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J. Biol. Chem., Vol. 280, Issue 19, 19364-19372, May 13, 2005

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Identification and Functional Characterization of a Novel Low Affinity Aromatic-preferring Amino Acid Transporter (arpAT)

ONE OF THE FEW PROTEINS SILENCED DURING PRIMATE EVOLUTION^*

Esperanza Fernández, David Torrents¶, Antonio Zorzano, Manuel Palacín, and Josep Chillarón, Senior researcher from the Programa Ramón y Cajal of the Spanish Ministry of Science and Technology||

From the Department of Biochemistry and Molecular Biology, Faculty of Biology, and Barcelona Science Park, University of Barcelona, E-08028 Barcelona, Spain and ^¶EMBL, Heidelberg 69117, Germany

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 µM, 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.

Received for publication, November 5, 2004 , and in revised form, February 17, 2005.

^* This work was supported in part by Spanish Ministry of Science and Technology Grant SAF2003-08940 (to M. P.), EC project Grant 502852 (EUGINDAT) (to M. P.), and the Institut de Salud Carlos III networks C3/08P and G03/054 (to M. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. A and B.

Supported by a postdoctoral contract from the Comissionat per a Universitats i Recerca.

^|| To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, New Bldg., Faculty of Biology, University of Barcelona, Av. Diagonal 645, E-08028, Barcelona, Spain. Tel.: 34-934034700; Fax: 34-934034717; E-mail: chillaro{at}worldonline.es.

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