Neuropharmacology 161 (2019) 107789

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Neuropharmacology

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Invited review Multifaceted regulation of the system A transporter Slc38a2 suggests T nanoscale regulation of amino acid metabolism and cellular signaling ∗ Robin Johansen Menchinia, , Farrukh Abbas Chaudhrya,b a Department of Molecular Medicine, University of Oslo, Oslo, Norway b Department of Plastic and Reconstructive Surgery, Oslo University Hospital, Oslo, Norway

HIGHLIGHTS

• Slc38a2 represents the classically described system A transport activity. • Slc38a2 accumulates small, neutral amino acids directly or indirectly by energizing ASCT1/2 and LAT1/2 transporters. • Slc38a2 is extensively regulated by cell stress, nutritional and hormonal signaling, and acts as an amino acid sensor upstream of mTOR. • Slc38a2 contributes to the pathology in a number of diseases such as cancer, epilepsy and diabetes mellitus.

ARTICLE INFO ABSTRACT

Keywords: Amino acids are essential for cellular protein synthesis, growth, metabolism, signaling and in stress responses. Slc38a1 Cell plasma membranes harbor specialized transporters accumulating amino acids to support a variety of cellular Slc38a2 biochemical pathways. Several transporters for neutral amino acids have been characterized. However, Slc38a2 SNAT2 (also known as SA1, SAT2, ATA2, SNAT2) representing the classical transport system A activity stands in a Glutamine unique position: Being a secondarily active transporter energized by the electrochemical gradient of Na+, it Osmoregulation creates steep concentration gradients for amino acids such as glutamine: this may subsequently drive the ac- Adaptive regulation cumulation of additional neutral amino acids through exchange via transport systems ASC and L. Slc38a2 is ubiquitously expressed, yet in a cell-specific manner. In this review, we show that Slc38a2 is regulated atthe transcriptional and translational levels as well as by ions and proteins through direct interactions. We describe how Slc38a2 senses amino acid availability and passes this onto intracellular signaling pathways and how it regulates protein synthesis, cellular proliferation and apoptosis through the mechanistic (mammalian) target of rapamycin (mTOR) and general control nonderepressible 2 (GCN2) pathways. Furthermore, we review how this extensively regulated transporter contributes to cellular osmoadaptation and how it is regulated by endoplasmic reticulum stress and various hormonal stimuli to promote cellular metabolism, cellular signaling and cell sur- vival. This article is part of the issue entitled ‘Special Issue on Neurotransmitter Transporters’.

1. Introduction metabolism and apoptosis (Wu, 2009). Glutamine is the most abundant amino acid in both plasma and cere- 1.1. Physiological roles of amino acids brospinal fluid (Curi et al., 2005). It plays a pivotal role in intermediary metabolism, as a nitrogen and carbon donor, in pH homeostasis and as a Amino acids play a number of roles in human metabolism. In addition substrate for biosynthetic pathways for neurotransmitters, glutathione, to their appearance as substrates for protein synthesis, amino acids act as proteins, nucleotides and amino sugars (Chaudhry et al., 2002a). Glutamine sources of energy, carbon, nitrogen, metabolic intermediaries as well as is the preferred nutrient for rapidly dividing cells such as immune cells, precursors for the synthesis of macromolecules such as hormones, he- enterocytes and cancer cells (Cruzat et al., 2018). Glutamine is a non-es- moglobin and cytochromes. In later years, it is recognized that amino acids sential amino acid, but it may become conditionally essential during cata- such as glutamine, leucine and arginine double as regulators of cell growth, bolic states as cellular demands increase.

∗ Corresponding author. E-mail address: [email protected] (R.J. Menchini). https://doi.org/10.1016/j.neuropharm.2019.107789 Received 19 April 2019; Received in revised form 16 September 2019; Accepted 20 September 2019 Available online 28 September 2019 0028-3908/ © 2019 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). R.J. Menchini and F.A. Chaudhry Neuropharmacology 161 (2019) 107789

1.2. Uptake systems for the neutral amino acids optic nerve pathology (Poulter et al., 2013; Perez et al., 2014; Toral et al., 2017). Slc38a7 is a lysosomal transporter suggested to be the In critically ill patients admitted to intensive care units, both low main carrier of glutamine across lysosomal membranes and required for and high plasma levels of glutamine are associated with a poor clinical cancer cell growth in periods of low glutamine availability (Verdon outcome (Oudemans-van Straaten et al., 2001; Rodas et al., 2012). et al., 2017). Slc38a9, like Slc38a7, is located on lysosomal membranes. Supplementing critically ill patients with parenteral glutamine sig- Recently, three independent laboratories showed that Slc38a9 is part of nificantly reduced hospital mortality, length of stay and rate ofin- the Rag GTPase-Ragulator amino acid-sensing machinery that controls fectious complications and more (Stehle et al., 2017). Thus, main- the activity of mechanistic target of rapamycin complex 1 (mTORC1) taining optimal concentrations of the amino acid plasma pool is (Rebsamen et al., 2015; Wang et al., 2015; Jung et al., 2015). In part, essential for homeostasis in healthy individuals. Several active and this is achieved through Slc38a9-mediated release of leucine, as concentrative transport systems for neutral amino acids across cell Slc38a9 is needed to transport several essential amino acids generated membranes have been demonstrated (Palacin et al., 1998). Glutamine is by proteolysis out of lysosomes (Wyant et al., 2017). Additionally, transported by the system A (alanine preferring) and system N (amide Slc38a9 transport activity may play a pivotal role in mTORC1 activa- preferring) activities originally described as Na+-dependent glutamine tion under conditions in which cells obtain amino acids by degrading transport activities in Ehrlich cells and hepatocytes, respectively extracellular proteins through macropinocytosis (Wyant et al., 2017). (Oxender and Christensen, 1963; Christensen et al., 1965; Kilberg et al., Within the Slc38 family, Slc38a9-a11 are the phylogenetically oldest 1980). Together with systems ASC (alanine, serine and cysteine pre- members with orthologues in C. elegans and D. melanogaster, while ferring) and L (leucine preferring), system A accounts for the majority Slc38a1-a5 have arisen later in evolutionary terms (Schioth et al., of amino acid uptake in mammalian cells (Palacin et al., 1998). Unlike 2013). As more Slc38 family amino acid transporters have been char- systems ASC and L, which are obligatory exchangers, system A cata- acterized, the dependence on Na+ and the division into systems A and lyzes the net uptake of a wider range of neutral amino acids, particu- N have become less distinct and additional spectacular mechanisms add larly alanine, serine, proline and glutamine. It is competitively inhibited to their function and regulation. by the non-metabolizable system A substrate analogue N-methyl-ami- noisobutyric acid (MeAIB) (Oxender and Christensen, 1963; 2. Slc38a2 characteristics Christensen et al., 1965). System N is characterized by transporting glutamine, histidine and asparagine, and tolerates Li+ substitution for 2.1. Slc38a2 structure Na+ (Kilberg et al., 1980). However, the molecular identity of the proteins responsible escaped discovery for decades (Barker and Ellory, The Slc38a2 gene consists of 16 exons and 15 introns in humans and 1990; Palacin et al., 1998). rodents (Palii et al., 2004). Exon 1 encodes most of the 5′-untranslated region (5′-UTR), while the start and stop codons are located in exon 2 1.3. Identification of the solute carrier 38 (Slc38) family of aminoacid and 16, respectively (Palii et al., 2004). Upon molecular characteriza- transporters tion, Slc38a2 was predicted to have (Reimer et al., 2000; Yao et al., 2000), and was recently demonstrated to have eleven transmembrane The field of system A and system N transporters was notopened domains, an intracellular N-terminus and an extracellular C-terminus until the group of Robert Edwards cloned the vesicular GABA trans- (Ge et al., 2018)(Fig. 1). Slc38a2 contains three N-glycosylation sites, porter (VGAT, also known as (aka) vesicular inhibitory amino acid Asn254, Asn258 and Asn272, which seem to play a part in protein ex- transporter (VIAAT)) (McIntire et al., 1997). Later, Chaudhry in colla- pression (Ge et al., 2018). The Slc38a2 protein also contains a disulfide boration with Edwards successfully identified and characterized the bridge between cysteine residues 245 and 279 situated in the third first system N transporter SN1 from its sequence homology toVGAT extracellular loop between transmembrane domains 5 and 6 (Chen (Chaudhry et al., 1999). Further molecular characterization revealed et al., 2016). Cys245 and Cys279 are highly conserved, and had reg- SN1 as the first identified member of the solute carrier (Slc) family 38of ulatory impact on Slc38a2 transport activity without them being re- amino acid transporters (Chaudhry et al., 2002a; Nissen-Meyer and quired for transport activity or trafficking to the plasma membrane Chaudhry, 2013; Broer, 2014). Together, the Slc32, Slc36 and Slc38 (Chen et al., 2016)(Fig. 1). Two other highly conserved cysteine re- families form a monophyletic group comprising the β-group of Slc sidues, Cys228 and Cys303, were shown to be pivotal for transport (Schioth et al., 2013). The Slc32 family contains the sole VGAT, the function (Chen et al., 2016). Slc36 family comprises the lysosomal proton-coupled amino acid transporters 1–4 (PAT1-4), while the Slc38 family consists of eleven 2.2. Kinetic properties of Slc38a2 members in the human genome, SLC38A1-A11 where SN1 is Slc38a3 (Schioth et al., 2013). Slc38a2 displays a preference for transport of short-chain neutral At the turn of the millennium, Slc38a1 (aka GlnT, ATA1, SAT1, SA2 amino acids across cell membranes, such as alanine, serine, proline and and SNAT1) and Slc38a2 (aka SA1, ATA2, SAT2 and SNAT2) were in glutamine (Reimer et al., 2000; Yao et al., 2000; Sugawara et al., 2000a; short succession independently cloned in rats by three laboratories and Gazzola et al., 2001; Hyde et al., 2001; Alfieri et al., 2001). Alpha demonstrated to underlie the system A transport activity (Varoqui et al., amino acid transport is competitively inhibited by the partial agonist 2000; Reimer et al., 2000; Yao et al., 2000; Sugawara et al., 2000a; MeAIB, a system A inhibitor commonly used as a model substrate for Chaudhry et al., 2002b). Based on screening for homologues to Slc38a1- system A (Christensen et al., 1965). It is a secondary active transporter Slc38a3, Slc38a4 (aka mNAT3, SAT3 and SNAT4) and Slc38a5 (aka SN2 in which Na+ drives the transport of neutral amino acids in symport and SNAT5) were identified as amino acid transporters (Sugawara with a 1:1 stoichiometry (Chaudhry et al., 2002b). This makes the et al., 2000b; Gu et al., 2001; Nakanishi et al., 2001; Hamdani et al., transporter electrogenic and able to generate steep substrate con- 2012). The tissue distribution and molecular function of these five centration gradients at resting potential (Reimer et al., 2000). Slc38a2 transporters comprising the classical system A and system N transport transport is strongly dependent on pH, and substrate transport is activities are comparably well characterized and reviewed previously markedly reduced as extracellular pH is lowered within the physiolo- (Nissen-Meyer and Chaudhry, 2013; Broer, 2014). gical range (Reimer et al., 2000). Slc38a2 partly allows replacement of In addition, Slc38a7 and Slc38a9 have been functionally char- Na+ with Li+ (Chaudhry et al., 2002b). We have demonstrated that acterized, while Slc38a6, Slc38a8, Slc38a10 and Slc38a11 remain elu- Slc38a2 has ordered binding, with Na+ binding first and the substrate + + sive ‘orphan’ transporters. However, mutations in the SLC38A8 gene second, and that H competes at the Na binding site increasing Km for cause foveal hypoplasia in several families with or without concurrent Na+ (Chaudhry et al., 2002b). Slc38a2 catalyzes a leak anion current,

2 R.J. Menchini and F.A. Chaudhry Neuropharmacology 161 (2019) 107789

Fig. 1. Putative structure of Slc38a2 with some important regulatory sites plotted. Slc38a2 has 11 putative transmembrane domains (TMDs) with an intracellular N-terminus and an ex- tracellular C-terminus. There are identified three N- glycosylation sites (Asn254, Asn258, Asn272) that play an important part in protein expression. Cys245 and Cys279 make a disulfide bridge in the third extra- cellular loop, while Cys228 and Cys303 are likely part of the 5th TMD. All four cysteines are highly con- served and regulate Slc38a2 transport. Asn82 and Thr384 are part of a Na+ binding site and control Na+ affinity, coupled Na+/substrate transport as well as the Slc38a2-associated anion leak current. Declining external pH impairs Slc38a2 transport ac- tivity through His504. In states of amino acid avail- ability, five lysyl residues in the Slc38a2 N-terminus gets accessible and targeted by a currently unknown E3-ligase for ubiquitin-proteasome-mediated de- gradation. which is augmented by binding of Na+ to Slc38a2 and inhibited to stress: the osmoregulatory and the amino acid regulatory responses varying degrees by substrate transport (Zhang and Grewer, 2007). This (Alfieri et al., 2001; Gazzola et al., 2001; Ling et al., 2001; Hyde et al., leak anion conductance is thermodynamically uncoupled from Na+ and 2001). Following both of these cell stress responses, Slc38a2 expression substrate transport. is dependent on the first common step of the integrated stress response A homology model predicted that Slc38a2 contains a conserved (ISR) pathway, eukaryotic translation initiation factor 2α (eIF2α) Na+ binding site formed by the central parts of transmembrane do- (Gaccioli et al., 2006; Krokowski et al., 2015). eIF2α is essential for mains 1 and 8 (Zhang et al., 2009). Mutation studies showed that start codon recognition and mRNA translation initiation (Jackson et al., mutating the conserved asparagine residue 82 in the highly conserved 2010). Upon its phosphorylation by one or more eIF2α kinases, such as transmembrane domain 1 and threonine residue 384 in transmembrane GCN2 and protein kinase R-like endoplasmic reticulum kinase (PERK), domain 8 markedly reduced the affinity of Slc38a2 for Na+ and thus phospho-eIF2α blocks 5′cap-dependent protein synthesis while indu- substrate transport (Zhang et al., 2008, 2009)(Fig. 1). These results cing transcription of selected genes that promote cell survival, such as implicate Asn82 and Thr384 in the control of the Na+ affinity of Slc38a2, activating transcription factor 4 (ATF4) and growth arrest and DNA consistent with these residues being part of a Na+ binding site. Muta- damage-inducible protein (GADD34) (Pakos-Zebrucka et al., 2016) tions of cysteine279 and tyrosine337 also inhibited Na+ binding, but to a (Fig. 2). Recently, it was demonstrated that Slc38a2 also is regulated by lesser extent (Zhang et al., 2008, 2009). Mutating either Asn82 or Thr384 intrinsic cell stresses, such as endoplasmic reticulum (ER) stress inhibited the leak anion current (Zhang et al., 2008, 2009), implying (Gjymishka et al., 2008). Thus, Slc38a2 may be a common target for that the Na+ binding site controls both Na+ affinity, coupled Na+/ induction or repression by the ISR. substrate transport and the anion leak current, which might pass through the same pore as the Na+ ions. Slc38a2 exhibits both progressive reduction in affinity for Na+ 4. Regulation of Slc38a2 by tonicity binding and progressive lowering of the Vmax for substrate transport as external pH declines (Chaudhry et al., 2002b; Baird et al., 2006). Mu- Mammalian cells adapt to hyperosmotic stress and osmotic tation studies show that extracellular pH affects Slc38a2 transport ac- shrinkage by regulatory volume increase (RVI), in which cellular vo- tivity partially through the conserved histidine 504 residue located on lume is restored by the rapid uptake of inorganic ions (Wehner et al., its extreme C-terminus (Baird et al., 2006). This seems to be achieved 2003). Although the RVI restores cell volume within minutes, the in- either directly by binding of H+ at an allosteric site, or indirectly by tracellular ionic strength becomes unnaturally high, perturbing the allosterically transmitting the effects of such H+ binding to the Na+ function of intracellular molecules. This is countered by accumulating binding site (Baird et al., 2006). Truncating the C-terminus containing compatible osmolytes such as betaine, taurine, and myo-inositol, re- the His504 residue greatly diminishes the pH sensitivity of Slc38a2 but ducing the intracellular ionic strength while maintaining cellular vo- does not abolish it (Zhang et al., 2011), pointing to the existence of lume (Burg et al., 2007). This osmoadaptation is to a large degree additional pH sensitive residues within the protein sequence. orchestrated by the nuclear factor of activated T cells 5 (NFAT5), aka Truncation studies demonstrate that the C-terminus is pivotal for tonicity-responsive enhancer binding protein (TonEBP), which stimu- Slc38a2-mediated amino acid transport by contributing to the control lates transcription of contributing genes such as the betaine GABA of voltage dependence for substrate transport (Zhang et al., 2011). This transporter (BGT1), taurine transporter (TauT) and myo-inositol trans- is achieved without affecting amino acid or Na+ affinity and/or binding porter (SMIT1) (Burg et al., 2007). However, accumulation of these (Zhang et al., 2011). The anion leak current induced by Na+ binding compatible osmolytes is slow. and inhibited by substrate binding was mostly unaffected by C-terminus Slc38a2 expression is regulated by tonicity through induction by truncation (Zhang et al., 2011). Taken together, these findings suggest NFAT5 (Trama et al., 2002), and contributes to the fast osmoadaptive that the C-terminus is important for modulating the rate and voltage response in all cell lines tested across mammalian species (Alfieri et al., dependence of amino acid translocation and/or relocation of the empty 2001, 2002; Nahm et al., 2002; Takanaga et al., 2002; Trama et al., Slc38a2 carrier to the extracellular surface of the plasma membrane. 2002; Lopez-Fontanals et al., 2003; Franchi-Gazzola et al., 2004, 2006; Bevilacqua et al., 2005; Ito et al., 2008; Nishimura et al., 2010). This is consistent with observations of a system A dependent fast regulatory 3. Universal regulatory features of Slc38a2 expression increase in neutral amino acids immediately after cellular exposure to hypertonicity (Bussolati et al., 2001). Upon hyperosmolar exposure, an It was early recognized that Slc38a2 exhibits two universal reg- increase in Slc38a2 mRNA expression (Alfieri et al., 2001) is followed ulatory response characteristics of system A activity following cell by a subsequent increase in Slc38a2 carrier expression on the plasma

3 R.J. Menchini and F.A. Chaudhry Neuropharmacology 161 (2019) 107789

Fig. 2. Slc38a2 and the amino acid response pathway. Starvation for one or more amino acids elicit in- tracellular accumulation of uncharged tRNAs. These are detected by GCN2 which activates cellular adaptation to an amino acid scarce environment through phosphorylation of the translation initiation factor eIF2α. Phospho-eIF2α reduces global protein synthesis. Simultaneously, it induces selective trans- lation of transcriptional regulators such as ATF4 and C/EBP. These regulators promote recovery of cellular amino acid homeostasis by inducing the expression of key proteins of amino acid biosynthesis and transport. One such protein is the Slc38a2 protein which is endowed with a C/EBP-ATF4 response ele- ment.

Fig. 3. Slc38a2 accumulates neutral amino acids (AA) that drive transport by the system ASC transporters ASCT1/2 and the system L trans- porters LAT1/2. The ASCT1/2 and LAT1/2 transporters are all ob- ligatory exchangers that energize uptake of some AA substrates against their concentration gradients with efflux of other AA down their concentration gra- dients. Slc38a2 is a unidirectional electrogenic system A transporter and therefore capable of accu- mulating AA and generating a high intracellular concentration, e.g, of glutamine. Glutamine and other Slc38a2 substrates subsequently power the ASCT1/2 and LAT1/2 by exchanging with their substrates, such as the BCAA and AAA. A, alanine; AAA, aromatic amino acids; BCAA, branched chain amino acids; C, cysteine; Q, glutamine; S, serine; T, threonine.

membrane (Franchi-Gazzola et al., 2004). This leads to increased response to hypertonicity overlaps with the comparably slower accu- system A transport activity and restoration of cell volume through an mulation of compatible osmolytes such as myo-inositol, taurine and expansion of the intracellular amino acid pool (Bussolati et al., 2001). betaine by SMIT1, TauT and BGT1, respectively (Franchi-Gazzola et al., System A is the only major transport activity for neutral amino acids in 2006). Accumulation of compatible osmolytes represents a more per- mesenchymal cells that uses the transmembrane sodium gradient to manent means for cellular osmoadaptation, but may also entail the risk concentrate its substrates intracellularly (Bussolati et al., 2001). Thus, of acquiring central pontine myelinolysis if hypo- or hypernatremia is Slc38a2 plays a pivotal role in the restoration of intracellular amino corrected too fast (Ito et al., 2008). acid stores as most other transport systems are exchangers whose in- The cellular pathways involved in the regulation of Slc38a2 in re- tracellular substrate concentration is coupled to substrates whose steep sponse to hypertonicity are less well understood. In rat skeletal muscle concentration gradient is fostered by the electrogenic Slc38a2 (Franchi- cells, the osmoregulatory response was shown to be inhibited by cy- Gazzola et al., 2006; Evans et al., 2007)(Fig. 3). In fact, the influx of cloheximide and actinomycin D, but not chloroquine (Kashiwagi et al., amino acids following increased system A transport activity is sufficient 2009). This demonstrates that the osmoadaptive response of Slc38a2 for volume recovery throughout the first hours of osmotic stress in consists of transcriptional upregulation without immediate transloca- human vascular endothelial cells (Dall'Asta et al., 1999). SLC38A2 tion of preformed Slc38a2 proteins to the plasma membrane. This in- suppression by RNA interference in cultured human hepatocytes se- terpretation is supported by SLC38A2 silencing experiments in cultured verely delayed cell volume recovery following osmotic stress by human hepatocytes indicating that the increase in system A transport blunting the compensatory increase in the intracellular amino acid activity in response to osmotic stress is accounted for by the synthesis of pool, particularly of glutamine (Bevilacqua et al., 2005). Glutamine is new SLC38A2 carriers (Bevilacqua et al., 2005). In mice thymocytes, the most abundant amino acid in both plasma and cerebrospinal fluid Slc38a2 induction in response to hyperosmotic stress is NFAT5 depen- and a good substrate for the widely expressed systems A, ASC and L dent, although an osmotic response element in the Slc38a2 sequence (Bussolati et al., 2001)(Fig. 3). It therefore plays an essential role in has not yet been described (Trama et al., 2002). In L6 myotubes and amino acid exchange via different transport systems. Glutamine in- Chinese hamster ovary (CHO) cells, the mitogen-activated protein tracellularly concentrated by Slc38a2 may secure the intracellular ac- (MAP) kinases c-Jun N-terminal kinase (JNK) and extracellular signal- cumulation of neutral amino acids, which are poor system A substrates, regulated kinase (ERK) did not contribute to the osmoregulatory re- through exchange via systems ASC and L. This fast osmoadaptive sponse (Lopez-Fontanals et al., 2003; Kashiwagi et al., 2009). In CHO

4 R.J. Menchini and F.A. Chaudhry Neuropharmacology 161 (2019) 107789 cells, inhibition of p38 partially inhibited the osmoregulatory response consists of both a rapid translocation of preformed Slc38a2 proteins in a mitogen-activated protein kinase kinase 3 (MKK3)-independent from perinuclear stores to the plasma membrane, and long-term tran- manner (Lopez-Fontanals et al., 2003), while in L6 myotubes, inhibition scriptional upregulation (Ling et al., 2001; Palii et al., 2006; Kashiwagi of p38 had no effect on the induction of Slc38a2 (Kashiwagi et al., et al., 2009). Reintroduction of extracellular system A substrates abol- 2009). ishes the transient increase in Slc38a2 expression that occurs in amino Slc38a2 induction by osmotic shock was reported to be eIF2α-in- acid starved cells in all cell lines tested across mammalian species dependent in mouse embryonic fibroblasts (Gaccioli et al., 2006). (Gazzola et al., 2001; Ling et al., 2001; Hyde et al., 2001; Bain et al., However, Krakowski and coworkers found the osmoadaptive response 2002; Lopez-Fontanals et al., 2003; Tanaka et al., 2005; Novak et al., of Slc38a2 to be inhibited in both mouse embryonic fibroblasts and 2006; Lopez et al., 2006; Wu et al., 2007; Nickel et al., 2010). human cervical cancer cells in mild hyperosmotic stress in response to high levels of phosphorylated eIF2α, a mediator of proapoptotic sig- 5.1. Slc38a2 and the GCN2 pathway naling (Krokowski et al., 2015). The osmoregulatory adaptation by Slc38a2 was rescued by protein phosphatase 1 (PP1) regulatory subunit Amino acid insufficiency is sensed by the eIF2α kinase GCN2. Lack GADD34 through the dephosphorylation of eIF2α (Krokowski et al., of one or several amino acids leads to intracellular accumulation of 2015). Hyperosmotic stress causes fragmentation of the Golgi apparatus uncharged tRNAs (Kilberg et al., 2009). These activate GCN2, leading and microtubule network, trapping immature SLC38A2 carriers in the to phosphorylation of eIF2α, the common integrator in the ISR (Pakos- cis-Golgi compartment and attenuating the osmoadaptive response of Zebrucka et al., 2016)(Fig. 2). Phosphorylation of eIF2α facilitates Slc38a2 (Krokowski et al., 2017). GADD34/PP1 counteracts this by adaptation to amino acid starvation by greatly reducing global protein maintaining Golgi and microtubule integrity during stress, promoting synthesis and thus cellular amino acid demand. Simultaneously, SLC38A2 maturation and trafficking to the plasma membrane in human phospho-eIF2α induces the translation of selected cellular stress re- corneal epithelial cells and mouse embryonic fibroblasts (Krokowski sponse proteins, such as the transcription factors ATF4 and CCAAT- et al., 2017). enhancer-binding protein β (C/EBPβ). In turn, they induce expression Osmotic stress contributes to the pathogenesis of many human of selected genes through cognate response elements, promoting re- diseases by triggering cell damaging processes such as cell shrinkage, covery of amino acid homeostasis (Fig. 2; Kilberg et al., 2009). oxidative stress and DNA damage (Brocker et al., 2012). Studies have The Slc38a2 gene contains such a highly conserved amino acid re- implicated Slc38a2-dependent osmoadaptation in heart failure, central sponse element (AARE), which controls its substrate-dependent tran- pontine myelinolysis and dry eye syndrome. Slc38a2 mRNA expression scriptional regulation and is located in intron 1 (Palii et al., 2004; Hyde was induced in cardiomyocytes and skeletal muscle cells in a TauT et al., 2007). Juxtaposed to this AARE, intron 1 also contains a CAAT- knockout model, which developed dilated cardiomyopathy (Ito et al., box (response element for C/EBPβ) that enhances the action of the 2008). This suggests that Slc38a2 may play an osmoprotective role in AARE, and a purine-rich (PuR) sequence that serves as a repressor congestive heart failure. Slc38a2 was induced in rat oligodendrocyte element to maintain a low basal transcription rate of Slc38a2 in the cell bodies, but not processes, throughout the brain in a model for absence of amino acid deficiency (Palii et al., 2004). The AARE is re- prolonged systemic hypertonicity (Maallem et al., 2008). Interestingly, quired for the induction of Slc38a2 by amino acid starvation, whereas Slc38a2 was not found to be expressed in oligodendrocytes, even mutating the CAAT-box can reduce induced transcription by approxi- marked by the same antibody under basal conditions (Gonzalez- mately 40% (Palii et al., 2006). Several of the proteins in the complexes Gonzalez et al., 2005; Jenstad et al., 2009). Additionally, Slc38a2 im- that bind the Slc38a2 AARE are shared with the juxtaposed CAAT-box munolabeling in diverse brain regions under basal conditions was not (Palii et al., 2006). Upon starvation for one or more amino acids, the recovered after 24 h of systemic hypertonicity (Maallem et al., 2008). CAAT-AARE sequences in the Slc38a2 gene are targeted by the GCN2/ Thus, Slc38a2 may play a part in the brain's osmoadaptive response to ATF4 pathway (Palii et al., 2006; Gaccioli et al., 2006). ATF4 binds to dystonicity to prevent central pontine myelinolysis, at least in a subset the CAAT-AARE complex as a heterodimer prior to RNA polymerase II of oligodendrocytes. SLC38A2 and GADD34/PP1 coexpression con- binding, promoting transiently enhanced Slc38a2 transcription by re- tributes to the osmoadaptive response in human corneal epithelial cells cruitment of additional transcription factors such as C/EBPα, C/EBPβ- as described above, protecting against the hyperosmotic tear film that LAP and c-Jun (Palii et al., 2006). In addition to recruitment of mem- causes dry eye syndrome (Krokowski et al., 2017). bers of the general transcription machinery, increased H3 acetylation Induction of system A transport activity has been proposed to be was evident at both the Slc38a2 promoter and AARE following amino involved in the volume increase needed to enter a new cell division acid limitation (Gjymishka et al., 2008; Thiaville et al., 2008). Slc38a2 cycle, either by supplying amino acids functioning as osmolytes directly induction in response to amino acid starvation is subject to regulation or through further substrate exchange via other amino acid transporters by a self-limiting program and subsequent binding of repressor proteins (Bussolati et al., 2001; Franchi-Gazzola et al., 2006). In CHO cells, in- such as ATF3, C/EBPβ-LIP and C/EBPδ slowly represses the AARE- hibition of cyclin-dependent kinase (CDK) 4-6-cyclin D complexes and mediated transcription (Palii et al., 2006). C/EBPγ had no effect on CDK4 lead to a slight decrease of basal system A activity, but not of the Slc38a2 transcription (Palii et al., 2006). osmotic response (Lopez-Fontanals et al., 2003). However, inhibition of In addition to the AARE, Slc38a2 contains an internal ribosome CDK2 repressed the Slc38a2-mediated osmotic response in a dose-de- entry site (IRES) at its 5′-UTR (Gaccioli et al., 2006). This IRES is pendent manner (Lopez-Fontanals et al., 2003). CDK2-cyclin E and constitutively active in both amino acid fed and starved cells. The IRES CDK2-cyclin A regulates entry into and progression through the S allows for cap-independent Slc38a2 translation during amino acid phase, contributing to the iso-osmotic volume increase that takes place starvation where global translation initiation might be restricted before mitosis. Whether Slc38a2 may contribute to cellular volume (Gaccioli et al., 2006). However, it neither stimulates translation nor is increase before mitosis in a CDK-dependent manner warrants further it regulated by adaptive regulation. The constitutively active nature of inquiry. its IRES testifies to the pivotal role of Slc38a2 in homeostasis and stress. In contrast to the transcriptional activation of Slc38a2, less is known 5. Regulation of Slc38a2 by amino acid availability about how preformed carriers are recruited from intracellular stores in response to amino acid deficiency. In rat skeletal muscle cells, inhibi- Another hallmark of Slc38a2 is its regulation in response to amino tion by chloroquine, cycloheximide and actinomycin D abolished the acid availability. This is achieved by the ability of Slc38a2 to adapt its adaptive response of Slc38a2 (Kashiwagi et al., 2009). This suggested activity to the availability of extracellular substrates, a response named that both transporter cycling from an intracellular store and de novo adaptive regulation (Gazzola et al., 1972). This adaptive response synthesis are involved in the response of Slc38a2 to amino acid

5 R.J. Menchini and F.A. Chaudhry Neuropharmacology 161 (2019) 107789 starvation. Further inhibition studies suggested that phosphoinositide from experiments with mouse livers, where increased Slc38a2 expres- 3-kinase (PI3K) is involved in transporter recruitment from an in- sion, achieved by an adenoviral delivery system, increased hepatic tracellular pool, while the MAP kinases ERK and JNK and the upstream amino acid concentrations and mTORC1/S6K activity (Uno et al., kinase MKK4 are involved in pathways for de novo synthesis in both L6 2015). myotubes and CHO cells (Lopez-Fontanals et al., 2003; Hyde et al., 2007; Kashiwagi et al., 2009). Inhibition of the p38 MAPK pathway, 7. Slc38a2 as a tranceptors MKK3 or mTOR pathway had no effect on adaptive regulation in rat skeletal muscle cells (Lopez-Fontanals et al., 2003; Hyde et al., 2007; Both the GCN2 and mTOR pathways are essential for amino acid Kashiwagi et al., 2009). Slc38a2 adaptive regulation is independent of sensing in mammalian cells (Broer and Broer, 2017). However, the CDK2, CDK4 (Lopez-Fontanals et al., 2003) and NFAT5 (Trama et al., amino acid sensing mechanisms upstream of GCN2 and mTOR are not 2002). However, it was recently demonstrated that CDK7 is induced in fully elucidated. In cultured myocytes, induction of Slc38a2 by amino a GCN2-dependent manner following amino acid starvation, and that acid starvation is inhibited upon introduction of a single amino acid inhibition of CDK7 blunts the adaptive response of Slc38a2 (Stretton while the medium remained starved for other amino acids (Hyde et al., et al., 2019). Slc38a2 is known to be stabilized during amino acid 2007). Moreover, the potency by which substrates repress Slc38a2 ex- limitation (Hyde et al., 2007). This stabilization is lost upon mutating pression is correlated to their transport Km (Hyde et al., 2007). In MCF- the N-terminal lysyl residues to alanine (Hoffmann et al., 2018)(Fig. 1). 7 human breast cancer cells, both acute and sustained incubation with Intriguingly, the adaptive upregulation of Slc38a2 was abolished in the MeAIB alone leads to increased mTOR-mediated p70 S6K1 phosphor- absence of Na+ (Hoffmann et al., 2018). Hoffmann and coworkers ylation (Pinilla et al., 2011). As MeAIB is not metabolized, this implies proposed a model in which Slc38a2 becomes downregulated in states of that Slc38a2 acts as an amino acid sensor upstream of mTOR (Hundal amino acid availability through ubiquitination of lysyl residues in its N- and Taylor, 2009). This observation lead Hyde and coworkers to pro- terminal tail, which become accessible to an unknown E3 ubiquitin li- pose that Slc38a2 acts as a transceptor, i.e., a hybrid transporter-re- gase upon conformational changes following substrate release ceptor (Hyde et al., 2007). According to this model, the Slc38a2 (Hoffmann et al., 2018)(Fig. 1). The binding of Na+ may stabilize transporter-substrate complex may sense changes in extracellular Slc38a2 through conformational changes making these lysyl residues amino acid availability and transmit this signal intracellularly, in ad- less accessible, while the absence of both Na+ and substrate may lead to dition to the ability of Slc38a2 to modulate the intracellular amino acid an N-terminal conformation that maximally exposes the lysyl residues, pool directly as a key transporter of glutamine (Hundal and Taylor, leading to protein instability and degradation. 2009). The mechanisms responsible for how substrate site occupancy is 6. Slc38a2 and the mTOR pathway sensed or how this signal is transduced downstream to allow Slc38a2 to autoregulate its own expression is currently unknown. One possibility is There has been special interest in the possibility that Slc38a2 may that the inward current coupled to substrate transport may lead to act in concert with the system L transporter LAT1 (Slc7a5) to in- membrane depolarization. This is suggested to increase intracellular tracellularly concentrate branched-chain amino acids, particularly Ca2+ and activate L-type voltage-sensitive calcium channels in response leucine (Fig. 3). In fact, Baird and coworkers have shown that coex- to increased amino acid concentration in the gut lumen in intestinal pression of Slc38a2 and LAT1 in Xenopus laevis oocytes leads to a endocrine cells (Young et al., 2010). The same mechanism was seen in considerable increase in intracellular accumulation of leucine as com- enteroendocrine GLUTag cells, where the transport-current associated pared with expression of LAT1 alone (Baird et al., 2009). Leucine is a with glutamine furnished by Slc38a2 has been proposed to contribute to potent stimulator of the mTOR pathway, and it is proposed that Slc38a2 membrane depolarization triggering GLP-1 release (Reimann et al., may indirectly influence amino acid sensing through LAT1 and mTOR 2014). Hundal and Taylor proposed that the trans-inhibition of Slc38a2 (Dodd and Tee, 2012). This endows the mTOR pathway with additional seen upon excess accumulation of Slc38a2 substrates in the cytoplasm, regulatory possibilities as Slc38a2 is extensively regulated. Additional which prevents Slc38a2 from completing its transport cycle and return evidence for cooperation between Slc38a2 and LAT1 for mTOR acti- to its outward facing position, may play a part (Hundal and Taylor, vation is the observation that Slc38a2 knockdown in rat myocytes leads 2009). Recently, members of the Slc36 family and the Slc38a2 homo- to a drop in intracellular concentrations of both glutamine and leucine, logue Slc38a9 have been proposed to act as amino acid transceptor followed by repression of mTORC1 signaling (Evans et al., 2008). This upstream of mTOR, further suggesting a transceptor role for Slc38a2 clearly implicates a dependency on Slc38a2 for furnishing the free in- (Fan and Goberdhan, 2018). tracellular amino acid pool with both amino acids (Evans et al., 2008). Additionally, glutamine has been demonstrated to be a rate limiting 8. Regulation of Slc38a2 by ER stress nutrient for mTOR activation (Nicklin et al., 2009). The exact mechanisms in which Slc38a2 and mTOR interact remain The ER is involved in manifold cellular functions such as protein elusive. In cultured primary trophoblasts isolated from normal human synthesis and processing, lipid synthesis and calcium regulation placentas, knockdown of either mTORC1 or mTORC2 greatly atte- (Almanza et al., 2019). Three ER stress signaling pathways collectively nuated system A and L transport activity, while knockdown of both known as the unfolded protein response (UPR) are triggered when mTORC1 and mTORC2 completely inhibited system A and L transport misfolded proteins accumulate in the ER lumen. The UPR aims to re- (Rosario et al., 2013). This was achieved through the specific down- store ER function through either the inositol-requiring enzyme 1 (IRE1), regulation of system A and L isoforms SLC38A2 and LAT1 in microvillus ATF6 or PERK pathways (Almanza et al., 2019). Similar to GCN2, PERK plasma membrane fractions (Rosario et al., 2013). Upon silencing of is an eIF2α kinase and part of the ISR (Pakos-Zebrucka et al., 2016). As both mTORC1 and mTORC2, growth factor-mediated stimulation of for the osmoregulatory and amino acid response pathways, phosphor- system A and L transport activity was abrogated (Rosario et al., 2013). ylation of eIF2α by PERK may suppress global protein synthesis aside Silencing mTORC1 leads to relocation of SLC38A2 from the plasma from the induction of selected proteins that facilitate the restoration of membrane to the cytosol in syncytial islands (Rosario et al., 2013). This ER homeostasis (Pakos-Zebrucka et al., 2016). suggests that mTORC1 and mTORC2 are involved in the posttransla- Following both amino acid limitation and ER stress in human he- tional regulation of Slc38a2 by regulating Slc38a2 trafficking, possibly patoma cells, ATF4 was synthesized and recruited to the Slc38a2 AARE through Nedd4-2 ubiquitination (Chen et al., 2015). This was later with subsequent synthesis and recruitment of ATF3 and C/EBPβ confirmed by Rosario and coworkers in primary human trophoblast (Gjymishka et al., 2008). Unlike for the AAR pathway, UPR stimulation cultures (Rosario et al., 2016). Additional evidence of interaction stems did not increase Slc38a2 transcription (Gjymishka et al., 2008). In fact,

6 R.J. Menchini and F.A. Chaudhry Neuropharmacology 161 (2019) 107789 responsiveness of the Slc38a2 gene to UPR pathway activation was cell This effect was not selective for Slc38a2, but part of a general cellular type specific. In response to UPR activators, human kidney and breast response with increased total content of ubiquitinated proteins (Nardi cancer cells exhibited increased Slc38a2 expression, whereas mouse et al., 2015). Nardi and coworkers propose that increased fatty acids and human liver cells and fibroblasts did not (Gjymishka et al., 2008). generated by increased lipolysis during fasting target intracellular Neither increased H3 acetylation nor recruitment of the general tran- proteins for proteolysis to free amino acids for hepatic gluconeogenesis scription machinery was seen at the Slc38a2 promoter or AARE fol- (Nardi et al., 2015). Fatty acid-induced downregulation of Slc38a2 also lowing UPR activation (Gjymishka et al., 2008). Mutation studies show decreases reuptake of free amino acids from the circulation into myo- that destruction of the Slc38a2 AARE at intron 1 abolishes both the cytes following proteolysis. Mutagenesis studies show that lysine re- transcriptional activation following AAR- and UPR-dependent tran- sidues on the intracellular N-terminal Slc38a2 are targeted for ubi- scription (Gjymishka et al., 2008). Amino acid limitation induced quitin-proteasome-mediated degradation through a currently unknown Slc38a2 transcription while activation of the UPR did not. However, E3-ligase (Nardi et al., 2015; Hoffmann et al., 2018)(Fig. 1). simultaneous activation of both pathways lead to almost complete SLC38A2 and ASCT2 (Slc1a5) are degraded through ubiquitination abolishment of Slc38a2 induction through the AAR (Gjymishka et al., by the E3 ubiquitin ligase RFN5 in a human breast cancer cell line 2008). Thus, it has been suggested that the lack of Slc38a2 induction by following paclitaxel-induced ER stress (Jeon et al., 2015). Many cancer the UPR is caused by a repressive signal that overrides ATF4 binding cells depend on glutamine to sustain glutaminolysis for synthesis of and prevents transcription (Gjymishka et al., 2008). It is unknown how proteins, nucleic acids and lipids (DeBerardinis and Cheng, 2010). the UPR-mediated repressive signal blocks Slc38a2 transcription, but it ASCT2 was recently shown to be responsible for most of the glutamine is not mediated by the UPR effectors ATF6 or XBP1 (Gjymishka et al., transport in human cervical cancer and osteosarcoma cells, while 2008). SLC38A2 expression in these cells was induced by the GCN2/ATF4 In another study, Slc38a2 was found to be necessary for arsenite- pathway following amino acid imbalance (Broer et al., 2016). De- induced ER stress in human embryonic kidney cells and mouse adipo- gradation of SLC38A2 and ASCT2 by RFN5 following paclitaxel-in- cytes (Oh et al., 2012). Slc38a2 expression and transport activity was duced ER stress ultimately decreases mTOR signaling and cellular induced by arsenite. This induction depended on ATF4 and both oxi- proliferation through reduced cellular glutamine uptake, thus setting dative and proteotoxic stress signals (Oh et al., 2012). Slc38a2 activity the stage for apoptosis and cell death (Jeon et al., 2015). seems to be specific for arsenite-mediated ER stress as it cannot bein- duced by the ER stressors tunicamycin or thapsigargin (Oh et al., 2012). 10. Slc38a2 is regulated by a novel K+ channel Arsenite can activate mTOR, and mTOR activity is augmented by raised levels of Slc38a2 carriers and transport activity during arsenite stress Two healthy born brothers developed ataxia, myoclonic jerks, lost (Oh et al., 2012). Oh and coworkers suggested that arsenite-induced the ability to walk and talk, and developed drug-resistant progressive activation of Slc38a2 by the PERK/ATF4 pathway increases system A myoclonus epilepsy (PME) during their first two years of life (Moen and L transport activity. This may supply the amino acids necessary for et al., 2016). Interestingly, cerebrospinal fluid (CSF) analyses revealed furnishing adaptive cell responses such as increased synthesis of cha- increased glutamine and reduced glutamate concentrations. As Slc38 perones and antioxidants. Import of leucine through the concerted ac- family transporters have been associated with replenishment of the fast tions of Slc38a2 and LAT1 activates mTOR. This increase in protein neurotransmitters glutamate and GABA (Chaudhry et al., 2002b, 2008; synthesis may ultimately overactivate the ER with subsequent devel- Jenstad et al., 2009; Nissen-Meyer and Chaudhry, 2013; Qureshi et al., opment of ER stress and activation of the UPR (Oh et al., 2012). To- 2019), we hypothesized that the observed glutamine and glutamate gether with LAT1, LAT3 (Slc43a1) and ATP-binding cassette sub-family levels and PME in the two children could be due to dysfunctional C member 4 (ABCC4), Slc38a2 was also found to be upregulated fol- Slc38a2 transport activity. To our surprise, whole exome sequencing lowing mercury exposure in mouse myoblasts in a PERK/ATF4-depen- did not reveal pathologic variants in Slc38a2 or any other Slc38 family dent manner (Usuki et al., 2017). transporter, but a novel frameshift mutation in a protein known as potassium channel tetramerization domain 7 (KCTD7). Although an 9. Slc38a2 degradation orphan protein, several studies indicated involvement of KCTD7 and its homologues in metabolism, cellular proliferation and differentiation Slc38a2 is extensively regulated and increases its expression and and had shown association with different forms of epilepsy (Liu et al., trafficking at the cell membrane in response to numerous stimuli. Thus, 2013; Van et al., 2007; Kousi et al., 2012; Farhan et al., 2014). In a mechanisms are needed for rapid Slc38a2 turnover to allow for fast series of electrophysiological experiments in Xenopus laevis oocytes, we downregulation in response to changes in the cellular microenviron- demonstrated that KCTD7 is a novel K+ channel hyperpolarizing and ment. There is increasing evidence of such regulatory control of Slc38a2 stabilizing the membrane potential (Moen et al., 2016). Notably, it also by the ubiquitin-proteasome system (UPS). In 3T3-L1 adipocytes, CHO regulates glutamine transport by Slc38a2 (Fig. 4), while it has no im- cells and Xenopus laevis oocytes, Slc38a2 plasma membrane activity and pact on Slc38a5 activity. Five pathogenic variants of KCTD7 depolarize expression are regulated by the E3 ubiquitin ligase Nedd4-2 (Hatanaka the cellular membrane potential and hamper glutamine transport. As et al., 2006a). Nedd4 and c-Cbl did not significantly affect Slc38a2 Slc38a2 is enriched in glutamatergic neurons and Slc38a2-mediated transport activity. Additionally, Slc38a2 stability seems to be subject to glutamine transport is required for synthesis of the neurotransmitter multi-monoubiquitination by a ubiquitin ligase other than Nedd4-2 glutamate (Jenstad et al., 2009), our data are thus consistent with the (Hatanaka et al., 2006a; Hoffmann et al., 2018). In cultured primary increased glutamine and reduced glutamate levels observed in the CSF . trophoblasts isolated from normal human placentas, Rosario and cow- Interaction between channels and transporters have recently been orkers show that mTORC1 regulates system A and L transport activity identified to be widespread. The sodium/myo-inositol cotransporter 1 by modulating SLC38A2 and LAT1 carrier expression on the plasma (SMIT1) forms a complex with potassium voltage-gated channel sub- membrane through ubiquitination by Nedd4-2 (Rosario et al., 2016). family Q member 1 (KCNQ1) and potassium voltage-gated channel The increased Slc38a2 carrier expression and transport activity subfamily E member 2 (KCNE2). This complex regulates CSF myo-in- following adaptive upregulation and osmotic stress, were strongly at- ositol levels and neuronal excitability (Roepke et al., 2011; Abbott tenuated following preincubation with polyunsaturated fatty acids in et al., 2014). The Ca2+-activated K+ channel (MaxiK) forms a complex rat L6 myotubes and human cervical cancer cells (Nardi et al., 2015). with the GABA transporter 3 (GAT3; Slc6a11) (Singh et al., 2016), This was not caused by Slc38a2 mRNA suppression, but rather by a while another K+ channel (KCNA2/Kv1.2) interacts with LAT1 decreased plasma membrane carrier expression as Slc38a2 transport (Baronas et al., 2018). In both cases, the channels and transporters proteins were targeted for degeneration by the UPS (Nardi et al., 2015). regulate reciprocal activity with impact on neurotransmission and

7 R.J. Menchini and F.A. Chaudhry Neuropharmacology 161 (2019) 107789

mellitus and metabolic syndrome (Hatanaka et al., 2006b). This may represent a chronic effect of the lack of insulin stimulus on Slc38a2, and testament to its importance for adipocyte function. Thus, Slc38a2 or its regulatory proteins may serve as therapeutic targets for new anti-dia- betic drugs. Recently, Medras and coworkers showed that cotreatment with oral glutamine supplementation and the GLP-1 analogue liraglu- tide leads to increased glycemic control. This is achieved through in- creased insulin production and reduced β-cell apoptosis in diabetic male rats associated with an upregulation of Slc38a2 in the endocrine pancreas (Medras et al., 2018). This is consistent with enrichment of Slc38a2 in pancreatic α-cells involved in release of glucagon and glu- tamate to stimulate insulin secretion (Gammelsaeter et al., 2011; Jenstad and Chaudhry, 2013).

12. Regulation of Slc38a2 by hormones, cytokines and vitamins

+ Fig. 4. Slc38a2 function is regulated by a novel K -channel. In addition to the previously mentioned regulatory mechanisms, The potassium channel tetramerization domain 7 (KCTD7) protein harbour a Slc38a2 expression is regulated in different organ systems by diverse bric-à-brac, tramtrack, broad complex/poxvirus and zinc finger (BTB/POZ) signal transduction pathways in response to changing physiological domain which is homologous to the highly conserved cytoplasmic N-terminal demands. assembly domain T1 of voltage-gated K+ (K ) channels. This motif supports v Slc38a2 is subject to regulation by a number of hormones. In rat protein-protein interactions. KCTD7 has been shown to regulate the transport function of Slc38a2. Pathologic variants of KCTD7 reduce glutamine transport hepatocytes, Slc38a2 expression is induced by glucagon through the and result in impaired glutamate synthesis. phosphorylation of transcription factor cAMP response element-binding protein (CREB) in a cAMP/PKA-dependent manner (Ortiz et al., 2011). CREB subsequently binds a cAMP response element (CRE) site located neuronal excitability. Thus, KCTD7 may be the first identified channel in the 5′-UTR promoter region of Slc38a2 (Ortiz et al., 2011). interacting with and regulating the function of Slc38a2. Other channels In rat dam mammary glands, Slc38a2 expression is induced by 17β- and/or proteins may regulate Slc38a2 in different ways. estradiol via the estrogen receptor (ER)-α, which in turn binds an es- trogen response element (ERE) in the Slc38a2 promoter (Velazquez- 11. Pharmacologic targeting Villegas et al., 2014). Slc38a2 mRNA and protein expression is also induced by prolactin in rat mammary gland explants and human breast Apart from the canonical competitive inhibitor of system A trans- cancer cells (Velazquez-Villegas et al., 2015). Recently, Morotti and port, MeAIB (Christensen et al., 1965), no pharmacological agent spe- coworkers demonstrated that Slc38a2 expression is induced by hy- cifically targeting Slc38a2 has been identified thus far. MeAIBisa poxia-inducible factor-1α (HIF-1α) in MCF-7 ERα+ human breast substrate analogue ousting other Slc38a2 substrates for transport by cancer cells, and that the downregulation of Slc38a2 by antiendocrine Slc38a2. treatment with the selective estrogen receptor degrader fulvestrant was In Xenopus laevis oocytes, Bröer and coworkers showed that Slc38a2 abolished under hypoxic conditions (Morotti et al., 2019). The binding is inhibited by the glutamine analogue y-glutamyl-p-nitroanilide sites for HIF-1α and ER-α overlap in a cis-regulatory element of (GPNA) (Broer et al., 2016). However, GPNA also inhibits ASCT2, Slc38a2, and there seems to be a switch between them for the regula- Slc38a1, Slc38a4, Slc38a5 and LAT1 (Broer et al., 2016; Chiu et al., tion of Slc38a2 in tumor hypoxia. In Slc38a2 knockdown experiments, 2017). The ASCT2 inhibitor benzylserine was also shown to inhibit tamoxifen-resistant human breast cancer cells showed reduced growth Slc38a2 as well as Slc38a1 and LAT1 (Broer et al., 2016; van et al., following decreased glutamine consumption, mitochondrial respiration 2018). Recently, 2-amino-4-bis(aryloxybenzyl)aminobutanoic acid and mTOR signaling (Morotti et al., 2019). As high tumor expression (AABA) was shown to block Slc38a2 and LAT1 (Broer et al., 2018). Due rates of Slc38a2 correlated with poor patient outcomes in analyzed to their lack of specificity, GPNA, AABA and benzylserine do not appear clinical data, Slc38a2 may furnish cancer cells with glutamine under to be useful inhibitors of Slc38a2 transport activity. stress conditions such as solid tumor hypoxia and be a possible target In rat skeletal muscle cells and adipocytes, Slc38a2 carriers are for novel antineoplastic agents (Morotti et al., 2019; Broer et al., 2019). translocated from their intracellular storage site in the trans-Golgi Placental Slc38a2 is subject to diverse hormonal regulation as part network to the plasma membrane in response to insulin signaling (Hyde of the maternal endocrine adaptation to pregnancy. In a human chor- et al., 2005; Hatanaka et al., 2006b). The saturated fatty acid ceramide iocarcinoma cell line, cortisol stimulates system A amino acid transport reduced system A transport activity by internalizing Slc38a2 and im- by translocating SLC38A2 to the plasma membrane from intracellular pairing insulin-stimulated translocation of Slc38a2 to the plasma stores through unknown mechanisms (Jones et al., 2009). At higher membrane (Hyde et al., 2005). Concomitantly, ceramide reduced pro- levels, cortisol induces Slc38a2 transcription. Daily testosterone injec- tein synthesis by reducing the intracellular amino acid pool and mTOR tions restricted fetal growth in pregnant rat dams by decreasing Slc38a2 pathway activity (Hyde et al., 2005). Hyde and coworkers speculated mRNA expression and carrier levels (Sathishkumar et al., 2011). Ma- that the use of sphingomyelinase/ceramide synthase inhibitors that ternal growth hormone treatment in pregnant pigs led to a significant reduce muscle ceramide levels may be beneficial in catabolic muscle- increase in Slc38a2 carrier levels in trophoblasts (Tung et al., 2012). wasting conditions such as uremic metabolic acidosis. In fact, inhibition Maternal infusion of exogenous insulin-like growth factor 1 (IGF1) in an of Slc38a2 by acidosis with subsequent reduction of the intracellular early pregnancy guinea pig model increased placental Slc38a2 expres- glutamine pool may play a pivotal role in the development of sarco- sion at midgestation (Sferruzzi-Perri et al., 2007). Globular adiponectin penia and cachexia in patients with diabetic nephropathy, end-stage increased SLC38A2 levels in cultured human trophoblasts (Jones et al., renal disease and uremic metabolic acidosis (Evans et al., 2008). Both 2010). As reviewed above, insulin increased Slc38a2 plasma membrane diabetes mellitus and metabolic syndrome are diseases characterized by levels by translocation from its trans-Golgi network stores (Hatanaka insulin resistance, which may influence Slc38a2 expression. Hatanaka et al., 2006b). In human cultured trophoblasts, full-length adiponectin and coworkers found that Slc38a2 mRNA expression is significantly abolishes this insulin-dependent increase in SLC38A2 levels (Jones downregulated in human and rodent adipose tissue in type 2 diabetes et al., 2010).

8 R.J. Menchini and F.A. Chaudhry Neuropharmacology 161 (2019) 107789

In the placenta, Slc38a2 and Slc38a1 are regulated by the key that Slc38a1 is enriched in PV+ GABAergic interneurons, where it is proinflammatory cytokines interleukin-1β (IL-1β), IL-6 and tumor ne- important for accumulation of glutamine for GABA synthesis and de- crosis factor α (TNFα). IL-6 and TNFα stimulated system A activity in termines the GABAergic vesicular load (Chaudhry et al., 2002b; Solbu cultured human trophoblasts by increasing mRNA expression and pro- et al., 2010; Qureshi et al., 2019). Indeed, Slc38a1 is essential for tein levels of Slc38a2 and Slc38a1 (Jones et al., 2009), while IL-1β cortical processing and plasticity (Qureshi et al., 2019). Altogether, reduced basal expression of Slc38a2 and Slc38a1 mRNA as well as Slc38a1 more selectively transports glutamine for neurotransmission system A transport activity (Thongsong et al., 2005). These findings and for aspects of heart physiology, while Slc38a2 is responsible for the may offer a link between proinflammatory cytokines and pregnancies general system A activity involved in hyperosmotic stress, amino acid complicated by obesity and/or diabetes mellitus, which exhibit ele- adaptation and ER stress and a target for regulation by hormones. vated levels of cytokines, insulin resistance, increased placental amino Hence when Slc38a1 and Slc38a2 are co-localized (Blot et al., 2009), acid uptake and fetal overgrowth. Slc38a2 and Slc38a1 expression and they may be differentially involved in cellular functions. Yet, they may system A transport activity were reduced in syncytiotrophoblasts in a partly compensate if one is dysfunctional (Qureshi et al., 2019). cohort of pregnant Malawian women with placental malaria and in- tervillousitis (Boeuf et al., 2013). Thus, Plasmodium falciparum may lead 14. Concluding remarks to fetal growth restriction in placental malaria by downregulating Slc38a2 and Slc38a1 through proinflammatory cytokines. Slc38a2 is Slc38a2 is regulated in numerous ways and by multitude of com- also regulated by the cytokine transforming growth factor-β1 (TGF-β1). ponents. We have demonstrated that KCTD7 regulates Slc38a2 and In rat aortic vascular smooth muscle cells, TGF-β1 induces Slc38a2 gene contributes to PME. However, as amino acid transport by Slc38a2 itself expression and increased Slc38a2-mediated proline transport, possibly also contributes to and regulates multitudinous metabolic and signaling contributing to wound healing and intimal thickening at sites of vas- pathways, Slc38a2 may contribute to the pathophysiology of a large cular injury (Ensenat et al., 2001). Interestingly, it has been discovered number of medical conditions, including cancer, neurological diseases, that dysfunctional TGF-β1 signaling underlies several hereditary vas- diabetes mellitus and more. cular syndromes such as the aortopaties Marfan syndrome and Loeys- Dietz syndrome (Takeda et al., 2016). Thus, the interplay between TGF- References β1 and Slc38a2 warrants further investigation.

Slc38a2 is also regulated by vitamins. 1,25-dihydroxy vitamin D3 Abbott, G.W., Tai, K.K., Neverisky, D.L., Hansler, A., Hu, Z., Roepke, T.K., Lerner, D.J., increases placental system A transport activity by increasing Slc38a2 Chen, Q., Liu, L., Zupan, B., Toth, M., Haynes, R., Huang, X., Demirbas, D., Buccafusca, R., Gross, S.S., Kanda, V.A., Berry, G.T., 2014. KCNQ1, KCNE2, and Na mRNA expression through the vitamin D receptor (VDR) in cultured +-coupled solute transporters form reciprocally regulating complexes that affect human primary trophoblast cells (Chen et al., 2017). neuronal excitability. Sci. Signal. 7, ra22. Alfieri, R.R., Cavazzoni, A., Petronini, P.G., Bonelli, M.A., Caccamo, A.E., Borghetti, A.F., Wheeler, K.P., 2002. Compatible osmolytes modulate the response of porcine en- 13. Differential function and regulation of Slc38a1 and Slc38a2 dothelial cells to hypertonicity and protect them from apoptosis. J. Physiol. 540, 499–508. It is interesting that the homologous transporter Slc38a1, which also Alfieri, R.R., Petronini, P.G., Bonelli, M.A., Caccamo, A.E., Cavazzoni, A., Borghetti, A.F., supports system A activity, lacks several of the functions and regulatory Wheeler, K.P., 2001. Osmotic regulation of ATA2 mRNA expression and amino acid transport System A activity. Biochem. Biophys. Res. Commun. 283, 174–178. mechanisms of Slc38a2. Slc38a1 is not involved in the same general Almanza, A., Carlesso, A., Chintha, C., Creedican, S., Doultsinos, D., Leuzzi, B., Luis, A., stress response pathways as Slc38a2 (Ling et al., 2001; Hyde et al., McCarthy, N., Montibeller, L., More, S., Papaioannou, A., Puschel, F., Sassano, M.L., 2001; Kashiwagi et al., 2009). The studies performed in organ systems Skoko, J., Agostinis, P., de, B.J., Eriksson, L.A., Fulda, S., Gorman, A.M., Healy, S., Kozlov, A., Munoz-Pinedo, C., Rehm, M., Chevet, E., Samali, A., 2019. Endoplasmic in which both transporters are expressed show that Slc38a1 is not reticulum stress signalling - from basic mechanisms to clinical applications. FEBS J. regulated by amino acid starvation (Novak et al., 2006; Jones et al., 286, 241–278. 2006) or hypertonic stress (Gaccioli et al., 2006; Maallem et al., 2008). Bain, P.J., LeBlanc-Chaffin, R., Chen, H., Palii, S.S., Leach, K.M., Kilberg, M.S., 2002.The mechanism for transcriptional activation of the human ATA2 transporter gene by However, Slc38a1 may play a part in arsenite-induced ER stress as it is amino acid deprivation is different than that for asparagine synthetase. J. Nutr. 132, reported to be moderately induced by arsenite in human embryonic 3023–3029. kidney cells, although this requires further inquiry (Oh et al., 2012). Baird, F.E., Bett, K.J., MacLean, C., Tee, A.R., Hundal, H.S., Taylor, P.M., 2009. Tertiary active transport of amino acids reconstituted by coexpression of System A and L Unlike Slc38a2, Slc38a1 is not known to be regulated by hormones, but transporters in Xenopus oocytes. Am. J. Physiol. Endocrinol. Metab. 297, E822–E829. as Slc38a2, its expression is downregulated by the proinflammatory Baird, F.E., Pinilla-Tenas, J.J., Ogilvie, W.L., Ganapathy, V., Hundal, H.S., Taylor, P.M., cytokines IL-1β, IL-6 and TNFα in human choriocarcinoma cells and 2006. Evidence for allosteric regulation of pH-sensitive System A (SNAT2) and System N (SNAT5) amino acid transporter activity involving a conserved histidine cultured human trophoblasts (Thongsong et al., 2005; Jones et al., residue. Biochem. J. 397, 369–375. 2009). Slc38a1 is upregulated following ischemia-reperfusion injury in Barker, G.A., Ellory, J.C., 1990. The identification of neutral amino acid transport sys- rat cardiomyocytes where it contributes to cysteine transport and car- tems. Exp. 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