Multifaceted regulation of the system A transporter Slc38a2 suggests nanoscale regulation of amino acid metabolism and cellular signaling

Journal Pre-proof Multifaceted regulation of the system A transporter Slc38a2 suggests nanoscale regulation of amino acid metabolism and cellular signaling Robin Johansen Menchini, Farrukh Abbas Chaudhry PII:

S0028-3908(19)30347-8

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https://doi.org/10.1016/j.neuropharm.2019.107789

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NP 107789

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Neuropharmacology

Received Date: 19 April 2019 Revised Date:

16 September 2019

Accepted Date: 20 September 2019

Please cite this article as: Menchini, R.J., Chaudhry, F.A., Multifaceted regulation of the system A transporter Slc38a2 suggests nanoscale regulation of amino acid metabolism and cellular signaling, Neuropharmacology (2019), doi: https://doi.org/10.1016/j.neuropharm.2019.107789. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 The Author(s). Published by Elsevier Ltd.

Multifaceted Regulation of the System A Transporter Slc38a2 Suggests Nanoscale Regulation of Amino Acid Metabolism and Cellular Signaling

Authors: Robin Johansen Menchini1,* and Farrukh Abbas Chaudhry1,2

1

Department of Molecular Medicine, University of Oslo, Oslo, Norway

2

Department of Plastic and Reconstructive Surgery, Oslo University Hospital, Norway

*Corresponding author: [email protected]

Running title: Regulation of Slc38a2 in Metabolism and Signaling

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Abstract Amino acids are essential for cellular protein synthesis, growth, metabolism, signaling and in stress responses. Cell plasma membranes harbor specialized transporters accumulating amino acids to support a variety of cellular biochemical pathways. Several transporters for neutral amino acids have been characterized. However, Slc38a2 (also known as SA1, SAT2, ATA2, SNAT2) representing the classical transport system A activity stands in a unique position: Being a secondarily active transporter energized by the electrochemical gradient of Na+, it creates steep concentration gradients for amino acids such as glutamine: this may subsequently drive the accumulation of additional neutral amino acids through exchange via transport systems ASC and L. Slc38a2 is ubiquitously expressed, yet in a cellspecific manner. In this review, we show that Slc38a2 is regulated at the 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 survival.

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Table of Contents Introduction

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Physiological roles of amino acids

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Uptake systems for the neutral amino acids

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Identification of the solute carrier 38 (Slc38) family of amino acid transporters Slc38a2 characteristics

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Slc38a2 structure

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Kinetic properties of Slc38a2

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Universal regulatory features of Slc38a2 expression

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Regulation of Slc38a2 by tonicity

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Regulation of Slc38a2 by amino acid availability

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Slc38a2 and the GCN2 pathway

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Slc38a2 and the mTOR pathway

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Slc38a2 as a transceptor

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Regulation of Slc38a2 by ER stress

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Slc38a2 degradation

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Slc38a2 is regulated by a novel K+ channel

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Pharmacologic targeting

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Regulation of Slc38a2 by hormones, cytokines and vitamins

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Differential function and regulation of Slc38a1 and Slc38a2

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Concluding remarks

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Reference list

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Introduction

Physiological roles of amino acids Amino acids play a number of roles in human metabolism. In addition to their appearance as substrates for protein synthesis, amino acids act as sources of energy, carbon, nitrogen, metabolic intermediaries as well as precursors for the synthesis of macromolecules such as hormones, hemoglobin and cytochromes. In later years, it is recognized that amino acids such as glutamine, leucine and arginine double as regulators of cell growth, metabolism and apoptosis (Wu, 2009). Glutamine is the most abundant amino acid in both plasma and cerebrospinal 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 substrate for biosynthetic pathways for neurotransmitters, glutathione, proteins, nucleotides and amino sugars (Chaudhry et al., 2002a). Glutamine is the preferred nutrient for rapidly dividing cells such as immune cells, enterocytes and cancer cells (Cruzat et al., 2018). Glutamine is a non-essential amino acid, but it may become conditionally essential during catabolic states as cellular demands increase.

Uptake systems for the neutral amino acids In critically ill patients admitted to intensive care units, both low and high plasma levels of glutamine are associated with a poor clinical outcome (Oudemansvan Straaten et al., 2001; Rodas et al., 2012). Supplementing critically ill patients with parental glutamine significantly reduced hospital mortality, length of stay and rate of infectious complications and more (Stehle et al., 2017). Thus, maintaining optimal concentrations of the amino acid plasma pool is essential for homeostasis in healthy

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individuals. Several active and concentrative transport systems for neutral amino acids across cell membranes have been demonstrated (Palacin et al., 1998). Glutamine is transported by the system A (alanine preferring) and system N (amide preferring) activities originally described as Na+-dependent glutamine transport activities in Ehrlich cells and hepatocytes, respectively (Oxender and Christensen, 1963; Christensen et al., 1965; Kilberg et al., 1980). Together with systems ASC (alanine, serine and cysteine preferring) and L (leucine preferring), system A accounts for the majority of amino acid uptake in mammalian cells (Palacin et al., 1998). Unlike systems ASC and L, which are obligatory exchangers, system A catalyzes the net uptake of a wider range of neutral amino acids, particularly alanine, serine, proline and glutamine. It is competitively inhibited by the non-metabolizable system A substrate analogue N-methyl-aminoisobutyric acid (MeAIB) (Oxender and Christensen, 1963; Christensen et al., 1965). System N is characterized by transporting glutamine, histidine and asparagine, and tolerates Li+ substitution for Na+ (Kilberg et al., 1980). However, the molecular identity of the proteins responsible escaped discovery for decades (Barker and Ellory, 1990; Palacin et al., 1998).

Identification of the solute carrier 38 (Slc38) family of amino acid transporters The field of system A and system N transporters was not opened until the group of Robert Edwards cloned the vesicular GABA transporter (VGAT, also known as (aka) vesicular inhibitory amino acid transporter (VIAAT)) (McIntire et al., 1997). Later, Chaudhry in collaboration with Edwards successfully identified and characterized the first system N transporter SN1 from its sequence homology to VGAT (Chaudhry et al., 1999). Further molecular characterization revealed SN1 as the first identified member of the solute carrier (Slc) family 38 of amino acid

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transporters (Chaudhry et al., 2002a; Nissen-Meyer and Chaudhry, 2013; Broer, 2014). Together, the Slc32, Slc36 and Slc38 families form a monophyletic group comprising the β-group of Slc (Schioth et al., 2013). The Slc32 family contains the sole VGAT, the Slc36 family comprises the lysosomal proton-coupled amino acid transporters 1-4 (PAT1-4), while the Slc38 family consists of eleven members in the human genome, SLC38A1-A11 where SN1 is Slc38a3 (Schioth et al., 2013). At the turn of the millennium, Slc38a1 (aka GlnT, ATA1, SAT1, SA2 and SNAT1) and Slc38a2 (aka SA1, ATA2, SAT2 and SNAT2) were in short succession independently cloned in rats by three laboratories and demonstrated to underly the system A transport activity (Varoqui et al., 2000; Reimer et al., 2000; Yao et al., 2000; Sugawara et al., 2000a; Chaudhry et al., 2002b). Based on screening for homologs to Slc38a1-Slc38a3, Slc38a4 (aka mNAT3, SAT3 and SNAT4) and Slc38a5 (aka SN2 and SNAT5) were identified as amino acid transporters (Sugawara et al., 2000b; Gu et al., 2001, Nakanishi et al., 2001; Hamdani et al., 2012). The tissue distribution and molecular function of these five transporters comprising the classical system A and system N transport activities are comparably well characterized and reviewed previously (Nissen-Meyer and Chaudhry, 2013; Broer, 2014). In addition, Slc38a7 and Slc38a9 have been functionally characterized, while Slc38a6, Slc38a8, Slc38a10 and Slc38a11 remain elusive ‘orphan’ transporters. However, mutations in the SLC38A8 gene cause foveal hypoplasia in several families with or without concurrent optic nerve pathology (Poulter et al., 2013; Perez et al., 2014; Toral et al., 2017). Slc38a7 is a lysosomal transporter suggested to be the main carrier of glutamine across lysosomal membranes and required for cancer cell growth in periods of low glutamine availability (Verdon et al., 2017). Slc38a9, like Slc38a7, is located on lysosomal membranes. Recently, three independent

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laboratories showed that Slc38a9 is part of the Rag GTPase-Ragulator amino acidsensing machinery that controls the activity of mechanistic target of rapamycin complex 1 (mTORC1) (Rebsamen et al., 2015; Wang et al., 2015; Jung et al., 2015). In part, this is achieved through Slc38a9-mediated release of leucine, as Slc38a9 is needed to transport several essential amino acids generated by proteolysis out of lysosomes (Wyant et al., 2017). Additionally, Slc38a9 transport activity may play a pivotal role in mTORC1 activation under conditions in which cells obtain amino acids by degrading extracellular proteins through macropinocytosis (Wyant et al., 2017). Within the Slc38 family, Slc38a9-a11 are the phylogenetically oldest members with orthologues in C. elegans and D. melanogaster, while Slc38a1-a5 have arisen later in evolutionary terms (Schioth et al., 2013). As more Slc38 family amino acid transporters have been characterized, the dependence on Na+ and the division into systems A and N have become less distinct and additional spectacular mechanisms add to their function and regulation.

Slc38a2 characteristics

Slc38a2 structure The Slc38a2 gene consists of 16 exons and 15 introns in humans and 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 and 16, respectively (Palii et al., 2004). Upon molecular characterization, Slc38a2 was predicted to have (Reimer et al., 2000; Yao et al., 2000), and was recently demonstrated to have eleven transmembrane domains, an intracellular N-terminus and an extracellular C-terminus (Ge et al., 2018). Slc38a2 contains three N-glycosylation sites, Asn254, Asn258 and

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Asn272, which seem to play a part in protein expression (Ge et al., 2018). The Slc38a2 protein also contains a disulfide bridge between cysteine residues 245 and 279 situated in the third extracellular loop between transmembrane domains 5 and 6 (Chen et al., 2016). Cys245 and Cys279 are highly conserved, and had regulatory impact on Slc38a2 transport activity without them being required for transport activity or trafficking to the plasma membrane (Chen et al., 2016). Two other highly conserved cysteine residues, Cys228 and Cys303, were shown to be pivotal for transport function (Chen et al., 2016).

Kinetic properties of Slc38a2 Slc38a2 displays a preference for transport of short-chain neutral amino acids across cell membranes, such as alanine, serine, proline and glutamine (Reimer et al., 2000; Yao et al., 2000; Sugawara et al., 2000a; Gazzola et al., 2001; Hyde et al., 2001; Alfieri et al., 2001). Alpha amino acid transport is competitively inhibited by the partial agonist MeAIB, a system A inhibitor commonly used as a model substrate for system A (Christensen et al., 1965). It is a secondary active transporter where Na+ drives the transport of neutral amino acids in symport with a 1:1 stoichiometry. This makes the transporter electrogenic and able to generate steep substrate concentration gradients at resting potential (Reimer et al., 2000; Chaudhry et al., 2002b). Slc38a2 transport is strongly dependent on pH, and substrate transport is markedly reduced as extracellular pH is lowered within the physiological range (Reimer et al., 2000). Slc38a2 partly allows replacement of Na+ with Li+ (Chaudhry et al., 2002b). We have demonstrated that Slc38a2 has ordered binding with Na+ binding first and the substrate second, and that H+ competes at the Na+ binding site increasing Km for Na+ (Chaudhry et al., 2002b). Slc38a2 catalyzes a leak anion

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current, which is augmented by binding of Na+ to Slc38a2 and inhibited to varying degrees by substrate transport (Zhang and Grewer, 2007). This leak anion conductance is thermodynamically uncoupled from Na+ and substrate transport. A homology model predicted that Slc38a2 contains a conserved Na+ binding site formed by the central parts of transmembrane domains 1 and 8 (Zhang et al., 2009). Mutation studies showed that mutating the conserved asparagine residue 82 in the highly conserved transmembrane domain 1 and threonine residue 384 in transmembrane domain 8 markedly reduced the affinity of Slc38a2 for Na+ and thus substrate transport (Zhang et al., 2008; Zhang et al., 2009). These results implicate Asn82 and Thr384 in the control of the Na+ affinity of Slc38a2, consistent with these residues being part of a Na+ binding site. Mutations of cysteine279 and tyrosine337 also inhibited Na+ binding, but to a lesser extent (Zhang et al., 2008; Zhang et al., 2009). Mutating either Asn82 or Thr384 inhibited the leak anion current (Zhang et al., 2008; Zhang et al., 2009), implying that the Na+ binding site controls both Na+ affinity, coupled Na+/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+ binding and progressive lowering of the Vmax for substrate transport as external pH declines (Chaudhry et al., 2002b; Baird et al., 2006). Mutation studies show that extracellular pH affects Slc38a2 transport activity partially through the conserved histidine 504 residue located on its extreme C-terminus (Baird et al., 2006). This seems to be achieved either directly by binding of H+ at an allosteric site, or indirectly by allosterically transmitting the effects of such H+ binding to the Na+ binding site (Baird et al., 2006). Truncating the C-terminus containing the His504 residue greatly diminishes the pH sensitivity of Slc38a2 but does not abolish it (Zhang et al., 2011),

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pointing to the existence of additional pH sensitive residues within the protein sequence. Truncation studies demonstrate that the C-terminus is pivotal for Slc38a2mediated amino acid transport by contributing to the control of voltage dependence for substrate transport (Zhang et al., 2011). This is achieved without affecting amino acid or Na+ affinity and/or binding (Zhang et al., 2011). The anion leak current induced by Na+ binding and inhibited by substrate binding was mostly unaffected by C-terminus truncation (Zhang et al., 2011). Taken together, these findings suggest that the C-terminus is important for modulating the rate and voltage dependence of amino acid translocation and/or relocation of the empty Slc38a2 carrier to the extracellular surface of the plasma membrane.

Universal regulatory features of Slc38a2 expression It was early recognized that Slc38a2 exhibits two universal regulatory response characteristics of system A activity following cell stress: the osmoregulatory and the amino acid regulatory responses (Alfieri et al., 2001; Gazzola et al., 2001; Ling et al., 2001; Hyde et al., 2001). Following both of these cell stress responses, Slc38a2 expression is dependent on the first common step of the integrated stress response (ISR) pathway, eukaryotic translation initiation factor 2α (eIF2α) (Gaccioli et al., 2006; Krokowski et al., 2015). eIF2α is essential for start codon recognition and mRNA translation initiation (Jackson et al., 2010). Upon its phosphorylation by one or more eIF2α kinases, such as GCN2 and protein kinase R-like endoplasmic reticulum kinase (PERK), phospho-eIF2α blocks 5’cap-dependent protein synthesis while inducing transcription of selected genes that promote cell survival, such as activating transcription factor 4 (ATF4) and growth arrest and DNA damage-inducible protein

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(GADD34) (Pakos-Zebrucka et al., 2016). Recently, it was demonstrated that Slc38a2 is also regulated by intrinsic cell stresses, such as endoplasmic reticulum (ER) stress (Gjymishka et al., 2008). Thus, Slc38a2 may be a common target for induction or repression by the ISR.

Regulation of Slc38a2 by tonicity Mammalian cells adapt to hyperosmotic stress and osmotic shrinkage by regulatory volume increase (RVI), in which cellular volume is restored by the rapid uptake of inorganic ions (Wehner et al., 2003). Although the RVI restores cell volume within minutes, the intracellular ionic strength becomes unnaturally high, perturbing the function of intracellular molecules. This is countered by accumulating compatible osmolytes such as betaine, taurine, and myo-inositol, reducing the intracellular ionic strength while maintaining cellular volume (Burg et al., 2007). This osmoadaptation is to a large degree orchestrated by the nuclear factor of activated T cells 5 (NFAT5), aka tonicity-responsive enhancer binding protein (TonEBP), which stimulates transcription of contributing genes such as the betaine GABA transporter (BGT1), taurine transporter (TauT) and myo-inositol transporter (SMIT1) (Burg et al., 2007). However, accumulation of these compatible osmolytes is slow. Slc38a2 expression is regulated by tonicity through induction by NFAT5 (Trama et al., 2002), and contributes to the fast osmoadaptive response in all cell lines tested across mammalian species (Alfieri et al., 2001; Nahm et al., 2002; Alfieri et al., 2002; Takanaga et al., 2002; Trama et al., 2002; Lopez-Fontanals et al., 2003; Franchi-Gazzola et al., 2006; Franchi-Gazzola et al., 2004; Bevilacqua et al., 2005; Ito et al., 2008; Nishimura et al., 2010). This is consistent with observations of a system A dependent fast regulatory increase in neutral amino acids immediately after

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cellular exposure to hypertonicity (Bussolati et al., 2001). Upon hyperosmolar exposure, an increase in Slc38a2 mRNA expression (Alfieri et al., 2001) is followed by a subsequent increase in Slc38a2 carrier expression on the plasma membrane (Franchi-Gazzola et al., 2004). This leads to increased system A transport activity and restoration of cell volume through an expansion of the intracellular amino acid pool (Bussolati et al., 2001). System A is the only major transport activity for neutral amino acids in mesenchymal cells that uses the transmembrane sodium gradient to concentrate its substrates intracellularly (Bussolati et al., 2001). Thus, Slc38a2 plays a pivotal role in the restoration of intracellular amino acid stores as most other transport systems are exchangers whose intracellular substrate concentration is coupled to substrates whose steep concentration gradient is fostered by the electrogenic Slc38a2 (Franchi-Gazzola et al., 2006; Evans et al., 2007). In fact, the influx of amino acids following increased system A transport activity is sufficient for volume recovery throughout the first hours of osmotic stress in human vascular endothelial cells (Dall'Asta et al., 1999). SLC38A2 suppression by RNA interference in cultured human hepatocytes severely delayed cell volume recovery following osmotic stress by blunting the compensatory increase in the intracellular amino acid pool, particularly of glutamine (Bevilacqua et al., 2005). As the most abundant amino acid in both plasma and cerebrospinal fluid, glutamine plays an important role in amino acid exchange between cells via different transport systems, as it is a good substrate for the widely expressed systems A, ASC and L (Bussolati et al., 2001). Glutamine intracellularly concentrated by Slc38a2 may secure the intracellular accumulation of neutral amino acids, which are poor system A substrates, through exchange via systems ASC and L. This fast osmoadaptive response to hypertonicity overlaps with the comparably slower accumulation of compatible osmolytes such as

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myo-inositol, taurine and betaine by SMIT1, TauT and BGT1, respectively (FranchiGazzola et al., 2006). Accumulation of compatible osmolytes represents a more permanent means for cellular osmoadaptation, but may also entail the risk of acquiring central pontine myelinolysis if hypo- or hypernatremia are corrected too fast (Ito et al., 2008). The cellular pathways involved in the regulation of Slc38a2 in response to hypertonicity are less well understood. In rat skeletal muscle cells, the osmoregulatory response was shown to be inhibited by cycloheximide and actinomycin D, but not chloroquine (Kashiwagi et al., 2009). This demonstrates that the osmoadaptive response of Slc38a2 consists of transcriptional upregulation without immediate translocation of preformed Slc38a2 proteins to the plasma membrane. This interpretation is supported by SLC38A2 silencing experiments in cultured human hepatocytes indicating that the increase in system A transport activity in response to osmotic stress is accounted for by the synthesis of new SLC38A2 carriers (Bevilacqua et al., 2005). In mice thymocytes, Slc38a2 induction in response to hyperosmotic stress is NFAT5 dependent, although an osmotic response element in the Slc38a2 sequence has not yet been described (Trama et al., 2002). In L6 myotubes and Chinese hamster ovary (CHO) cells, the mitogen-activated protein (MAP) kinases c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) did not contribute to the osmoregulatory response (Lopez-Fontanals et al., 2003; Kashiwagi et al., 2009). In CHO cells, inhibition of p38 partially inhibited the osmoregulatory response in a mitogen-activated protein kinase kinase 3 (MKK3)independent manner (Lopez-Fontanals et al., 2003), while in L6 myotubes, inhibition of p38 had no effect on the induction of Slc38a2 (Kashiwagi et al., 2009).

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Slc38a2 induction by osmotic shock was reported to be eIF2α-independent in mouse embryonic fibroblasts (Gaccioli et al., 2006). However, Krakowski and coworkers found the osmoadaptive response of Slc38a2 to be inhibited in both mouse embryonic fibroblasts and human cervical cancer cells in mild hyperosmotic stress in response to high levels of phosphorylated eIF2α, a mediator of proapoptotic signaling (Krokowski et al., 2015). The osmoregulatory adaptation by Slc38a2 was rescued by protein phosphatase 1 (PP1) regulatory subunit GADD34 through the dephosphorylation of eIF2α (Krokowski et al., 2015). Hyperosmotic stress causes fragmentation of the Golgi apparatus and microtubule network, trapping immature SLC38A2 carriers in the cis-Golgi compartment and attenuating the osmoadaptive response of Slc38a2 (Krokowski et al., 2017). GADD34/PP1 counteracts this by maintaining Golgi and microtubule integrity during stress, promoting SLC38A2 maturation and trafficking to the plasma membrane in human corneal epithelial cells and mouse embryonic fibroblasts (Krokowski et al., 2017). Osmotic stress contributes to the pathogenesis of many human diseases by triggering cell damaging processes such as cell shrinkage, oxidative stress and DNA damage (Brocker et al., 2012). Studies have implicated Slc38a2-dependent osmoadaptation in heart failure, central pontine myelinolysis and dry eye syndrome. Slc38a2 mRNA expression was induced in cardiomyocytes and skeletal muscle cells in a TauT knockout model, which developed dilated cardiomyopathy (Ito et al., 2008). This suggests that Slc38a2 may play an osmoprotective role in congestive heart failure. Slc38a2 was induced in rat oligodendrocyte cell bodies, but not processes, throughout the brain in a model for prolonged systemic hypertonicity (Maallem et al., 2008). Interestingly, Slc38a2 was not found to be expressed in oligodendrocytes marked by the same antibody under basal conditions (Gonzalez-Gonzalez et al.,

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2005; Jenstad et al., 2009). Additionally, Slc38a2 immunolabeling in diverse brain regions under basal conditions was not recovered after 24 hours of systemic hypertonicity (Maallem et al., 2008). Thus, Slc38a2 may play a part in the brain’s osmoadaptive response to dystonicity to prevent central pontine myelinolysis, at least in a subset of oligodendrocytes. SLC38A2 and GADD34/PP1 coexpression contributes to the osmoadaptive response in human corneal epithelial cells as described above, protecting against the hyperosmotic tear film that causes dry eye syndrome (Krokowski et al., 2017). Induction of system A transport activity has been proposed to be involved in the volume increase needed to enter a new cell division cycle, either by supplying amino acids functioning as osmolytes directly or through further substrate exchange via other amino acid transporters (Bussolati et al., 2001; Franchi-Gazzola et al., 2006). In CHO cells, inhibition of cyclin-dependent kinase (CDK) 4-6-cyclin D complexes and CDK4 lead to a slight decrease of basal system A activity, but not of the osmotic response (Lopez-Fontanals et al., 2003). However, inhibition of CDK2 repressed the Slc38a2-mediated osmotic response in a dose-dependent manner (Lopez-Fontanals et al., 2003). CDK2-cyclin E and CDK2-cyclin A regulates entry into and progression through the S phase, contributing to the iso-osmotic volume increase that takes place before mitosis. Whether Slc38a2 may contribute to cellular volume increase before mitosis in a CDK-dependent manner warrants further inquiry.

Regulation of Slc38a2 by amino acid availability Another hallmark of Slc38a2 is its regulation in response to amino acid availability. This is achieved by the ability of Slc38a2 to adapt its activity to the availability of extracellular substrates, a response named adaptive regulation 16

(Gazzola et al., 1972). This adaptive response consists of both a rapid translocation of preformed Slc38a2 proteins from perinuclear stores to the plasma membrane, and long-term transcriptional upregulation (Ling et al., 2001; Palii et al., 2006; Kashiwagi et al., 2009). Reintroduction of extracellular system A substrates abolishes the transient increase in Slc38a2 expression that occurs in amino acid starved cells in all cell lines tested across mammalian species (Gazzola et al., 2001; Ling et al., 2001; Hyde et al., 2001; Bain et al., 2002; Lopez-Fontanals et al., 2003; Tanaka et al., 2005; Novak et al., 2006; Lopez et al., 2006; Wu et al., 2007; Nickel et al., 2010).

Slc38a2 and the GCN2 pathway Amino acid insufficiency is sensed by the eIF2α kinase GCN2. Lack of one or several amino acids leads to intracellular accumulation of uncharged tRNAs (Kilberg et al., 2009). These activate GCN2, leading to phosphorylation of eIF2α, the common integrator in the ISR (Pakos-Zebrucka et al., 2016). Phosphorylation of eIF2α facilitates adaptation to amino acid starvation by greatly reducing global protein synthesis and thus cellular amino acid demand. Simultaneously, phospho-eIF2α induces the translation of selected cellular stress response proteins, such as the transcription factors ATF4 and CCAAT-enhancer-binding protein β (C/EBPβ). In turn, they induce expression of selected genes through cognate response elements, promoting recovery of amino acid homeostasis (Figure 1; Kilberg et al., 2009). The Slc38a2 gene contains such a highly conserved amino acid response element (AARE), which controls its substrate-dependent transcriptional regulation and is located in intron 1 (Palii et al., 2004; Hyde et al., 2007). Juxtaposed to this AARE, intron 1 also contains a CAAT-box (response element for C/EBPβ) that enhances the action of the AARE, and a purine-rich (PuR) sequence that serves as a

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repressor element to maintain a low basal transcription rate of Slc38a2 in the absence of amino acid deficiency (Palii et al., 2004). The AARE is required for the induction of Slc38a2 by amino acid starvation, whereas mutating the CAAT-box can reduce induced transcription by approximately 40% (Palii et al., 2006). Several of the proteins in the complexes that bind the Slc38a2 AARE are shared with the juxtaposed CAAT-box (Palii et al., 2006). Upon starvation for one or more amino acids, the CAAT-AARE sequences in the Slc38a2 gene is targeted by the GCN2/ATF4 pathway (Palii et al., 2006; Gaccioli et al., 2006). ATF4 binds to the CAAT-AARE complex as a heterodimer prior to RNA polymerase II binding, promoting transiently enhanced Slc38a2 transcription by recruitment of additional transcription factors such as C/EBPα, C/EBPβ-LAP and c-Jun (Palii et al., 2006). In addition to recruitment of members of the general transcription machinery, increased H3 acetylation was evident at both the Slc38a2 promoter and AARE following amino acid limitation (Gjymishka et al., 2008; Thiaville et al., 2008). Slc38a2 induction in response to amino acid starvation is subject to regulation by a self-limiting program and subsequent binding of repressor proteins such as ATF3, C/EBPβ-LIP and C/EBPδ slowly represses the AARE-mediated transcription (Palii et al., 2006). C/EBPγ had no effect on Slc38a2 transcription (Palii et al., 2006). In addition to the AARE, Slc38a2 contains an internal ribosome entry site (IRES) at its 5’-UTR (Gaccioli et al., 2006). This IRES is constitutively active in both amino acid fed and starved cells. The IRES allows for cap-independent Slc38a2 translation during amino acid starvation where global translation initiation might be restricted (Gaccioli et al., 2006). However, it neither stimulates translation nor is it regulated by adaptive regulation. The constitutively active nature of its IRES testifies to the pivotal role of Slc38a2 in homeostasis and stress.

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In contrast to the transcriptional activation of Slc38a2, less is known about how preformed carriers are recruited from intracellular stores in response to amino acid deficiency. In rat skeletal muscle cells, inhibition by chloroquine, cycloheximide and actinomycin D abolished the adaptive response of Slc38a2 (Kashiwagi et al., 2009). This suggested that both transporter cycling from an intracellular store and de novo synthesis are involved in the response of Slc38a2 to amino acid starvation. Further inhibition studies suggested that phosphoinositide 3-kinase (PI3K) is involved in transporter recruitment from an intracellular pool, while the MAP kinases ERK and JNK and the upstream kinase MKK4 are involved in pathways for de novo synthesis in both L6 myotubes and CHO cells (Lopez-Fontanals et al., 2003; Hyde et al., 2007; Kashiwagi et al., 2009). Inhibition of the p38 MAPK pathway, MKK3 or mTOR pathway had no effect on adaptive regulation in rat skeletal muscle cells (LopezFontanals et al., 2003; Hyde et al., 2007; Kashiwagi et al., 2009). Slc38a2 adaptive regulation is independent of CDK2, CDK4 (Lopez-Fontanals et al., 2003) and NFAT5 (Trama et al., 2002). However, it was recently demonstrated that CDK7 is induced in a GCN2-dependent manner following amino acid starvation, and that inhibition of CDK7 blunts the adaptive response of Slc38a2 (Stretton et al., 2019). Slc38a2 is known to be stabilized during amino acid limitation (Hyde et al., 2007). This stabilization is lost upon mutating the N-terminal lysyl residues to alanine (Hoffmann et al., 2018). Intriguingly, the adaptive upregulation of Slc38a2 was abolished in the absence of Na+ (Hoffmann et al., 2018). Hoffmann and coworkers proposed a model in which Slc38a2 becomes downregulated in states of amino acid availability through ubiquitination of lysyl residues in its N-terminal tail, which become accessible to an unknown E3 ubiquitin ligase upon conformational changes following substrate release (Hoffmann et al., 2018). The binding of Na+ may stabilize Slc38a2 through

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conformational changes making these lysyl residues less accessible, while the absence of both Na+ and substrate may lead to an N-terminal conformation that maximally exposes the lysyl residues, leading to protein instability and degradation.

Slc38a2 and the mTOR pathway

There has been special interest in the possibility that Slc38a2 may act in concert with the system L transporter LAT1 (Slc7a5) to intracellularly concentrate branched-chain amino acids, particularly leucine. In fact, Baird and coworkers have shown that coexpression of Slc38a2 and LAT1 in Xenopus laevis oocytes lead to a considerable increase in intracellular accumulation of leucine as compared with expression of LAT1 alone (Baird et al., 2009). Leucine is a potent stimulator of the mTOR pathway, and it is proposed that Slc38a2 may indirectly influence amino acid sensing through LAT1 and mTOR (Dodd and Tee, 2012). This endows the mTOR pathway with additional regulatory possibilities as Slc38a2 is extensively regulated. Additional evidence for cooperation between Slc38a2 and LAT1 for mTOR activation is the observation that Slc38a2 knockdown in rat myocytes leads to a drop in intracellular concentrations of both glutamine and leucine, followed by repression of mTORC1 signaling (Evans et al., 2008). This clearly implicates a dependency on Slc38a2 for furnishing the free intracellular amino acid pool with both amino acids (Evans et al., 2008). Additionally, glutamine has been demonstrated to be a rate limiting nutrient for mTOR activation (Nicklin et al., 2009). The exact mechanisms in which Slc38a2 and mTOR interact remain elusive. In cultured primary trophoblasts isolated from normal human placentas, knockdown of either mTORC1 or mTORC2 greatly attenuated system A and L transport activity,

20

while knockdown of both mTORC1 and mTORC2 completely inhibited system A and L transport (Rosario et al., 2013). This was achieved through the specific downregulation of system A and L isoforms SLC38A2 and LAT1 in microvillus plasma membrane fractions (Rosario et al., 2013). Upon silencing of both mTORC1 and mTORC2, growth factor-mediated stimulation of system A and L transport activity was abrogated (Rosario et al., 2013). Silencing mTORC1 leads to relocation of SLC38A2 from the plasma membrane to the cytosol in syncytial islands (Rosario et al., 2013). This suggests that mTORC1 and mTORC2 are involved in the posttranslational regulation of Slc38a2 by regulating Slc38a2 trafficking, possibly through Nedd4-2 ubiquitination (Chen et al., 2015). This was later confirmed by Rosario and coworkers in primary human trophoblast cultures (Rosario et al., 2016). Additional evidence of interaction stems from experiments with mouse livers, where increased Slc38a2 expression, achieved by an adenoviral delivery system, increased hepatic amino acid concentrations and mTORC1/S6K activity (Uno et al., 2015).

Slc38a2 as a tranceptor

Both the GCN2 and mTOR pathways are essential for amino acid sensing in mammalian cells (Broer and Broer, 2017). However, the amino acid sensing mechanisms upstream of GCN2 and mTOR are not fully elucidated. In cultured myocytes, induction of Slc38a2 by amino acid starvation is inhibited upon introduction of a single amino acid while the medium remained starved for other amino acids (Hyde et al., 2007). Moreover, the potency by which substrates repress Slc38a2 expression is correlated to their transport Km (Hyde et al., 2007). In MCF-7 human breast cancer cells, both acute and sustained incubation with MeAIB alone leads to

21

increased mTOR-mediated p70 S6K1 phosphorylation (Pinilla et al., 2011). As MeAIB is not metabolized, this implies that Slc38a2 acts as an amino acid sensor upstream of mTOR (Hundal and Taylor, 2009). This observation lead Hyde and coworkers to propose that Slc38a2 acts as a tranceptor, i.e., a hybrid transporterreceptor (Hyde et al., 2007). According to this model, the Slc38a2 transportersubstrate complex may sense changes in extracellular amino acid availability and transmit this signal intracellularly, in addition to the ability of Slc38a2 to modulate the intracellular amino acid pool directly as a key transporter of glutamine (Hundal and Taylor, 2009). The mechanisms responsible for how substrate site occupancy is sensed or how this signal is transduced downstream to allow Slc38a2 to autoregulate its own expression is currently unknown. One possibility is that the inward current coupled to substrate transport may lead to membrane depolarization. This is suggested to increase intracellular Ca2+ and activate L-type voltage-sensitive calcium channels in response to increased amino acid concentration in the gut lumen in intestinal endocrine cells (Young et al., 2010). The same mechanism was seen in enteroendocrine GLUTag cells, where the transport-current associated with glutamine furnished by Slc38a2 has been proposed to contribute to membrane depolarization triggering GLP-1 release (Reimann et al., 2014). Hundal and Taylor proposed that the trans-inhibition of Slc38a2 seen upon excess accumulation of Slc38a2 substrates in the cytoplasm, which prevents Slc38a2 from completing its transport cycle and return to its outward facing position, may play a part (Hundal and Taylor, 2009). Recently, members of the Slc36 family and the Slc38a2 homologue Slc38a9 have been proposed to act as amino acid tranceptors upstream of mTOR, further suggesting a tranceptor role for Slc38a2 (Fan and Goberdhan, 2018).

22

Regulation of Slc38a2 by ER stress

The ER is involved in manifold cellular functions such as protein synthesis and processing, lipid synthesis and calcium regulation (Almanza et al., 2019). Three ER stress signaling pathways collectively known as the unfolded protein response (UPR) are triggered when misfolded proteins accumulate in the ER lumen. The UPR aims to restore ER function through either the inositol-requiring enzyme 1 (IRE1), ATF6 or PERK pathways (Almanza et al., 2019). Similar to GCN2, PERK is an eIF2α kinase and part of the ISR (Pakos-Zebrucka et al., 2016). As for the osmoregulatory and amino acid response pathways, phosphorylation of eIF2α by PERK may suppress global protein synthesis aside from the induction of selected proteins that facilitate the restoration of ER homeostasis (Pakos-Zebrucka et al., 2016). Following both amino acid limitation and ER stress in human hepatoma cells, ATF4 was synthesized and recruited to the Slc38a2 AARE with subsequent synthesis and recruitment of ATF3 and C/EBPβ (Gjymishka et al., 2008). Unlike for the AAR pathway, UPR stimulation did not increase Slc38a2 transcription (Gjymishka et al., 2008). In fact, responsiveness of the Slc38a2 gene to UPR pathway activation was cell type specific. In response to UPR activators, human kidney and breast cancer cells exhibited increased Slc38a2 expression, whereas mouse and human liver cells and fibroblasts did not (Gjymishka et al., 2008). Neither increased H3 acetylation nor recruitment of the general transcription machinery was seen at the Slc38a2 promoter or AARE following UPR activation (Gjymishka et al., 2008). Mutation studies show that destruction of the Slc38a2 AARE at intron 1 abolishes both the transcriptional activation following AAR- and UPR-dependent transcription (Gjymishka et al., 2008).

23

Amino acid limitation induced Slc38a2 transcription while activation of the UPR did not. However, simultaneous activation of both pathways lead to almost complete abolishment of Slc38a2 induction through the AAR (Gjymishka et al., 2008). Thus, it has been suggested that the lack of Slc38a2 induction by the UPR is caused by a repressive signal that overrides ATF4 binding and prevents transcription (Gjymishka et al., 2008). It is unknown how the UPR-mediated repressive signal blocks Slc38a2 transcription, but it is not mediated by the UPR effectors ATF6 or XBP1 (Gjymishka et al., 2008). In another study, Slc38a2 was found to be necessary for arsenite-induced ER stress in human embryonic kidney cells and mouse adipocytes (Oh et al., 2012). Slc38a2 expression and transport activity was induced by arsenite. This induction depended on ATF4 and both oxidative and proteotoxic stress signals (Oh et al., 2012). Slc38a2 activity seems to be specific for arsenite-mediated ER stress as it cannot be induced by the ER stressors tunicamycin or thapsigargin (Oh et al., 2012). Arsenite can activate mTOR, and mTOR activity is augmented by raised levels of Slc38a2 carriers and transport activity during arsenite stress (Oh et al., 2012). Oh and coworkers suggested that arsenite-induced activation of Slc38a2 by the PERK/ATF4 pathway increases system A and L transport activity. This may supply the amino acids necessary for furnishing adaptive cell responses such as increased synthesis of chaperones and antioxidants. Import of leucine through the concerted actions of Slc38a2 and LAT1 activates mTOR. This increase in protein synthesis may ultimately overactivate the ER with subsequent development of ER stress and activation of the UPR (Oh et al., 2012). Together with LAT1, LAT3 (Slc43a1) and ATP-binding cassette sub-family C member 4 (ABCC4), Slc38a2 was also found to

24

be upregulated following mercury exposure in mouse myoblasts in a PERK/ATF4dependent manner (Usuki et al., 2017).

Slc38a2 degradation

Slc38a2 is extensively regulated and increases its expression and trafficking at the cell membrane in response to numerous stimuli. Thus, mechanisms are needed for rapid Slc38a2 turnover to allow for fast downregulation in response to changes in the cellular microenvironment. There is increasing evidence of such regulatory control of Slc38a2 by the ubiquitin-proteasome system (UPS). In 3T3-L1 adipocytes, CHO cells and Xenopus laevis oocytes, Slc38a2 plasma membrane activity and expression are regulated by the E3 ubiquitin ligase Nedd4-2 (Hatanaka et al., 2006a). Nedd4 and cCbl did not significantly affect Slc38a2 transport activity. Additionally, Slc38a2 stability seems to be subject to multi-monoubiquitination by a ubiquitin ligase other than Nedd4-2 (Hatanaka et al., 2006a; Hoffmann et al., 2018). In cultured primary trophoblasts isolated from normal human placentas, Rosario and coworkers show that mTORC1 regulates system A and L transport activity by modulating SLC38A2 and LAT1 carrier expression on the plasma membrane through ubiquitination by Nedd4-2 (Rosario et al., 2016). The increased Slc38a2 carrier expression and transport activity following adaptive upregulation and osmotic stress were strongly attenuated following preincubation with polyunsaturated fatty acids in rat L6 myotubes and human cervical cancer cells (Nardi et al., 2015). This was not caused by Slc38a2 mRNA suppression, but rather by a decreased plasma membrane carrier expression as Slc38a2 transport proteins were targeted for degeneration by the UPS (Nardi et al.,

25

2015). This effect was not selective for Slc38a2, but part of a general cellular response with increased total content of ubiquitinated proteins (Nardi et al., 2015). Nardi and coworkers propose that increased fatty acids generated by increased lipolysis during fasting targets intracellular proteins for proteolysis to free amino acids for hepatic gluconeogenesis (Nardi et al., 2015). Fatty acid-induced downregulation of Slc38a2 also decreases reuptake of free amino acids from the circulation into myocytes following proteolysis. Mutagenesis studies show that lysine residues on the intracellular N-terminal Slc38a2 are targeted for ubiquitin-proteasome-mediated degradation through a currently unknown E3-ligase (Nardi et al., 2015; Hoffmann et al., 2018). SLC38A2 and ASCT2 (Slc1a5) are degraded through ubiquitination by the E3 ubiquitin ligase RFN5 in a human breast cancer cell line following paclitaxel-induced ER stress (Jeon et al., 2015). Many cancer cells depend on glutamine to sustain glutaminolysis for synthesis of proteins, nucleic acids and lipids (DeBerardinis and Cheng, 2010). ASCT2 was recently shown to be responsible for most of the glutamine transport in human cervical cancer and osteosarcoma cells, while SLC38A2 expression in these cells was induced by the GCN2/ATF4 pathway following amino acid imbalance (Broer et al., 2016). Degradation of SLC38A2 and ASCT2 by RFN5 following paclitaxel-induced ER stress ultimately decreases mTOR signaling and cellular proliferation through reduced cellular glutamine uptake, thus setting the stage for apoptosis and cell death (Jeon et al., 2015).

Slc38a2 is regulated by a novel K+ channel

26

Two healthy born brothers developed ataxia, myoclonic jerks, lost the ability to walk and talk, and developed drug-resistant progressive myoclonus epilepsy (PME) during their first two years of life (Moen et al., 2016). Interestingly, cerebrospinal fluid (CSF) analyses revealed increased glutamine and reduced glutamate concentrations. As Slc38 family transporters have been associated with replenishment of the fast neurotransmitters glutamate and GABA (Chaudhry et al., 2002b; Chaudhry et al., 2008; Jenstad et al., 2009; Nissen-Meyer and Chaudhry, 2013; Qureshi et al., 2019), we hypothesized that the observed glutamine and glutamate levels and PME in the two children could be due to dysfunctional Slc38a2 transport activity. To our surprise, whole exome sequencing did not reveal pathologic variants in Slc38a2 or any other Slc38 family transporter, but a novel frameshift mutation in a protein known as potassium channel tetramerization domain 7 (KCTD7). Although an orphan protein, several studies indicated involvement of KCTD7 and its homologues in metabolism, cellular proliferation and differentiation and had shown association with different forms of epilepsy (Liu et al., 2013; Van et al., 2007; Kousi et al., 2012; Farhan et al., 2014). In a series of electrophysiological experiments in Xenopus laevis oocytes, we demonstrated that KCTD7 is a novel K+ channel hyperpolarizing and stabilizing the membrane potential (Moen et al., 2016). Notably, it also regulates glutamine transport by Slc38a2 (Figure 2), while it has no impact on Slc38a5 activity. Five pathogenic variants of KCTD7 depolarize the cellular membrane potential and hamper glutamine transport. As Slc38a2 is enriched in glutamatergic neurons and Slc38a2-mediated

glutamine

transport

is

required

for

synthesis

of

the

neurotransmitter glutamate (Jenstad et al., 2009), our data are thus consistent with the increased glutamine and reduced glutamate levels observed in the CSF.

27

Interaction between channels and transporters have recently been identified to be widespread. The sodium/myo-inositol cotransporter 1 (SMIT1) forms a complex with potassium voltage-gated channel subfamily Q member 1 (KCNQ1) and potassium voltage-gated channel subfamily E member 2 (KCNE2) and regulates CSF myo-inositol levels and neuronal excitability (Roepke et al., 2011; Abbott et al., 2014). The Ca2+-activated K+ channel (MaxiK) forms a complex with the GABA transporter 3 (GAT3; Slc6a11) (Singh et al., 2016), while another K+ channel (KCNA2/Kv1.2) interacts with LAT1 (Baronas et al., 2018). In both cases, the channels and transporters regulate reciprocal activity with impact on neurotransmission and neuronal excitability. Thus, KCTD7 may be the first identified channel interacting with and regulating the function of Slc38a2. Other channels and/or proteins may regulate Slc38a2 in different ways.

Pharmacologic targeting

Apart from the canonical competitive inhibitor of system A transport, MeAIB, (Christensen et al., 1965), no pharmacological agent specifically targeting Slc38a2 has been identified thus far. MeAIB is a substrate analogue ousting other Slc38a2 substrates for transport by Slc38a2. In Xenopus laevis oocytes, Bröer and coworkers showed that Slc38a2 is inhibited by the glutamine analogue y-glutamyl-p-nitroanilide (GPNA) (Broer et al., 2016). However, GPNA also inhibits ASCT2, Slc38a1, Slc38a4, Slc38a5 and LAT1 (Broer et al., 2016; Chiu et al., 2017). The ASCT2 inhibitor benzylserine was also shown to inhibit Slc38a2 as well as Slc38a1 and LAT1 (Broer et al., 2016; van et al., 2018). Recently, 2-amino-4-bis(aryloxybenzyl)aminobutanoic acid (AABA) was

28

shown to block Slc38a2 and LAT1 (Broer et al., 2018). Due to their lack of specificity, GPNA, AABA and benzylserine do not appear to be useful inhibitors of Slc38a2 transport activity. In rat skeletal muscle cells and adipocytes, Slc38a2 carriers are translocated from their intracellular storage site in the trans-Golgi network to the plasma membrane in response to insulin signaling (Hyde et al., 2005; Hatanaka et al., 2006b). The saturated fatty acid ceramide reduced system A transport activity by internalizing Slc38a2 and impairing insulin-stimulated translocation of Slc38a2 to the plasma membrane (Hyde et al., 2005). Concomitantly, ceramide reduced protein synthesis by reducing the intracellular amino acid pool and mTOR pathway activity (Hyde

et

al.,

2005).

Hyde

and

coworkers

speculated

that

the

use

of

sphingomyelinase/ceramide synthase inhibitors that reduce muscle ceramide levels may be beneficial in catabolic muscle-wasting conditions such as uremic metabolic acidosis. In fact, inhibition of Slc38a2 by acidosis with subsequent reduction of the intracellular glutamine pool may play a pivotal role in the development of sarcopenia and cachexia in patients with diabetic nephropathy, end-stage renal disease and uremic metabolic acidosis (Evans et al., 2008). Both diabetes mellitus and metabolic syndrome are diseases characterized by insulin resistance, which may influence Slc38a2 expression. Hatanaka and coworkers found that Slc38a2 mRNA expression is significantly downregulated in human and rodent adipose tissue in type 2 diabetes 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-diabetic drugs. Recently, Medras and coworkers showed that cotreatment with oral glutamine supplementation and the GLP-1

29

analogue liraglutide leads to increased glycemic control through increased 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 glutamate to stimulate insulin secretion (Gammelsaeter et al., 2011; Jenstad and Chaudhry, 2013).

Regulation of Slc38a2 by hormones, cytokines and vitamins In addition to the previously mentioned regulatory mechanisms, Slc38a2 expression is regulated in different organ systems by diverse signal transduction pathways in response to changing physiological demands. Slc38a2 is subject to regulation by a number of hormones. In rat hepatocytes, Slc38a2 expression is induced by glucagon through the phosphorylation of transcription factor cAMP response element-binding protein (CREB) in a cAMP/PKAdependent manner (Ortiz et al., 2011). CREB subsequently binds a cAMP response element (CRE) site located in the 5’-UTR promoter region of Slc38a2 (Ortiz et al., 2011). In rat dam mammary glands, Slc38a2 expression is induced by 17β-estradiol via the estrogen receptor (ER)-α, which in turn binds an estrogen response element (ERE) in the Slc38a2 promoter (Velazquez-Villegas et al., 2014). Slc38a2 mRNA and protein expression is also induced by prolactin in rat mammary gland explants and human breast cancer cells (Velazquez-Villegas et al., 2015). Recently, Morotti and coworkers demonstrated that Slc38a2 expression is induced by hypoxia-inducible factor-1α (HIF-1α) in MCF-7 ERα+ human breast cancer cells, and that the downregulation of Slc38a2 by antiendocrine treatment with the selective estrogen

30

receptor degrader fulvestrant was abolished under hypoxic conditions (Morotti et al., 2019). The binding sites for HIF-1α and ER-α overlaps in a cis-regulatory element of Slc38a2, and there seems to be a switch between them for the regulation of Slc38a2 in tumor hypoxia. In Slc38a2 knockdown experiments, tamoxifen-resistant human breast cancer cells showed reduced growth following decreased glutamine consumption, mitochondric respiration and mTOR signaling (Morotti et al., 2019). As high tumor expression rates of Slc38a2 correlated with worse patient outcomes in analyzed clinical data, Slc38a2 may furnish cancer cells with glutamine under stress conditions such as solid tumor hypoxia and be a possible target for novel antineoplastic agents (Morotti et al., 2019; Broer et al., 2019). Placental Slc38a2 is subject to diverse hormonal regulation as part of the maternal endocrine adaptation to pregnancy. In a human choriocarcinoma cell line, cortisol stimulates system A amino acid transport by translocating SLC38A2 to the plasma membrane from intracellular stores through unknown mechanisms (Jones et al., 2009). At higher levels, cortisol induces Slc38a2 transcription. Daily testosterone injections restricted fetal growth in pregnant rat dams by decreasing Slc38a2 mRNA expression and carrier levels (Sathishkumar et al., 2011). Maternal growth hormone treatment in pregnant pigs led to a significant increase in Slc38a2 carrier levels in trophoblasts (Tung et al., 2012). Maternal infusion of exogenous insulin-like growth factor 1 (IGF1) in an early pregnancy guinea pig model increased placental Slc38a2 expression at midgestation (Sferruzzi-Perri et al., 2007). Globular adiponectin increased SLC38A2 levels in cultured human trophoblasts (Jones et al., 2010). As reviewed above, insulin increased Slc38a2 plasma membrane levels by translocation from its trans-Golgi network stores (Hatanaka et al., 2006b). In human cultured

31

trophoblasts, full-length adiponectin abolishes this insulin-dependent increase in SLC38A2 levels (Jones et al., 2010). In

the

placenta,

Slc38a2

and

Slc38a1

are

regulated

by

the

key

proinflammatory cytokines interleukin-1β (IL-1β), IL-6 and tumor necrosis factor α (TNFα). IL-6 and TNFα stimulated system A activity in cultured human trophoblasts by increasing mRNA expression and protein levels of Slc38a2 and Slc38a1 (Jones et al., 2009), while IL-1β reduced basal expression of Slc38a2 and Slc38a1 mRNA as well as system A transport activity (Thongsong et al., 2005). These findings may offer a link between proinflammatory cytokines and pregnancies complicated by obesity and/or diabetes mellitus, which exhibit elevated levels of cytokines, insulin resistance, increased placental amino acid uptake and fetal overgrowth. Slc38a2 and Slc38a1 expression and system A transport activity were reduced in syncytiotrophoblasts in a cohort of pregnant Malawian women with placental malaria and intervillousitis (Boeuf et al., 2013). Thus, Plasmodium falciparum may lead to fetal growth restriction in placental malaria by downregulating Slc38a2 and Slc38a1 through proinflammatory cytokines. Slc38a2 is also regulated by the cytokine transforming growth factor-β1 (TGF-β1). In rat aortic vascular smooth muscle cells, TGF-β1 induces Slc38a2 gene expression and increased Slc38a2-mediated proline transport, possibly contributing to wound healing and intimal thickening at sites of vascular injury (Ensenat et al., 2001). Interestingly, it has been discovered that dysfunctional TGF-β1 signaling underlies several hereditary vascular syndromes such as the aortopaties Marfan syndrome and Loeys-Dietz syndrome (Takeda et al., 2016). Thus, the interplay between TGF-β1 and Slc38a2 warrants further investigation. Slc38a2 is also regulated by vitamins. 1,25-dihydroxy vitamin D3 increases placental system A transport activity by increasing Slc38a2 mRNA expression

32

through the vitamin D receptor (VDR) in cultured human primary trophoblast cells (Chen et al., 2017).

Differential function and regulation of Slc38a1 and Slc38a2 It is interesting that the homologous transporter Slc38a1, which also supports system A activity, lacks several of the functions and regulatory mechanisms of Slc38a2. Slc38a1 is not involved in the same general stress response pathways as Slc38a2 (Ling et al., 2001; Hyde et al., 2001; Kashiwagi et al., 2009). The studies done in organ systems in which both transporters are expressed show that Slc38a1 is not regulated by amino acid starvation (Novak et al., 2006; Jones et al., 2006) or hypertonic stress (Gaccioli et al., 2006; Maallem et al., 2008). However, Slc38a1 may play a part in arsenite-induced ER stress as it is reported to be moderately induced by arsenite in human embryonic kidney cells, although this requires further inquiry (Oh et al., 2012). Unlike Slc38a2, Slc38a1 is not known to be regulated by hormones, but as Slc38a2 its expression is downregulated by the proinflammatory cytokines IL1β, IL-6 and TNFα in human choriocarcinoma cells and cultured human trophoblasts (Thongsong et al., 2005; Jones et al., 2009). Slc38a1 is upregulated following ischemia-reperfusion injury in rat cardiomyocytes where it contributes to cysteine transport and cardiomyocyte glutathione synthesis (King et al., 2011). Both Slc38a1 and Slc38a2 is upregulated following hypoxia by HIF-1α (Horie et al., 2018; Morotti et al., 2019). In mice primary cultures of cerebral cortical neurons, neuronal differentiation induced by brain-derived neurotrophic factor (BDNF) upregulates Slc38a1, which contributes to carry the nutritional burden associated with dendritic growth and cortical neuronal branching (Burkhalter et al., 2007).

33

Slc38a1 has selectively higher affinity for glutamine than Slc38a2 has (Km 0.37 mM and 2.3 mM, respectively, in Xenopus laevis oocytes at -50 mV; Chaudhry 2002). The opposite is the case when it comes to the Km for MeAIB and Na+: It is higher in Slc38a1 than in Slc38a2. Slc38a1 also has a restricted distribution. The two original reports on Slc38a1 characterization have shown that Slc38a1 mRNA is enriched in brain and heart, while it was not detectable in spleen, lung, liver, muscle, kidney and testes (Varoqui et al., 2000; Chaudhry et al., 2002b). However, low mRNA or protein levels of Slc38a1 were later demonstrated in some other organs as well. In the brain, we have shown that Slc38a1 is enriched in PV+ GABAergic interneurons, where it is important for accumulation of glutamine for GABA synthesis and determines the GABAergic vesicular load (Chaudhry et al., 2002b; Solbu et al., 2010; Qureshi et al., 2019). Indeed, Slc38a1 is essential for cortical processing and plasticity (Qureshi et al.,

2019).

Altogether,

Slc38a1

more

selectively

transports

glutamine

for

neurotransmission and for aspects of heart physiology, while Slc38a2 is responsible for the general system A activity involved in hyperosmotic stress, amino acid adaptation and ER stress and a target for regulation by hormones. Hence when Slc38a1 and Slc38a2 are co-localized (Blot et al., 2009), they may be differentially involved in cellular functions. Yet, they may partly compensate if one is dysfunctional (Qureshi et al., 2019).

Concluding remarks Slc38a2 is regulated in numerous ways and by multitude of components. We have demonstrated that KCTD7 regulates Slc38a2 and contributes to PME. However, as amino acid transport by Slc38a2 itself also contributes to and regulates multitudinous metabolic and signaling pathways, Slc38a2 may contribute to the

34

pathophysiology of a large number of medical conditions, including cancer, neurological diseases, diabetes mellitus and more.

35

Figure 1. Putative structure of Slc38a2 with some important regulatory sites plotted Slc38a2 has 11 putative transmembrane domains (TMDs) with an intracellular Nterminus and an extracellular 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 extracellular loop, while Cys228 and Cys303 are likely part of the 5th TMD. All four cysteines are highly conserved 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 activity through His504. In states of amino acid availability, five lysyl residues in the Slc38a2 N-terminus gets accessible and targeted by a currently unknown E3-ligase for ubiquitin-proteasomemediated degradation.

Figure 2. Slc38a2 accumulates neutral amino acids (AA) that drive transport by the system ASC transporters ASCT1/2 and the system L transporters LAT1/2 The ASCT1/2 and LAT1/2 transporters are all obligatory exchangers that energize uptake of some AA substrates against their concentration gradients with efflux of other AA down their concentration gradients. Slc38a2 is a unidirectional electrogenic system A transporter and therefore capable of accumulating 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.

36

A, alanine; AAA, aromatic amino acids; BCAA, branched chain amino acids; C, cysteine; Q, glutamine; S, serine; T, threonine.

Figure 3. Slc38a2 and the amino acid response pathway Starvation for one or more amino acids elicit intracellular 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 translation 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 element.

Figure 4. Slc38a2 function is regulated by a novel K+-channel. The potassium channel tetramerization domain 7 (KCTD7) protein harbour a bric-àbrac, tramtrack, broad complex/poxvirus and zinc finger (BTB/POZ) domain which is homologous to the highly conserved cytoplasmic N-terminal assembly domain T1 of voltage-gated K+ (Kv) channels. This motif supports protein-protein interactions. KCTD7 has been shown to regulate the transport function of Slc38a2. Pathologic variants of KCTD7 reduce glutamine transport and result in impaired glutamate synthesis.

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Highlights    

Slc38a2 represents the classically described system A transport activity. Through electrogenic transport, Slc38a2 furnish cells with small, neutral amino acids. We review how Slc38a2 is extensively regulated by cell stress, nutritional and hormonal signaling. Slc38a2 may contribute to the pathology in a number of diseases such as cancer, epilepsy, diabetes mellitus and more.