Effects of sodium and amino acid substrate availability upon the expression and stability of the SNAT2 (SLC38A2) amino acid transporter

Thorsten Hoffmann, Emma Cwiklinski, Dinesh Shah, Clare Stretton, Russell Hyde, Peter Taylor, Harinder Hundal

Research output: Contribution to journalArticle

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Abstract

The SNAT2 (SLC38A2) System A amino acid transporter mediates Na+-coupled cellular uptake of small neutral α-amino acids (AA) and is extensively regulated in response to humoral and nutritional cues. Understanding the basis of such regulation is important given that AA uptake via SNAT2 has been linked to activation of mTORC1; a major controller of many important cellular processes including, for example, mRNA translation, lipid synthesis and autophagy and whose dysregulation has been implicated in the development of cancer and conditions such as obesity and type 2 diabetes. Extracellular AA withdrawal induces an adaptive upregulation of SNAT2 gene transcription and SNAT2 protein stability but, as yet, the sensing mechanism(s) that initiate this response remain poorly understood although interactions between SNAT2 and its substrates may play a vital role. Herein, we have explored how changes in substrate (AA and Na+) availability impact upon the adaptive regulation of SNAT2 in HeLa cells. We show that whilst AA deprivation induces SNAT2 gene expression, this induction was not apparent if extracellular Na+ was removed during the AA withdrawal period. Furthermore, we show that the increase in SNAT2 protein stability associated with AA withdrawal is selectively repressed by provision of SNAT2 AA substrates (Me-AIB and glutamine), but not non-substrates. This stabilization and substrate-induced repression was critically dependent upon the cytoplasmic N-terminal tail of SNAT2 (containing lysyl residues which are putative targets of the ubiquitin-proteosome system), because “grafting” this tail onto SNAT5, a related SLC38 family member that does not exhibit adaptive regulation, confers substrate-induced changes in stability of the SNAT2-5 chimeric transporter. In contrast, expression of SNAT2 in which the N-terminal lysyl residues were mutated to alanine rendered the transporter stable and insensitive to substrate-induced changes in protein stability. Intriguingly, SNAT2 protein stability was dramatically reduced in the absence of extracellular Na+ irrespective of whether substrate AAs were present or absent. Our findings indicate that the presence of extracellular Na+ (and potentially its binding to SNAT2) may be crucial for not only sensing SNAT2 AA occupancy and consequently for initiating the adaptive response under AA insufficient conditions, but for enabling substrate-induced changes in SNAT2 protein stability.
Original languageEnglish
Article number63
Pages (from-to)1-9
Number of pages9
JournalFrontiers in Pharmacology
Volume9
DOIs
Publication statusPublished - 7 Feb 2018

Fingerprint

Amino Acid Transport Systems
Sodium
Amino Acids
Protein Stability
Neutral Amino Acids
Autophagy
Protein Biosynthesis
Ubiquitin
Glutamine
HeLa Cells
Alanine
Type 2 Diabetes Mellitus
Cues
Up-Regulation
Obesity
Lipids
Gene Expression

Keywords

  • Adaptive regulation
  • Amino acid sensing
  • NAT5
  • Sodium ion
  • System A
  • Transporter

Cite this

@article{95f251cb574a4859972585868348d4e3,
title = "Effects of sodium and amino acid substrate availability upon the expression and stability of the SNAT2 (SLC38A2) amino acid transporter",
abstract = "The SNAT2 (SLC38A2) System A amino acid transporter mediates Na+-coupled cellular uptake of small neutral α-amino acids (AA) and is extensively regulated in response to humoral and nutritional cues. Understanding the basis of such regulation is important given that AA uptake via SNAT2 has been linked to activation of mTORC1; a major controller of many important cellular processes including, for example, mRNA translation, lipid synthesis and autophagy and whose dysregulation has been implicated in the development of cancer and conditions such as obesity and type 2 diabetes. Extracellular AA withdrawal induces an adaptive upregulation of SNAT2 gene transcription and SNAT2 protein stability but, as yet, the sensing mechanism(s) that initiate this response remain poorly understood although interactions between SNAT2 and its substrates may play a vital role. Herein, we have explored how changes in substrate (AA and Na+) availability impact upon the adaptive regulation of SNAT2 in HeLa cells. We show that whilst AA deprivation induces SNAT2 gene expression, this induction was not apparent if extracellular Na+ was removed during the AA withdrawal period. Furthermore, we show that the increase in SNAT2 protein stability associated with AA withdrawal is selectively repressed by provision of SNAT2 AA substrates (Me-AIB and glutamine), but not non-substrates. This stabilization and substrate-induced repression was critically dependent upon the cytoplasmic N-terminal tail of SNAT2 (containing lysyl residues which are putative targets of the ubiquitin-proteosome system), because “grafting” this tail onto SNAT5, a related SLC38 family member that does not exhibit adaptive regulation, confers substrate-induced changes in stability of the SNAT2-5 chimeric transporter. In contrast, expression of SNAT2 in which the N-terminal lysyl residues were mutated to alanine rendered the transporter stable and insensitive to substrate-induced changes in protein stability. Intriguingly, SNAT2 protein stability was dramatically reduced in the absence of extracellular Na+ irrespective of whether substrate AAs were present or absent. Our findings indicate that the presence of extracellular Na+ (and potentially its binding to SNAT2) may be crucial for not only sensing SNAT2 AA occupancy and consequently for initiating the adaptive response under AA insufficient conditions, but for enabling substrate-induced changes in SNAT2 protein stability.",
keywords = "Adaptive regulation, Amino acid sensing, NAT5, Sodium ion, System A, Transporter",
author = "Thorsten Hoffmann and Emma Cwiklinski and Dinesh Shah and Clare Stretton and Russell Hyde and Peter Taylor and Harinder Hundal",
note = "This work was supported by Diabetes UK and the Biotechnology and Biological Sciences Research Council.",
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Effects of sodium and amino acid substrate availability upon the expression and stability of the SNAT2 (SLC38A2) amino acid transporter. / Hoffmann, Thorsten; Cwiklinski, Emma; Shah, Dinesh; Stretton, Clare; Hyde, Russell; Taylor, Peter; Hundal, Harinder.

In: Frontiers in Pharmacology, Vol. 9, 63, 07.02.2018, p. 1-9.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Effects of sodium and amino acid substrate availability upon the expression and stability of the SNAT2 (SLC38A2) amino acid transporter

AU - Hoffmann, Thorsten

AU - Cwiklinski, Emma

AU - Shah, Dinesh

AU - Stretton, Clare

AU - Hyde, Russell

AU - Taylor, Peter

AU - Hundal, Harinder

N1 - This work was supported by Diabetes UK and the Biotechnology and Biological Sciences Research Council.

PY - 2018/2/7

Y1 - 2018/2/7

N2 - The SNAT2 (SLC38A2) System A amino acid transporter mediates Na+-coupled cellular uptake of small neutral α-amino acids (AA) and is extensively regulated in response to humoral and nutritional cues. Understanding the basis of such regulation is important given that AA uptake via SNAT2 has been linked to activation of mTORC1; a major controller of many important cellular processes including, for example, mRNA translation, lipid synthesis and autophagy and whose dysregulation has been implicated in the development of cancer and conditions such as obesity and type 2 diabetes. Extracellular AA withdrawal induces an adaptive upregulation of SNAT2 gene transcription and SNAT2 protein stability but, as yet, the sensing mechanism(s) that initiate this response remain poorly understood although interactions between SNAT2 and its substrates may play a vital role. Herein, we have explored how changes in substrate (AA and Na+) availability impact upon the adaptive regulation of SNAT2 in HeLa cells. We show that whilst AA deprivation induces SNAT2 gene expression, this induction was not apparent if extracellular Na+ was removed during the AA withdrawal period. Furthermore, we show that the increase in SNAT2 protein stability associated with AA withdrawal is selectively repressed by provision of SNAT2 AA substrates (Me-AIB and glutamine), but not non-substrates. This stabilization and substrate-induced repression was critically dependent upon the cytoplasmic N-terminal tail of SNAT2 (containing lysyl residues which are putative targets of the ubiquitin-proteosome system), because “grafting” this tail onto SNAT5, a related SLC38 family member that does not exhibit adaptive regulation, confers substrate-induced changes in stability of the SNAT2-5 chimeric transporter. In contrast, expression of SNAT2 in which the N-terminal lysyl residues were mutated to alanine rendered the transporter stable and insensitive to substrate-induced changes in protein stability. Intriguingly, SNAT2 protein stability was dramatically reduced in the absence of extracellular Na+ irrespective of whether substrate AAs were present or absent. Our findings indicate that the presence of extracellular Na+ (and potentially its binding to SNAT2) may be crucial for not only sensing SNAT2 AA occupancy and consequently for initiating the adaptive response under AA insufficient conditions, but for enabling substrate-induced changes in SNAT2 protein stability.

AB - The SNAT2 (SLC38A2) System A amino acid transporter mediates Na+-coupled cellular uptake of small neutral α-amino acids (AA) and is extensively regulated in response to humoral and nutritional cues. Understanding the basis of such regulation is important given that AA uptake via SNAT2 has been linked to activation of mTORC1; a major controller of many important cellular processes including, for example, mRNA translation, lipid synthesis and autophagy and whose dysregulation has been implicated in the development of cancer and conditions such as obesity and type 2 diabetes. Extracellular AA withdrawal induces an adaptive upregulation of SNAT2 gene transcription and SNAT2 protein stability but, as yet, the sensing mechanism(s) that initiate this response remain poorly understood although interactions between SNAT2 and its substrates may play a vital role. Herein, we have explored how changes in substrate (AA and Na+) availability impact upon the adaptive regulation of SNAT2 in HeLa cells. We show that whilst AA deprivation induces SNAT2 gene expression, this induction was not apparent if extracellular Na+ was removed during the AA withdrawal period. Furthermore, we show that the increase in SNAT2 protein stability associated with AA withdrawal is selectively repressed by provision of SNAT2 AA substrates (Me-AIB and glutamine), but not non-substrates. This stabilization and substrate-induced repression was critically dependent upon the cytoplasmic N-terminal tail of SNAT2 (containing lysyl residues which are putative targets of the ubiquitin-proteosome system), because “grafting” this tail onto SNAT5, a related SLC38 family member that does not exhibit adaptive regulation, confers substrate-induced changes in stability of the SNAT2-5 chimeric transporter. In contrast, expression of SNAT2 in which the N-terminal lysyl residues were mutated to alanine rendered the transporter stable and insensitive to substrate-induced changes in protein stability. Intriguingly, SNAT2 protein stability was dramatically reduced in the absence of extracellular Na+ irrespective of whether substrate AAs were present or absent. Our findings indicate that the presence of extracellular Na+ (and potentially its binding to SNAT2) may be crucial for not only sensing SNAT2 AA occupancy and consequently for initiating the adaptive response under AA insufficient conditions, but for enabling substrate-induced changes in SNAT2 protein stability.

KW - Adaptive regulation

KW - Amino acid sensing

KW - NAT5

KW - Sodium ion

KW - System A

KW - Transporter

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DO - 10.3389/fphar.2018.00063

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JO - Frontiers in Pharmacology

JF - Frontiers in Pharmacology

SN - 1663-9812

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ER -