Cloning, expression and functional characterization of the putative regeneration and tolerance factor (RTF/TJ6) as a functional vacuolar ATPase proton pump regulatory subunit with a conserved sequence of immunoreceptor tyrosine-based activation motif.
In an attempt to identify new immunoreceptor tyrosine-based activation motif (ITAM)-containing human molecules that may regulate hitherto unknown immune cell functions, we BLAST searched the National Center for Biotechnology Information database for ITAM-containing sequences. A human expressed sequence tag showing partial homology to the murine TJ6 (mTJ6) gene and encoding a putative ITAM sequence has been identified and used to clone the human TJ6 (hTJ6) gene from an HL-60-derived cDNA library. hTJ6 was found to encode a protein of 856 residues with a calculated mass of 98 155 Da. Immunolocalization and sequence analysis revealed that hTJ6 is a membrane protein with predicted six transmembrane-spanning regions, typical of ion channels, and a single putative ITAM (residues 452-466) in a juxtamembrane or hydrophobic intramembrane region. hTJ6 is highly homologous to Bos taurus 116-kDa subunit of the vacuolar proton-translocating ATPase. Over-expression of hTJ6 in HEK 293 cells increased H+ uptake into intracellular organelles, an effect that was sensitive to inhibition by bafilomycin, a selective inhibitor of vacuolar H+ pump. Northern blot analysis demonstrated three different hybridizing mRNA transcripts corresponding to 3.2, 5.0 and 7.3 kb, indicating the presence of several splice variants. Significant differences in hTJ6 mRNA levels in human tissues of different origins point to possible tissue-specific function. Although hTJ6 was found to be a poor substrate for tyrosine-phosphorylating enzymes, suggesting that its ITAM sequence is non-functional in protein tyrosine kinase-mediated signaling pathways, its role in organellar H+ pumping suggests that hTJ6 function may participate in protein trafficking/processing.
Babichev
Yael
Y
Department of Microbiology and Immunology, Cancer Research Center, Faculty of Health Sciences, Ben Gurion University of the Negev, PO Box 653, Beer Sheva 84105, Israel.
Tamir
Ami
A
Park
Meeyoug
M
Muallem
Shmuel
S
Isakov
Noah
N
D020816:Amino Acid Motifs / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D017124:Conserved Sequence; D006801:Humans; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D011257:Pregnancy Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000276:immunology; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D021381:Protein Transport / Q000276:immunology; D011971:Receptors, Immunologic / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013491:Suppressor Factors, Immunologic / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D006180:Proton-Translocating ATPases
D011257:Pregnancy Proteins; D021122:Protein Subunits; D011971:Receptors, Immunologic; D013491:Suppressor Factors, Immunologic; D014443:Tyrosine; D025262:Vacuolar Proton-Translocating ATPases; C475700:regeneration and tolerance factor; C525845:ATP6V0A2 protein, human; D006180:Proton-Translocating ATPases