Identification and functional characterization of a novel low affinity aromatic-preferring amino acid transporter (arpAT). One of the few proteins silenced during primate evolution.
We have identified in silico arpAT, a gene encoding a new member of the LSHAT family, and cloned it from kidney. Co-expression of arpAT with the heavy subunits rBAT or 4F2hc elicited a sodium-independent alanine transport activity in HeLa cells. L-tyrosine, l-3,4-dihydroxyphenylalanine (L-DOPA), L-glutamine, L-serine, L-cystine, and L-arginine were also transported. Kinetic and cis-inhibition studies showed a K(m) = 1.59 +/- 0.24 mM for L-alanine or IC50 in the millimolar range for most amino acids, except L-proline, glycine, anionic and D-amino acids, which were not inhibitory. L-DOPA and L-tyrosine were the most effective competitive inhibitors of L-alanine transport, with IC50 values of 272.2 +/- 57.1 and 716.3 +/- 112.4 microM, respectively. In the small intestine, arpAT mRNA was located at the enterocytes, in a decreasing gradient from the crypts to the tip of the villi. It was also expressed in neurons from different brain areas. Finally, we show that while the arpAT gene is conserved in rat, dog, and chicken, it has become silenced in humans and chimpanzee. Actually, it has been recently reported that it is one of the 33 recently inactivated genes in the human lineage. The evolutionary implications of the silencing process and the roles of arpAT in transport of L-DOPA in the brain and in aromatic amino acid absorption are discussed.
Fernández
Esperanza
E
Department of Biochemistry and Molecular Biology, Faculty of Biology, and Barcelona Science Park, University of Barcelona, E-08028 Barcelona, Spain.
Torrents
David
D
Zorzano
Antonio
A
Palacín
Manuel
M
Chillarón
Josep
J
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D026905:Amino Acid Transport Systems; D026921:Amino Acid Transport Systems, Neutral / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D000596:Amino Acids / Q000737:chemistry; D024322:Amino Acids, Aromatic / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001692:Biological Transport; D015152:Blotting, Northern; D015153:Blotting, Western; D019070:Cell Lineage; D002645:Chickens; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D019143:Evolution, Molecular; D015723:Gene Library; D020868:Gene Silencing; D005973:Glutamine / Q000737:chemistry; D005998:Glycine / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D017403:In Situ Hybridization; D020128:Inhibitory Concentration 50; D007421:Intestine, Small / Q000378:metabolism; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D007980:Levodopa / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D002679:Pan troglodytes; D010802:Phylogeny; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012694:Serine / Q000737:chemistry; D012964:Sodium / Q000737:chemistry; D014162:Transfection
D026905:Amino Acid Transport Systems; D026921:Amino Acid Transport Systems, Neutral; D000596:Amino Acids; D024322:Amino Acids, Aromatic; D018076:DNA, Complementary; D012333:RNA, Messenger; C501858:Slc7a15 protein, mouse; D005973:Glutamine; D012694:Serine; D007980:Levodopa; D001120:Arginine; D011392:Proline; D012964:Sodium; D003545:Cysteine; D000409:Alanine; D005998:Glycine