Location of the human PLD2 (hPLD2) gene is chromosome 17: 4710391 ~ 4726729 (forward strand) (Ensembl database: gene: PLD2, ENSG00000129219). hPLD2 has 25 exons and 24 coding exons.

Two transcripts of hPLD2 (hPLD2A and hPLD2B) code long-range proteins. As shown in figure 1, splicing variation in exon 23 gives rise to an 11 amino acid difference between the two. hPLD2A has a 3503 bp mRNA (933 amino acids) and hPLD2B has a 3391 bp mRNA (922 amino acids) (Ensembl database: PLD2A (transcript: ENST00000263088, protein: ENSP0000026308), PLD2B (transcript: ENST00000572940, protein: ENSP00000459571)).

Two products of hPLD2 can be generated by two transcripts (splicing variants). hPLD2A has 933 amino acids and hPLD2B has 922 amino acids. hPLD2B contains 11 amino acid deletions as compared with hPLD2A. These two products have evolutionally conserved domains. Like other members of the PLD superfamily, PLD2 also has a HKD domain (HxKxxxxDxxxxxxGSxN, x = any amino acid), which is essential for mediating PLD enzymatic activity (Frohman et al., 1999; Exton, 2002; Jenkins and Frohman, 2005). The phox homology (PX) domain and the pleckstrin homology (PH) domain are known to be involved in interactions with lipids and other proteins (Frohman et al., 1999; Sung et al., 1999; Exton, 2002). In particular, the PLD2-PX domain has been reported to interact with a variety of proteins, such as, dynamin, PLCγ, Cdk5, Grb2, and munc18 (Lee et al., 2009). In addition, the PLD2-PH domain is also involved in the interaction with Src and Rac2 (Ahn et al., 2003; Mahankali et al., 2011). The PLD2-PX domain can act as a guanine nucleotide exchange factor (GEF) for dynamin to enhance endocytosis and as a GEF for RhoA to induce LPA-mediated stress fiber formation (Lee et al., 2006; Jeon et al., 2011). Furthermore, the PLD2-PH domain can act as a GEF for Rac2 (Mahankali et al., 2011). Ckd5 has been reported to phosphorylate the PLD2-PX domain (Ser 134), and thus, mediate PLD2 activation and the secretion of insulin in a pancreatic β cell line (Lee et al., 2008a). Src also can interact with the PLD2-PH domain and phosphorylate PLD2 to mediate EGF-induced Src activation (Ahn et al., 2003). The Y169/Y179 residues of PLD2-PX domain are critically implicated in the interaction with Grb2 (Di Fulvio et al., 2006). This interaction is known to be important for the activation and intracellular localization of PLD2. In addition, the dissociation of munc-18 from the PLD2-PX domain is essential for EGF-induced PLD2 activation (Lee et al., 2004). The PLD2-PX domain interacts with the PLCγ-SH3 domain to mediate the EGF-induced activations of PLD2 and PLCγ (Jang et al., 2003). In addition to interacting with proteins, the PLD2-PH domain can interact with phosphoinositide 4,5-bisphosphate (PtdIns (4,5)P2), and this interaction is known to be important for the intracellular localization of PLD2 (Honda et al., 1999; Hodgkin et al., 2000; Sciorra et al., 2002). In the primary structures, the main difference between PLD1 and PLD2 is a loop region, that is, PLD1 has this region, whereas PLD2 does not (Colley et al., 1997; Hammond et al., 1997; Du et al., 2000; Peng and Frohman, 2012). It is considered that the loop region in PLD1 has autoinhibitory activity.

PLD2 has been reported to be expressed in a variety type of tissues, such as, ovary, placenta, prostate, spinal cord, trachea, thymus, and thyroid. Specially, PLD2 mRNA has been detected in many different brain regions (Peng and Rhodes, 2000). During rat brain development, PLD2 mRNA levels are elevated and peak in the adult brain. In addition, PLD2 mRNA levels are transiently reduced during cerebral hypoxic-ischemic injury (Peng et al., 2006). Vessel occlusion-induced hypoxia in the hippocampus has been reported to increase PLD2 levels (Min do et al., 2007). Furthermore, the expression of PLD2 is known to be significantly elevated in many cancers, including breast, renal, and colon cancer (Zhao et al., 2000; Saito et al., 2007; Wood et al., 2007).

Most studies have found PLD2 is mainly localized in the plasma membrane (Du et al., 2004) and that PLD1 is primarily localized in specialized vesicles, such as, endoplasmic reticulum (ER), Golgi apparatus, secretory vesicles, endosomes, and lysosomes (Brown et al., 1998; Freyberg et al., 2001), and several reports indicate that PLD2 is also localized in cytoplasmic vesicles (Divecha et al., 2000). In addition, PLD2 can be translocated into the submembranous vesicles by serum and ruffling membranes by epidermal growth factor (EGF) (Colley et al., 1997; Honda et al., 1999). As mentioned above, the interaction between the PLD2-PH domain and PtdIns (4,5)P2 is important for the localization of PLD2. A PLD2-PH domain mutant incapable of interacting with PtdIns (4,5)P2 was not localized to the plasma membrane like wild-type PLD2, but localized to punctuate structures in cytoplasm (Sciorra et al., 2002).

PLD2 is a phosphatidylcholine (PC)-hydrolyzing enzyme and generates choline and phosphatidic acid (PA) (Jenkins and Frohman, 2005). PLD2 can be activated by multiple extracellular ligands and the major roles of PLD2 can be achieved by PA generation. PA is considered as a second messenger that mediates a variety of cellular functions such as, proliferation, cell growth, vesicle trafficking (endocytosis and exocytosis), and actin-cytoskeletal arrangement (Park et al., 2012). Furthermore, animal studies have shown that PLD2 can serve as a key mediator of in vivo pathophysiologic functions (Oliveira et al., 2010). Regardless PA generation, PLD2 protein can act as a GTPase-activating protein (GAP) for dynamin and a GEF for RhoA and Rac2 (Lee et al., 2006; Jeon et al., 2011; Mahankali et al., 2011). It is known that these functions of PLD2 and PA can be mediated by their binding partners (Jang et al., 2012). Currently, PLD2 and PA are known to have about 40 and 50 binding partners, respectively. Interacting partners include various classes of proteins (kinase, phosphatase, GTPase, structural protein, transporter, adapter, phospholipase, transcription factor), and phospholipids (PA, PtdIns5P, PtdIns(4,5)P2, and PtdIns(3,4,5)P3).

Proliferative signaling
Multiple extracellular mitogenic signals, such as, EGF, platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF) can activate PLD2 to generate PA (Lee et al., 2009; Park et al., 2012). Specially, PLD2 is known to be implicated in EGF-mediated cell proliferation (Ahn et al., 2003). Furthermore, it has been well established that EGF signaling can be regulated via dynamic interactions between PLD2 (PA) and its binding partners (Lee et al., 2009). In addition, EGF can induce dissociation between PLD2 and munc-18 to activate PLD2 (Lee et al., 2004). Activated EGFR can recruit PLCγ to EGFR complex, and PLD2 can activate PLCγ which can generate IP3 and DAG for the activation of PKC (Jang et al., 2003). PLCγ can also serve as a GEF for dynamin (Choi et al., 2004). GTP-loaded dynamin and PKC can activate PLD2, which can act as a GAP for dynamin to potentiate EGFR endocytosis (Park et al., 2004; Lee et al., 2006). PA, generated by PLD activation, can recruit SOS to the plasma membrane. SOS acting as a GEF for Ras can activate the MAP kinase cascade (a key proliferative signaling pathway) and eventually induce cell proliferation and transformation (Zhao et al., 2007).

Cell growth signaling
Cells regulate cellular homeostasis by using extracellular nutrients and growth signals, and malfunctions in this process cause severe diseases, such as, cancer and diabetes. PLD2 acts as a key regulator of growth signaling mainly via the control of the mammalian target of rapamycin (mTOR), which is a key target for cancer treatment (Ha et al., 2006). Both PLD2 and PA can affect mTor signaling. PA can directly interact with the FRB domain of mTOR and activate mTOR (Toschi et al., 2009). Rapamycin (an anti-cancer drug) can inhibit mTOR activation by competing with PA for mTOR (Fang et al., 2001). In addition, PLD can bind Raptor, which complex strongly with mTOR, and interact with Rheb (an upstream GTPase of mTOR) in a GTP-dependent manner (Sun et al., 2008). Furthermore, these interactions are required for the activation of mTOR complex and the mediation of growth signaling.

Vesicle trafficking (endocytosis, and exocytosis)
PLD is known to be implicated in vesicle trafficking, such as, in intracellular vesicle trafficking, endocytosis, and exocytosis (Jones et al., 1999). In addition, PA generation by PLD has been reported to be involved in vesicle fusion by mediating inner membrane curvature (Jenkins and Frohman, 2005). Many reports have suggested that PLD1 is essentially involved in secretion and exocytosis (Vitale et al., 2001; Vitale et al., 2002), but recently, PLD2 was also found to be required for exocytosis. For example, Lee et al. reported that PLD2 is critically involved in insulin secretion by EGF in primary pancreatic islets and in a pancreatic beta cell line (Lee et al., 2008b). Also, PLD2 can induce angiotensin II-mediated aldosterone secretion from adrenal glomerulosa cells (Qin et al., 2010), and in addition to exocytosis, PLD2 is a well known essential mediator of receptor-mediated endocytosis and phagocytosis (Shen et al., 2001; Iyer et al., 2004). Furthermore, the overexpression of PLD2 wild-type can increase EGFR endocytosis, whereas the catalytic inactive PLD2 mutant does not (Shen et al., 2001). Moreover, mu-opioid receptor endocytosis and constitutive metabotropic glutamate receptor endocytosis can be affected by PLD2 activation (Koch et al., 2003; Bhattacharya et al., 2004). PLD2 activity can also regulate the phagocytosis of complement-opsonized zymosan in macrophages (Iyer et al., 2004). Many authors have suggested that PA generation by PLD2 activation is critically implicated in endocytosis and exocytosis. However, recently, it was reported that PLD2 itself, and not PLD2 activity, regulates endocytosis and phagocytosis (Lee et al., 2006; Mahankali et al., 2011). As we addressed above, PLD2 has GAP and GEF functions (PLD2-PX domain, which acts as a GAP for dynamin, and a PLD2-PH domain, which acts as a GEF for Rac2). Furthermore, the PLD2-GAP function for dynamin can accelerate EGF-mediated EGFR endocytosis and the PLD2-GEF function for Rac2 can increase phagocytosis in RAW264.7 macrophages.

Cytoskeletal rearrangement
Cells can undertake cytoskeletal changes by utilizing the dynamics of actin and tubulin (Berepiki et al., 2011; de Forges et al., 2012). These changes play a vital role in mediating a variety of cellular processes, such as, adhesion, spreading, migration, phagocytosis, and cytokinesis (Hynes, 2002). These processes are essential for pathophysiological functions, such as, organ morphogenesis and metastasis (Fletcher and Mullins, 2010). Several authors have suggested that PLD2 has a close relationship with cytoskeletal dynamics. For example, PLD2 can directly interact with kinases (PKC and PtdIns(4)P 5-Kinase) and small G proteins (Ral, Arf1, Arf4, and Arf6) that modulate cytoskeletal dynamics (Jang et al., 2012). Furthermore, cytoskeletal proteins, such as, actin and tubulin, can direct bind to PLD2 and inhibit its activity (Chae et al., 2005). Also, PA generation by PLD2 activation can activate PtdIns(4)P 5-Kinase to generate PtdIns(4,5)P2, which is involved in actin polymerization (Moritz et al., 1992). Also, integrin-mediated PA generation by PLD can recruit GTP-loaded Rac1 to the plasma membrane and be involved in activating Rac1 to mediate cell spreading and migration (Chae et al., 2008). Recently, in addition to PA generation by PLD2 activation, PLD2 (acting as a GEF) was found to be critically implicated in actin dynamics, for example, PLD2 can serve as a GEF for RhoA and Rac2 (Jeon et al., 2011; Mahankali et al., 2011). Furthermore, it has been reported that PLD2, acting as a GEF for RhoA, participates in LPA-mediated stress fiber formation and that PLD2, acting as a GEF for Rac2, is important for the mediations of migration and phagocytosis.

We used PLD2A protein sequences (protein ID : ENSP00000263088) and aligned sequences with Multiple Sequence Alignment program at the website of the European Bioinformatics Institute (EMBL-EBI), and found the following:
- 55% sequence identity with PLD1a of Homo sapiens (protein ID: ENSP00000342793).
- 89% sequence identity with PLD2 of Mouse (protein ID: ENSMUSP00000018429).
- 85% sequence identity with PLD2 of Rat (protein ID: ENSRNOP00000053831).
- 57% sequence identity with PLD2 of Zebrafish (protein ID: ENSDARP00000122561).