Model driven site-directed mutagenesis of the L-type amino acid transporter 2 reveals sensitive sites of the thyroid hormone 3,3'-T2 uptake

The brush-border membrane of the small intestine and kidney proximal tubule are the major sites for the absorption and re-absorption of nutrients in the body respectively. Transport of amino acids is mediated through the action of numerous secondary active transporters. In the mouse, neutral amino acids are transported by B0AT1 [broad neutral (0) amino acid transporter 1; SLC6A19 (solute carrier family 6 member 19)] in the intestine and by B0AT1 and B0AT3 (SLC6A18) in the kidney. Immunoprecipitation and Blue native electrophoresis of intestinal brush-border membrane proteins revealed that B0AT1 forms complexes with two peptidases, APN (aminopeptidase N/CD13) and ACE2 (angiotensin-converting enzyme 2). Physiological characterization of B0AT1 expressed together with these peptidases in Xenopus laevis oocytes revealed that APN increased the substrate affinity of the transporter up to 2.5-fold and also increased its surface expression (Vmax). Peptide competition experiments, in silico modelling and site-directed mutagenesis of APN suggest that the catalytic site of the peptidase is involved in the observed changes of B0AT1 apparent substrate affinity, possibly by increasing the local substrate concentration. These results provide evidence for the existence of B0AT1-containing digestive complexes in the brush-border membrane, interacting differentially with various peptidases, and responding to the dynamic needs of nutrient absorption in the intestine and kidney.

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Involvement of the L-Type Amino Acid Transporter Lat2 in the Transport of 3,3′-Diiodothyronine across the Plasma Membrane

European Thyroid Journal ◽

10.1159/000381542 ◽

2015 ◽

Vol 4 (Suppl. 1) ◽

pp. 42-50 ◽

Cited By ~ 13

Author(s):

Anita Kinne ◽

Melanie Wittner ◽

Eva K. Wirth ◽

Katrin M. Hinz ◽

Ralf Schülein ◽

...

Keyword(s):

Amino Acid ◽

Thyroid Hormone ◽

Thyroid Hormones ◽

Surface Expression ◽

Amino Acid Transporter ◽

Cell System ◽

Monocarboxylate Transporter ◽

Cell Surface Expression ◽

Xenopus Laevis Oocytes ◽

Acid Transporter

Thyroid hormones are transported across cell membranes by transmembrane transporter proteins, for example by members of the monocarboxylate transporter (MCT) and the L-type amino acid transporter (LAT) families. LATs consist of a light chain (e.g. LAT2) and a heavy chain (CD98), which is essential for their cell surface expression and functionality. The specificity of Lat2 for thyroid hormones and their metabolites and its role in their transport was not fully clear. This fact motivated us to establish a cell system to elucidate the uptake of thyroid hormones and their metabolites by mouse Lat2. The coinjection of cRNA coding for Lat2 and CD98 into Xenopus laevis oocytes resulted in a markedly increased level of 3,3′-diiodo-L-thyronine (3,3′-T2) and to some extent also enhanced T3 transport. To gain insight into properties of thyroid hormones and their metabolites transported by Lat2, we inhibited 3,3′-T2 uptake by various iodothyronine derivatives. T1 and T2 derivatives as well as 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid strongly competed with 3,3′-T2 uptake. In addition, we performed T2 uptake measurements with the thyroid hormone-specific transporter MCT8. For both Lat2 and MCT8, Km values in a low micromolar range were calculated. We demonstrated that oocytes are a suitable system for thyroid hormone transport studies mediated by Lat2. Our data indicates that Lat2 compared to other thyroid hormone transporters prefers 3,3′-T2 as the substrate. Thus, Lat2 might contribute to the availability of thyroid hormone by importing and/or exporting 3,3′-T2, which is generated either by T3 inactivation or by rapid deiodinase 1-mediated rT3 degradation.

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A novel role of the C-terminus of b0,+AT in the ER–Golgi trafficking of the rBAT–b0,+AT heterodimeric amino acid transporter

Biochemical Journal ◽

10.1042/bj20081798 ◽

2008 ◽

Vol 417 (2) ◽

pp. 441-448 ◽

Cited By ~ 12

Author(s):

Shinichi Sakamoto ◽

Arthit Chairoungdua ◽

Shushi Nagamori ◽

Pattama Wiriyasermkul ◽

Kanyarat Promchan ◽

...

Keyword(s):

Amino Acid ◽

Light Chain ◽

Amino Acid Transporter ◽

Site Directed Mutagenesis ◽

Amino Acid Transporters ◽

Hek 293 ◽

C Terminus ◽

Heterodimeric Complex ◽

Acid Transporter ◽

Membrane Spanning

The heterodimeric complex composed of rBAT (related to b0,+ amino acid transporter), a single-membrane-spanning glycosylated heavy chain, and b0,+AT, a putative 12-membrane-spanning non-glycosylated light chain, is an amino acid transporter that mediates the activity of system b0,+, a major apical transport system for cystine and dibasic amino acids in renal proximal tubule and small intestine. The C-terminus of b0,+AT has been proposed to play an important role in the functional expression of the heterodimeric transporters. In the present study, to reveal the roles of the C-terminus, we analysed b0,+AT mutants whose C-termini were sequentially deleted or replaced by site-directed mutagenesis in polarized MDCKII (Madin–Darby canine kidney II), non-polarized HEK-293 (human embryonic kidney-293) and HeLa cells. Although the deletion of C-terminus of b0,+AT did not affect the formation of a heterodimer with rBAT, it resulted in the loss of apparent transport function, owing to the failure of the plasma-membrane targeting of rBAT–b0,+AT heterodimeric complex associated with incomplete glycosylation of rBAT. A motif-like sequence Val480-Pro481-Pro482 was identified in the C-terminus of b0,+AT to be responsible for the C-terminus action in promoting the trafficking of rBAT–b0,+AT heterodimeric complex from the ER (endoplasmic reticulum) to Golgi apparatus. This is, to our knowledge, the first demonstration of the active contribution of the C-terminus of a light-chain subunit to the intracellular trafficking of heterodimeric transporters. Because the motif-like sequence Val480-Pro481-Pro482 is well conserved among the C-termini of light-chain subunits, common regulatory mechanisms could be proposed among heterodimeric amino acid transporters.

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Chemical Approaches for Studying the Biology and Pharmacology of Membrane Transporters: The Histidine/Large Amino Acid Transporter SLC7A5 as a Benchmark

Molecules ◽

10.3390/molecules26216562 ◽

2021 ◽

Vol 26 (21) ◽

pp. 6562

Author(s):

Mariafrancesca Scalise ◽

Raffaella Scanga ◽

Lara Console ◽

Michele Galluccio ◽

Lorena Pochini ◽

...

Keyword(s):

Amino Acid ◽

Essential Amino Acids ◽

Amino Acid Transporter ◽

Membrane Transporters ◽

Site Directed Mutagenesis ◽

General Applicability ◽

Regulatory Domains ◽

Acid Transporter ◽

Function Relationship ◽

Basic Features

The localization of membrane transporters at the forefront of natural barriers makes these proteins very interesting due to their involvement in the absorption and distribution of nutrients and xenobiotics, including drugs. Over the years, structure/function relationship studies have been performed employing several strategies, including chemical modification of exposed amino acid residues. These approaches are very meaningful when applied to membrane transporters, given that these proteins are characterized by both hydrophobic and hydrophilic domains with a different degree of accessibility to employed chemicals. Besides basic features, the chemical targeting approaches can disclose information useful for pharmacological applications as well. An eminent example of this picture is the histidine/large amino acid transporter SLC7A5, known as LAT1 (Large Amino Acid Transporter 1). This protein is crucial in cell life because it is responsible for mediating the absorption and distribution of essential amino acids in peculiar body districts, such as the blood brain barrier and placenta. Furthermore, LAT1 can recognize a large variety of molecules of pharmacological interest and is also considered a hot target for drugs due to its over-expression in virtually all human cancers. Therefore, it is not surprising that the chemical targeting approach, coupled with bioinformatics, site-directed mutagenesis and transport assays, proved fundamental in describing features of LAT1 such as the substrate binding site, regulatory domains and interactions with drugs that will be discussed in this review. The results on LAT1 can be considered to have general applicability to other transporters linked with human diseases.

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