Abstract
We have isolated a cDNA from a human placental choriocarcinoma cell cDNA library which, when expressed in HeLa cells, induces a Na+-dependent amino acid transport system with preference for zwitterionic amino acids. Anionic amino acids, cationic amino acids, imino acids, and N-methylated amino acids are excluded by this system. These characteristics are identical to those described for the amino acid transporter Bo. When expressed in Xenopus laevis oocytes that do not have detectable endogenous activity of the amino acid transporter Bo, the cloned transporter increases alanine transport in the oocytes severalfold and induces alanine-evoked inward currents in the presence of Na+. The cDNA codes for a polypeptide containing 541 amino acids with 10 putative transmembrane domains. Amino acid sequence homology predicts this transporter (hATBo) to be a member of a superfamily consisting of the glutamate transporters, the neutral amino acid transport system ASCT, and the insulin-activable neutral/anionic amino acid transporter. Chromosomal assignment studies with somatic cell hybrid analysis and fluorescent in situ hybridization have located the ATBo gene to human chromosome 19q13.3.
Highlights
We have isolated a cDNA from a human placental choriocarcinoma cell cDNA library which, when expressed in HeLa cells, induces a Na؉-dependent amino acid transport system with preference for zwitterionic amino acids
Construction of cDNA Library from JAR Cell mRNA—cDNA library was constructed with SuperScript Plasmid System (Life Technologies, Inc.) using poly(A)ϩ RNA isolated from confluent cultures of JAR cells
Isolation and Initial Characterization of Positive Clones— When the JAR cell cDNA library was screened under medium stringency conditions using the ASCT1 cDNA as the probe, several positive colonies were identified
Summary
Amino acid transport system ASC, which accepts alanine, serine, and cysteine as substrates, has been reported by two groups of investigators [11, 12] This system, cloned from human brain cDNA libraries, has been shown to be expressed in a wide variety of tissues, including intestine, kidney, and placenta. We report here on the cloning, functional expression, and chromosomal localization of this amino acid transport system from a human placental choriocarcinoma cell line (JAR) This represents the first successful cloning of an epithelial amino acid transport system that is likely to be of major importance in the transport of neutral amino acids in the placenta, intestine, and kidney
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