Nutrient signalling by the mTOR (mammalian target of rapamycin) pathway involves upstream sensing of free AA (amino acid) concentrations. Several AA-regulated kinases have recently been identified as putative intracellular AA sensors. Their activity will reflect the balance between AA flows through underlying mechanisms which together determine the size of the intracellular free AA pool. For indispensable AAs, these mechanisms are primarily (i) AA transport across the cell membrane, and (ii) protein synthesis/breakdown. The System L AA transporter is the primary conduit for cellular entry of indispensable neutral AAs (including leucine and phenylalanine) and potentially a key modulator of AA-sensitive mTOR signalling. Coupling of substrate flows through System L and other AA transporters (e.g. System A) may extend the scope for sensing nutrient abundance. Factors influencing AA transporter activity (e.g. hormones) may affect intracellular AA concentrations and hence indirectly mTOR pathway activity. Several AA transporters are themselves regulated by AA availability through ‘adaptive regulation’, which may help to adjust the gain of AA sensing. The substrate-binding sites of AA transporters are potentially direct sensors of AA availability at both faces of the cell surface, and there is growing evidence that AA transporters of the SNAT (sodium-coupled neutral AA transporter) and PAT (proton-assisted AA transporter) families may operate, at least under some circumstances, as transporter-like sensors (or ‘transceptors’) upstream of mTOR.
It is now well-established that AAs (amino acids) act through the co-ordination of several signalling pathways to modulate vital processes, including protein synthesis, proteolysis, mRNA turnover, hormone action and release, as well as the transport and metabolism of AAs themselves (see, e.g., [1–4] for reviews). The two best-studied nutrient signalling cascades in higher eukaryotes are the GCN (general control non-derepressible) and TOR (target of rapamycin) pathways, both of which are regulated by mechanisms which include upstream sensing of intracellular AA concentrations [3,5–7]. The GCN2 protein kinase appears to monitor intracellular AA abundance from the level of tRNA charging: uncharged tRNA accumulation in AA-deprived cells activates GCN2, which phosphorylates and inactivates the initiation factor eIF2α (eukaryotic initiation factor 2α) [1,8], suppressing global mRNA translation, but permitting translation of the transcription factor ATF4 (activating transcription factor 4) with the consequent induction of genes for AA biosynthesis and transport [1,7]. In contrast, the TOR pathway is stimulated by cellular AA supplementation and activates mRNA translation, promoting cell growth [3,9]. We have shown that the Xenopus oocyte (a lower-vertebrate cell in which the highly conserved TOR pathway is responsive to both growth factors and nutrients) has an AA-sensing mechanism upstream of TOR which responds directly to intracellular microinjection of a range of large AAs (e.g. leucine), consistent with the proposal that the size of the intracellular free AA pool regulates TOR signalling . The AA-sensing mechanism upstream of TOR is poorly defined, although AA-regulated kinases, including the lipid kinase Vps34 (vacuolar protein sorting 34) [10,11] and the protein kinase MAP4K3 (mitogen-activated protein kinase kinase kinase kinase 3) , as well as Rags (Ras-related GTPases) , are candidate components.
The activity of the AA-responsive signalling pathways described above will reflect the size of the intracellular free AA pool as determined by the balance between AA flows through underlying processes which include (i) AA transport across the cell membrane, (ii) protein synthesis/breakdown, and (iii) AA biosynthesis and catabolism [4,14] (Figure 1). Inhibitor and gene-silencing studies have demonstrated a key role for particular AA transporters in the maintenance of ‘steady-state’ free AA concentrations in animal cells (e.g. [15,16]), implying that signalling pathways regulated by intracellular AA concentration may be intrinsically linked to AA transporter activity. The present review focuses on the mechanisms by which AA transporters help determine neutral (zwitterionic) AA concentrations in body fluid pools and how these putative ‘housekeeping’ activities may have unrecognized importance in regulation of nutrient signals such as those generated by the TOR pathway. It also examines the hypothesis that certain AA transporters expressed at the surface of mammalian cells may also function directly as extracellular AA sensors (or ‘transceptors’).
Schematic diagram illustrating processes which contribute to establishment of ‘steady-state’ intracellular AA concentrations and their interactions with major intracellular nutrient signalling pathways
AA transporters as modulators of intracellular free AA concentrations
The fluxes of a particular AA across the plasma membrane of a specific type of animal cell are likely to occur via several different AA transport ‘systems’. The molecular biology and functional properties of these various systems have been reviewed extensively (e.g. [17–20]). The System L transporter is the primary route for cellular entry of indispensable large neutral AAs in many mammalian cell types and, given that several of these AAs (notably leucine) are known to be potent activators of the TOR pathway, it is potentially a key modulator of AA-sensitive TOR signalling. System L transporters are composed of heterodimers of an AA permease [LAT (L-type amino acid transporter) 1 and 2] and the 4F2hc/CD98 glycoprotein . The LAT1 transporter is predominantly expressed in tissues with high rates of growth and protein turnover (including many proliferating tumours [21,22]) and is therefore of particular interest in the context of TOR signalling. We have demonstrated that overexpression of System L in Xenopus oocytes confers sensitivity of their TOR pathway to extracellular AAs , although only inasmuch as it acts as a conduit to facilitate AA delivery to the intracellular AA-sensor mechanism.
System L operates as an obligatory 1:1 AA exchanger which can couple the cellular uptake of indispensible AA substrates with the efflux (by heteroexchange) of cytoplasmic AAs ; thus net cellular accumulation (or depletion) of specific System L substrates is possible, without any overall change in the total AA concentration on either side of the cell membrane. The concentration gradients of exchange AA substrates are generally assumed to be generated at least partly by activity of secondary active AA transporters such as the widely expressed System A. SNAT2 (sodium-coupled neutral AA transporter 2) is the predominant System A isoform in most extraneural tissues  and is able to concentrate AA substrates intracellularly as a consequence of its unidirectional Na+–AA-coupled transport cycle . The AA substrate ranges of Systems A and L overlap to only a limited extent, therefore coupling of AA substrate flows through the two transport systems may indirectly extend the scope for sensing nutrient abundance [23–25] by generating cellular accumulation of TOR-pathway activators such as leucine (System L substrate) in exchange for cytoplasmic AAs such as glutamine (an AA substrate for both Systems A and L, which is itself unable to activate TOR directly when injected into oocytes ). We now have direct evidence that this type of coupling mechanism (termed tertiary active AA transport [23,24]) may develop, in that net accumulation of System L substrates (mainly leucine and isoleucine) by Xenopus oocytes incubated in buffer containing an AA mixture at physiological plasma concentrations is enhanced by 90% overall when System A is co-expressed alongside System L (F.E. Baird, K. Bett, H.S. Hundal and P.M. Taylor, unpublished work). The fact that downstream consequences of pharmacological blockade or suppression of System A include reduced cellular levels of System L substrates such as leucine [15,16,26] is further evidence for the occurrence of tertiary active AA transport of this type. Expression of both CD98/LAT1 [27,28] and SNAT2  show positive correlation with TOR pathway activation, and their functional coupling may help to explain why, in certain circumstances, glutamine exerts a similar effect to that of leucine on TOR pathway activity  or is at least required for the leucine effect .
An indirect effect of intracellular AA accumulation by transporters such as SNAT2 is the accompanying cell swelling owing to osmotic water movement. Cell swelling is itself an anabolic signal in tissues such as liver  and skeletal muscle , and, at least in muscle, there is evidence that swelling is detected by an adhesion-dependent mechanism involving integrins which is able to generate an activating signal to the TOR pathway [33,34]. The activity of AA transporters associated with net uptake of cations (Na+, cationic AAs) will also tend to depolarize the cell membrane. Such depolarizing effects may trigger a rise in intracellular Ca2+, an effect associated with AA-dependent activation of TORC1 (TOR complex 1) and Vps34 (at least in certain cell types) .
The intracellular AA sensor upstream of TOR is able to detect small (<10%) changes in intracellular leucine concentration , as might be produced by relatively small perturbations of leucine transport (see, e.g., [14,35] for reviews). Humoral factors influencing AA transporter activity, e.g. insulin stimulation of System A , may generate detectable changes in intracellular AA concentrations and consequently modulate (or even enhance) AA signalling. Certain growth factors and cytokines also up-regulate System L, which may provide an important component of the overall signal-directing cells such as activated lymphocytes to grow and progress through the cell cycle [37,38], as well as the necessary increase in provision of AAs for protein synthesis. There is evidence for tight control of leucine transport activity in non-growing cells, possibly in order to prevent inappropriate activation of signalling pathways such as TOR .
AA transporters as nutrient sensors: the transceptor concept
The substrate-binding sites of AA transporters are well positioned to act as direct sensors of AA availability at both faces of the cell surface, given a suitable mechanism for transducing the AA ligand-binding event to a change in activity of a downstream intracellular signalling cascade. AA transporters exhibiting a dual transport/receptor function (so-called ‘transceptors’) have been demonstrated in lower eukaryotes such as yeast or Drosophila. AA transport through the yeast AA permease Gap1p stimulates the cAMP/PKA (protein kinase A) signalling pathway and elicits changes in metabolism and expression of stress-responsive genes . A yeast AA permease homologue with limited transport capability, Ssy1p, has been implicated in the sensing of extracellular AA availability, so that, in the presence of AAs, it stimulates a proteolysis-dependent signalling pathway which increases the expression of a variety of AA and peptide permeases [40,41]. Two AA transporter genes which lie upstream of TOR signalling in Drosophila have been identified by genetic studies: slimfast is a component of TSC (tuberous sclerosis complex)/TOR signalling in the fat body , whereas the PAT (proton-assisted AA transporter)-type transporter PATH (pathetic) has been described as a high-affinity AA ‘sensor’, because it apparently has minimal transport activity . Gap1, Ssy1, slimfast and PATH are all members of the APC (AA/polyamine/organocation) transporter superfamily (, see also http://www.tcdb.org), which includes the AA transporter families to which LAT1 and SNAT2 belong [19,20]. Two recent lines of evidence indicate that SNAT2 may also operate, at least under some circumstances, as a transporter-like sensor (or transceptor).
(i) Global AA deprivation induces cellular SNAT2 gene expression (a process termed ‘adaptive regulation’) in a manner that can be repressed by resupply of a single AA substrate for System A. The potency of this repressive effect is correlated directly with the transport Km for System A substrates including MeAIB (α-methylaminoisobutyric acid), a specific non-metabolizable System A substrate. Furthermore, SNAT2 gene silencing increases the transcription of a SNAT2 reporter gene, whereas SNAT2 overexpression decreases it, leading us to propose that SNAT2 represses its own expression through a signal at least partly responsive to the occupancy of the substrate-binding site on SNAT2 . Adaptive regulation of SNAT2 (which is apparently initiated in part by a GCN2-dependent mechanism [46,47]) may help to adjust the gain of AA sensing in response to altered external AA availability.
(ii) SNAT2 gene silencing in L6 skeletal muscle cells appears to impair insulin signalling though phosphoinositide 3-kinase  and hence potentially to downstream targets including TOR. We also have preliminary evidence (in MCF7 cells; J.J. Pinilla-Tenas, H.S. Hundal and P.M. Taylor, unpublished work) that SNAT2 may be able to signal directly through to TOR, because MeAIB is able to promote rapamycin-sensitive phosphorylation of the TOR target p70S6K (p70 S6 kinase) despite actually decreasing intracellular AA concentrations by competing out uptake of natural AA substrates.
How might an AA transporter such as SNAT2 generate an intracellular chemical signal? Kielland-Brandt and colleagues have proposed a mechanistic model for transporter-like sensors such as Ssy1p, which distinguishes between signalling and non-signalling conformations based on the orientation of the substrate-binding site within the membrane . An important property of the model is that intracellular binding of AA substrate locks the transceptor in an inward-facing (non-signalling) conformation [41,49]. This property bears at least a superficial resemblance to an unusual characteristic of System A transport, termed trans-inhibition, a rapidly invoked kinetic effect by which cytoplasmic AAs inhibit the uptake of extracellular AAs through the transporter [45,50]. Studies using SNAT2 overexpressed in oocytes indicate that trans-inhibition may involve ‘trapping’ of SNAT2 in an inwardly facing direction by binding of cytoplasmic AAs, which prevents the conformational change necessary for the unloaded transporter to complete the transport cycle by returning to the extracellular face of the membrane. Trans-inhibition of SNAT2 is suggested to allow dynamic ‘feedback’ regulation of the cellular AA uptake rate by substrate abundance on either side of the membrane  and may in itself be an underappreciated mechanism for regulation of the intracellular AA pool size (particularly in cells lacking other Na+-dependent AA transporters). Very recent work has revealed a surprising complexity in the substrate-translocation pathway of archetypal nutrient transporters, including the presence of (i) primary and secondary substrate-binding sites (which may be occupied simultaneously) and/or (ii) occluded states in which bound substrates are temporarily shut off from both entry and exit pathways [52–54]. Within such complexity, it is tempting to speculate on on/off signalling conformations and those in which it may not even be necessary for bound substrate to be translocated through a transporter protein for signalling to be initiated.
Protein–protein interactions are likely to be of great importance for early signal transduction, and transporters are reported to interact with a variety of other proteins which influence their trafficking, localization and functional activity (see, e.g., [14,55,56] for reviews). Domains have been identified in the intracellular hydrophilic regions of yeast Gap1p and Ssy1p that propagate ligand-induced signalling  and a domain within the N-terminus of the SNAT2 protein allows it to be stabilized in an AA-dependent manner, conceivably by changes in phosphorylation  or ubiquitination  of this region of the SNAT2 protein. The hepatic System A transporter closely associates with integrin α3β1 dimers  and the 4F2hc/CD98 regulatory subunit of the System L transporter has been implicated in integrin signalling [60,61], although in neither case has any direct link with nutrient sensing been established. Fodrin (a structural protein which may have important roles in signalling from integrins and focal adhesions) has also been shown to associate with hepatic System A transporter activity  as well as CAT1 (cationic amino acid transporter 1) . The glutamate transporter EAAT1 (excitatory amino acid transporter 1) is required for the activation of glial p42/44 MAPK (mitogen-activated protein kinase) signalling in response to extracellular glutamate , although the mechanism is not known. GAT1 [GABA (γ-aminobutyric acid) transporter 1] is one of several neurotransmitter transporters regulated by syntaxin 1A, a component of the synaptic vesicle docking and fusion apparatus. Direct interaction between the N-terminal cytoplasmic domain of GAT1 and syntaxin 1A decreases substrate flux through the transporter , but this inhibition is relieved by GAT1 substrates, providing a mechanism by which to increase GABA transport when synaptic GABA concentrations become elevated [66,67]. GAT1 substrates also enable tyrosine phosphorylation of the transporter , reducing its rate of internalization from the plasma membrane and conceivably acting as the trigger for activation of an intracellular signal.
AA transporters are required to deliver AAs to intracellular AA sensors such as those of the TOR and GCN2 signalling pathways and may also operate subtly at various levels (as éminences grises, or ‘powers behind the throne’) to help to modulate nutrient signalling in animal cells under normal circumstances. They are also likely to have an impact on other mammalian signalling proteins that are responsive to changes in AA availability, such as nitric oxide synthase, p42/44 MAPK and JNK (c-Jun N-terminal kinase) (see, e.g., [4,14]). Up-regulation of AA transporter expression and/or activity helps to amplify the anabolic signal during periods of rapid cell growth and proliferation (e.g. during the immune response). These properties contribute to making AA transporters attractive targets (as well as delivery systems) for AA-based therapeutics inhibiting cell growth (e.g. immunosuppressant or anticancer treatments ) or alternatively in promoting growth of wasted tissues (e.g. to counteract sarcopenia [16,45]).
mTOR Signalling, Nutrients and Disease: Biochemical Society Focused Meeting held at Medical Sciences Teaching Centre, University of Oxford, U.K., 15–16 September 2008. Organized and Edited by Richard Boyd (Oxford, U.K.), Deborah Goberdhan (Oxford, U.K.) and Richard Lamb (Cancer Research UK, London, U.K.).
GABA transporter 1
general control non-derepressible
L-type amino acid transporter
mitogen-activated protein kinase
sodium-coupled neutral AA transporter 2
target of rapamycin
vacuolar protein sorting 34
I thank my colleagues in Dundee, particularly Hari Hundal, for their valuable comments and discussion.
Work is supported by the Biotechnology and Biological Sciences Research Council [grant numbers C19477 and NUF12], Tenovus Tayside [grant number T02/14], The Nuffield Foundation and Anonymous Trust.