Molecular cloning, primary structure, and expression of the human growth factor-activatable Na+/H+ antiporter.
We present the complete sequence of a cDNA encoding the human amiloride-sensitive Na+/H+ antiporter. After functional complementation of a mouse fibroblast mutant by gene transfer, we isolated a 0.8 kb genomic probe from a third-cycle mouse transformant. The probe detects gene amplification in Na+/H+ antiporter "overexpressers" and a single class of mRNA of ca. 5.6 kb in human, mouse, and hamster cells. With this probe we isolated a 4 kb cDNA from a library constructed from a mouse transformant in which the transfected human gene was amplified. This cDNA includes a noncoding leader of 407 bp, a 2682 bp open reading frame, and a 3' noncoding sequence containing a mouse B1 repeated element. The amino acid sequence predicts a protein of Mr = 99,354 with an N-terminal amphipathic domain that contains 10 putative transmembrane-spanning segments and two potential glycosylation sites, followed by a hydrophilic stretch of 395 residues, presumably cytoplasmic. Stable expression of the transfected cDNA in Na+/H+ antiporter-deficient cells restored the key functional features of this transporter: H+i-activated Na+ influx, amiloride sensitivity, and pHi regulation.
Sardet
C
C
Centre de Biochimie-CNRS, Université de Nice, France.
Franchi
A
A
Pouysségur
J
J
D000584:Amiloride / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D005796:Genes; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D017923:Sodium-Hydrogen Exchangers; D013045:Species Specificity; D014162:Transfection
D002352:Carrier Proteins; D012333:RNA, Messenger; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride