C-terminal region regulates the functional expression of human noradrenaline transporter splice variants

Chiharu Sogawa, Kei Kumagai, Norio Sogawa, Katsuya Morita, Toshihiro Dohi, Shigeo Kitayama

Research output: Contribution to journalArticle

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Abstract

The NET [noradrenaline (norepinephrine) transporter], an Na +/Cl--dependent neurotransmitter transporter, has several isoforms produced by alternative splicing in the C-terminal region, each differing in expression and function. We characterized the two major isoforms of human NET, hNET1, which has seven C-terminal amino acids encoded by exon 15, and hNET2, which has 18 amino acids encoded by exon 16, by site-directed mutagenesis in combination with NE (noradrenaline) uptake assays and cell surface biotinylation. Mutants lacking one third or more of the 24 amino acids encoded by exon 14 exhibited neither cell surface expression nor NE uptake activity, with the exception of the mutant lacking the last eight amino acids of hNET2, whose expression and uptake resembled that of the WT (wild-type). A triple alanine replacement of a candidate motif (ENE) in this region mimicked the influences of the truncation. Deletion of either the last three or another four amino acids of the C-terminus encoded by exon 15 in hNET1 reduced the cell surface expression and NE uptake, whereas deletion of all seven residues reduced the transport activity but did not affect the cell surface expression. Replacement of RRR, an endoplasmic reticulum retention motif, by alanine residues in the C-terminus of hNET2 resulted in a similar expression and function compared with the WT, while partly recovering the effects of the mutation of ENE. These findings suggest that in addition to the function of the C-terminus, the common proximal region encoded by exon 14 regulates the functional expression of splice variants, such as hNET1 and hNET2.

Original languageEnglish
Pages (from-to)185-195
Number of pages11
JournalBiochemical Journal
Volume401
Issue number1
DOIs
Publication statusPublished - Jan 1 2007

Fingerprint

Norepinephrine Plasma Membrane Transport Proteins
Exons
Norepinephrine
Amino Acids
Alanine
Protein Isoforms
Neurotransmitter Transport Proteins
Biotinylation
Mutagenesis
Alternative Splicing
Site-Directed Mutagenesis
Endoplasmic Reticulum
Assays
Mutation

Keywords

  • Alternative splicing
  • Membrane trafficking
  • Neurotransmitter
  • Noradrenaline (norepinephrine) transporter (NET)
  • Noradrenaline uptake

ASJC Scopus subject areas

  • Biochemistry

Cite this

C-terminal region regulates the functional expression of human noradrenaline transporter splice variants. / Sogawa, Chiharu; Kumagai, Kei; Sogawa, Norio; Morita, Katsuya; Dohi, Toshihiro; Kitayama, Shigeo.

In: Biochemical Journal, Vol. 401, No. 1, 01.01.2007, p. 185-195.

Research output: Contribution to journalArticle

Sogawa, Chiharu ; Kumagai, Kei ; Sogawa, Norio ; Morita, Katsuya ; Dohi, Toshihiro ; Kitayama, Shigeo. / C-terminal region regulates the functional expression of human noradrenaline transporter splice variants. In: Biochemical Journal. 2007 ; Vol. 401, No. 1. pp. 185-195.
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N2 - The NET [noradrenaline (norepinephrine) transporter], an Na +/Cl--dependent neurotransmitter transporter, has several isoforms produced by alternative splicing in the C-terminal region, each differing in expression and function. We characterized the two major isoforms of human NET, hNET1, which has seven C-terminal amino acids encoded by exon 15, and hNET2, which has 18 amino acids encoded by exon 16, by site-directed mutagenesis in combination with NE (noradrenaline) uptake assays and cell surface biotinylation. Mutants lacking one third or more of the 24 amino acids encoded by exon 14 exhibited neither cell surface expression nor NE uptake activity, with the exception of the mutant lacking the last eight amino acids of hNET2, whose expression and uptake resembled that of the WT (wild-type). A triple alanine replacement of a candidate motif (ENE) in this region mimicked the influences of the truncation. Deletion of either the last three or another four amino acids of the C-terminus encoded by exon 15 in hNET1 reduced the cell surface expression and NE uptake, whereas deletion of all seven residues reduced the transport activity but did not affect the cell surface expression. Replacement of RRR, an endoplasmic reticulum retention motif, by alanine residues in the C-terminus of hNET2 resulted in a similar expression and function compared with the WT, while partly recovering the effects of the mutation of ENE. These findings suggest that in addition to the function of the C-terminus, the common proximal region encoded by exon 14 regulates the functional expression of splice variants, such as hNET1 and hNET2.

AB - The NET [noradrenaline (norepinephrine) transporter], an Na +/Cl--dependent neurotransmitter transporter, has several isoforms produced by alternative splicing in the C-terminal region, each differing in expression and function. We characterized the two major isoforms of human NET, hNET1, which has seven C-terminal amino acids encoded by exon 15, and hNET2, which has 18 amino acids encoded by exon 16, by site-directed mutagenesis in combination with NE (noradrenaline) uptake assays and cell surface biotinylation. Mutants lacking one third or more of the 24 amino acids encoded by exon 14 exhibited neither cell surface expression nor NE uptake activity, with the exception of the mutant lacking the last eight amino acids of hNET2, whose expression and uptake resembled that of the WT (wild-type). A triple alanine replacement of a candidate motif (ENE) in this region mimicked the influences of the truncation. Deletion of either the last three or another four amino acids of the C-terminus encoded by exon 15 in hNET1 reduced the cell surface expression and NE uptake, whereas deletion of all seven residues reduced the transport activity but did not affect the cell surface expression. Replacement of RRR, an endoplasmic reticulum retention motif, by alanine residues in the C-terminus of hNET2 resulted in a similar expression and function compared with the WT, while partly recovering the effects of the mutation of ENE. These findings suggest that in addition to the function of the C-terminus, the common proximal region encoded by exon 14 regulates the functional expression of splice variants, such as hNET1 and hNET2.

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