title: Structural insights into thyroid hormone transport mechanisms of the L-type amino acid transporter 2.
abstract: Thyroid hormones (THs) are transported across cell membranes by different transmembrane transporter proteins. In previous studies, we showed marked 3,3'-diiodothyronine (3,3'-T2) but moderate T3 uptake by the L-type amino acid transporter 2 (Lat2). We have now studied the structure-function relationships of this transporter and TH-like molecules. Our Lat2 homology model is based on 2 crystal structures of the homologous 12-transmembrane helix transporters arginine/agmatine antiporter and amino acid/polyamine/organocation transporter. Model-driven mutagenesis of residues lining an extracellular recognition site and a TH-traversing channel identified 9 sensitive residues. Using Xenopus laevis oocytes as expression system, we found that side chain shortening (N51S, N133S, N248S, and Y130A) expanded the channel and increased 3,3'-T2 transport. Side chain enlargements (T140F, Y130R, and I137M) decreased 3,3'-T2 uptake, indicating channel obstructions. The opposite results with mutations maintaining (F242W) or impairing (F242V) uptake suggest that F242 may have a gating function. Competitive inhibition studies of 14 TH-like compounds revealed that recognition by Lat2 requires amino and carboxylic acid groups. The size of the adjacent hydrophobic group is restricted. Bulky substituents in positions 3 and 5 of the tyrosine ring are allowed. The phenolic ring may be enlarged, provided that the whole molecule is flexible enough to fit into the distinctly shaped TH-traversing channel of Lat2. Taken together, the next Lat2 features were identified 1) TH recognition site; 2) TH-traversing channel in the center of Lat2; and 3) switch site that potentially facilitates intracellular substrate release. Together with identified substrate features, these data help to elucidate the molecular mechanisms and role of Lat2 in T2 transport.
authors.affiliation: Leibniz-Institut für Molekulare Pharmakologie (FMP) (K.M.H., K.M., A.K., R.S., G.K.), 13125 Berlin, Germany; and Institut für Experimentelle Endokrinologie (J.K.), Charité-Universitätsmedizin Berlin, 13353 Berlin, Germany.
authors.forename: Katrin M
authors.initials: KM
authors.lastname: Hinz
authors.affiliation: Leibniz-Institut für Molekulare Pharmakologie (FMP) (K.M.H., K.M., A.K., R.S., G.K.), 13125 Berlin, Germany; and Institut für Experimentelle Endokrinologie (J.K.), Charité-Universitätsmedizin Berlin, 13353 Berlin, Germany.
authors.forename: Katja
authors.initials: K
authors.lastname: Meyer
authors.affiliation: Leibniz-Institut für Molekulare Pharmakologie (FMP) (K.M.H., K.M., A.K., R.S., G.K.), 13125 Berlin, Germany; and Institut für Experimentelle Endokrinologie (J.K.), Charité-Universitätsmedizin Berlin, 13353 Berlin, Germany.
authors.forename: Anita
authors.initials: A
authors.lastname: Kinne
authors.affiliation: Leibniz-Institut für Molekulare Pharmakologie (FMP) (K.M.H., K.M., A.K., R.S., G.K.), 13125 Berlin, Germany; and Institut für Experimentelle Endokrinologie (J.K.), Charité-Universitätsmedizin Berlin, 13353 Berlin, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Schülein
authors.affiliation: Leibniz-Institut für Molekulare Pharmakologie (FMP) (K.M.H., K.M., A.K., R.S., G.K.), 13125 Berlin, Germany; and Institut für Experimentelle Endokrinologie (J.K.), Charité-Universitätsmedizin Berlin, 13353 Berlin, Germany.
authors.forename: Josef
authors.initials: J
authors.lastname: Köhrle
authors.affiliation: Leibniz-Institut für Molekulare Pharmakologie (FMP) (K.M.H., K.M., A.K., R.S., G.K.), 13125 Berlin, Germany; and Institut für Experimentelle Endokrinologie (J.K.), Charité-Universitätsmedizin Berlin, 13353 Berlin, Germany.
authors.forename: Gerd
authors.initials: G
authors.lastname: Krause
mesh_terms: D027182:Amino Acid Transport System y+ / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D018360:Crystallography, X-Ray; D027301:Fusion Regulatory Protein 1, Light Chains / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D010649:Phenylalanine / Q000378:metabolism; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D013963:Thyroid Hormones / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D027182:Amino Acid Transport System y+; D027301:Fusion Regulatory Protein 1, Light Chains; C518286:SLC7A8 protein, mouse; D013963:Thyroid Hormones; D010649:Phenylalanine