title: C-terminal region regulates the functional expression of human noradrenaline transporter splice variants.
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.
authors.affiliation: Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8525, Japan.
authors.forename: Chiharu
authors.initials: C
authors.lastname: Sogawa
authors.forename: Kei
authors.initials: K
authors.lastname: Kumagai
authors.forename: Norio
authors.initials: N
authors.lastname: Sogawa
authors.forename: Katsuya
authors.initials: K
authors.lastname: Morita
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Dohi
authors.forename: Shigeo
authors.initials: S
authors.lastname: Kitayama
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000235:genetics; D004285:Dogs; D005091:Exons; D005786:Gene Expression Regulation; D014644:Genetic Variation; D006801:Humans; D007668:Kidney; D016297:Mutagenesis, Site-Directed; D050484:Norepinephrine Plasma Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D050484:Norepinephrine Plasma Membrane Transport Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins