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J. Biol. Chem., Vol. 283, Issue 39, 26428-26435, September 26, 2008

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Identification and Characterization of a Diamine Exporter in Colon Epithelial Cells^*

Takeshi Uemura, Hagit F. Yerushalmi, George Tsaprailis^¶, David E. Stringer, Kirk E. Pastorian^||, Leo Hawel, III^||, Craig V. Byus^||, and Eugene W. Gerner¹

From the Arizona Cancer Center, University of Arizona, Tucson, Arizona 85724, Faculty of Medicine, Technion, Haifa 31096, Israel, ^¶Center for Toxicology, College of Pharmacy, Tucson, Arizona 85721, and ^||Department of Biomedical Sciences, University of California, Riverside, California 92521-0121

SLC3A2, a member of the solute carrier family, was identified by proteomics methods as a component of a transporter capable of exporting the diamine putrescine in the Chinese hamster ovary (CHO) cells selected for resistance to growth inhibition by high exogenous concentrations of putrescine. Putrescine transport was increased in inverted plasma membrane vesicles prepared from cells resistant to growth inhibition by putrescine compared with transport in inverted vesicles prepared from non-selected cells. Knockdown of SLC3A2 in human cells, using short hairpin RNA, caused an increase in putrescine uptake and a decrease in arginine uptake activity. SLC3A2 knockdown cells accumulated higher polyamine levels and grew faster than control cells. The growth of SLC3A2 knockdown cells was inhibited by high concentrations of putrescine. Knockdown of SLC3A2 reduced export of polyamines from cells. Expression of SLC3A2 was suppressed in human HCT116 colon cancer cells, which have an activated K-RAS, compared with their isogenic clone, Hkh2 cells, which lack an activated K-RAS allele. Spermidine/spermine N¹-acetyltransferase (SAT1) was co-immunoprecipitated by an anti-SLC3A2 antibody as was SLC3A2 with an anti-SAT1 antibody. SLC3A2 and SAT1 colocalized on the plasma membrane. These data provide the first molecular characterization of a polyamine exporter in animal cells and indicate that the diamine putrescine is exported by an arginine transporter containing SLC3A2, whose expression is negatively regulated by K-RAS. The interaction between SLC3A2 and SAT1 suggests that these proteins may facilitate excretion of acetylated polyamines.

Received for publication, June 20, 2008 , and in revised form, July 24, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant CA123065. 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 Table S1 and sequence data.

¹ To whom correspondence should be addressed: Arizona Cancer Center, 1515 N. Campbell Ave., Tucson, AZ 85724. Tel.: 520-626-2197; Fax: 520-626-4480; E-mail: egerner{at}azcc.arizona.edu.

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