) (Akiduki &

nonphysiological conditions (Cao et al., 2007; Huang et al., 2006a, 2006b; T. Zhu et al., 2005). Addicsin is upregulated after exposure to the pro-oxidants benzo[α]pyrene and hydrogen peroxide through activation of the nuclear transcription factor I (NFI) (R. Chen et al., 2007). Addicsin facilitates DNA repair by interacting with X-ray cross-complementing group 1 protein, a regulator of the DNA base excision repair processes that translocates to the nucleus in response to oxidative stress (R. Chen et al., 2007; Wang et al., 2009). Thus, NFImediated addicsin upregulation protects against DNA damage induced by benzo[α]pyrene and hydrogen peroxide. Third, addicsin also inhibits cancer cell migration as was observed in HeLa, B16, and HCCLM3 cancer cells. (H. Chen et al., 2007). Addicsin has an important role in maintaining the stability of F-actin and in the initiation of actin cytoskeletal rearrangements. Moreover, knockdown of addicsin results in the inactivation of the MEK– ERK signaling cascade. Thus, addicsin inhibits cell migration by activating the mitogenactivated protein kinase (MAPK) cascade and regulating the rearrangement of the F-actin cytoskeleton (H. Chen et al., 2007). Fourth, addicsin participates in the regulation of GSH synthesis; the association of addicsin with EAAC1 at the plasma membrane inhibits the uptake of cysteine for GSH synthesis and thus determines the intracellular GSH content *in vitro* and *in vivo* (Watabe et al., 2007, 2008). This suggests that addicsin is a therapeutic target for enhancing GSH levels in patients with neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases, associated with oxidative stress. Fifth, addicsin significantly inhibits neurite growth in differentiated CAD cells by inactivating Rab1, a positive regulator of ER-to-Golgi trafficking (Maier et al., 2009). Finally, addicsin participates in the regulation of EAAC1-mediated glutamate uptake (Akiduki & Ikemoto, 2008) and ER protein trafficking (Liu et al., 2008; Ruggiero et al., 2008). We discuss these

The "ADP-ribosylation factor-like 6 interacting protein 1 (Arl6ip1)" is the new name assigned to three independently described factors: the original Arl6ip, apoptotic regulator in the membrane of the ER (ARMER), and protein KIAA0069. The Arl6ip1 protein was first identified by yeast two-hybrid screening using mouse ARL6 as bait (Ingley et al., 1999) and as a negative regulatory factor during myeloid differentiation by differential display (Pettersson et al., 2000). Moreover, a novel protein, designated ARMER, initially discovered as a false-positive clone by yeast two-hybrid screening using Bcl-xL as bait, is also Arl6ip1 (Lui et al., 2003). In addition, Arl6ip1 has more than 96% homology with the human protein KIAA0069, the product of a cDNA isolated from the human myeloblast cell line KG-1 during a systematic effort to characterize complete cDNAs (Nomura et al., 1994). Amino acid analysis of Arl6ip1 demonstrates that it is composed of 203 amino acids and encodes a 23-kDa protein with four putative transmembrane segments (Pettersson et al., 2000). Several studies indicate that Arl6ip1 is an integral membrane protein localized to the ER (Lui et al., 2003; Pettersson et al., 2000). Furthermore, computational analysis of the topology of Arl6ip1 demonstrates that the N- and C-terminal ends are both exposed to the cytoplasm (Lui et al., 2003). Consistent with these results, Arl6ip1 has two putative casein kinase II phosphorylation motifs (amino acids 18–21 and 128–131), three putative PKC phosphorylation motifs (amino acids 94–96, 115–117, and 128–130), a *N*-glycosylation motif (amino acids 6–9), a prenyl group-binding motif (amino acids 72–75), and an ER retention signal in the C-terminal cytoplasmic region (amino acids 200–203) (Akiduki &

latter two physiological functions in detail (Section 2).

**1.3 Arl6ip1** 

Ikemoto, 2008; Lui et al., 2003) (Fig. 1). Thus, Arl6ip1 function may be controlled by diverse intracellular cell signals, but it is unknown whether these motifs are physiologically functional.

The functions of Arl6ip1 remain largely unknown, but culture studies have provided several intriguing possibilities. For example, Arl6ip1 protects HT1080 fibrosarcoma cells from apoptosis induced by serum starvation, doxorubicin, UV irradiation, tumor necrosis factor , and ER stressors by inhibiting caspase-9 activity (Lui et al., 2003). In addition, Arl6ip1 suppresses cisplatin-induced apoptosis in CaSki human cervical cancer cells by regulating the expression of apoptosis-related proteins caspase-3, caspase-9, p53, NF-B, MAPK, Bcl-2, Bcl-xL, and Bax (Guo et al., 2010a). Furthermore, Arl6ip1 is involved in cell growth, cell cycle progression, and invasion of cancer cells. Downregulation of Arl6ip1 suppresses cell proliferation and colony formation, arrests cell cycling at the G0/G1 phase, and inhibits migration of CaSki human cervical cancer cells (Guo et al., 2010b). Most relevant to the present discussion, Arl6ip1 is involved in the regulation of EAAC1. Recently, we demonstrated that Arl6ip1 is a novel addicsin-associating factor that indirectly promotes PKC-dependent EAAC1-mediated glutamate uptake by decreasing the number of addicsin molecules available for suppression of EAAC1 (Akiduki & Ikemoto, 2008).
