Alternative splicing of the Menkes copper Atpase (Atp7a) transcript in the rat intestinal epithelium.
The intestinal Menkes copper Atpase (Atp7a) gene is strongly induced by iron deficiency in the rat intestine. We sought to develop an in vitro model to understand the mechanism of this induction by performing molecular studies in native rat intestine and in intestinal epithelial (IEC-6) cells. IEC-6 cells express Atp7a, and induction was noted with iron deprivation. 5' Rapid amplification of cDNA ends and PCR experiments revealed three splice variants in rat intestine and IEC-6 cells; all variants were strongly induced during iron deprivation (five- to sevenfold). The splice variants presumably encode proteins that would either contain the extreme NH(2) terminus of the protein (containing copper binding domain 1) or not. We thus hypothesized that more than one version of Atp7a protein exists. Antibodies against this NH(2)-terminal region of the protein were developed (named N-term) and used along with previously reported antibodies (against more COOH-terminal regions, termed 54-10) to perform immunoblotting and immunolocalization studies. Results with the 54-10 antiserum revealed an Atp7a protein variant of approximately 190 kDa that localized to the trans-Golgi network of IEC-6 cells and trafficked to the plasma membrane with copper loading. Using the N-term antiserum, however, we noted protein of approximately 97 and 64 kDa. The 97-kDa protein was cytosolic and nuclear, whereas the 64-kDa protein was nuclear specific. Immunolocalization analyses with the N-term antiserum showed strong staining of nuclei in IEC-6 and Caco-2 cells and in rat intestine. We conclude that novel Atp7a protein variants may exist in rat and human intestinal epithelial cells, with different intracellular locations and potentially distinct physiological functions.
Collins
James F
JF
Food Science and Human Nutrition Department, Institute for Food and Agricultural Sciences, University of Florida, Gainesville, Florida 32611, USA. jfcollins@ufl.edu
Hua
Ping
P
Lu
Yan
Y
Ranganathan
P N
PN
D000251:Adenosine Triphosphatases / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017398:Alternative Splicing; D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D015153:Blotting, Western; D018938:Caco-2 Cells; D027682:Cation Transport Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D002467:Cell Nucleus / Q000201:enzymology; D003300:Copper / Q000378:metabolism*; D000073840:Copper-Transporting ATPases; D003600:Cytosol / Q000201:enzymology; D004790:Enzyme Induction; D004847:Epithelial Cells / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D006056:Golgi Apparatus / Q000201:enzymology; D006801:Humans; D007150:Immunohistochemistry; D007413:Intestinal Mucosa / Q000201:enzymology*; D000090463:Iron Deficiencies; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D021381:Protein Transport; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D000099236:Solute Carrier Family 11, Member 2
D000251:Adenosine Triphosphatases; D027682:Cation Transport Proteins; D003300:Copper; D000073840:Copper-Transporting ATPases; D012333:RNA, Messenger; D000099236:Solute Carrier Family 11, Member 2; C081184:ATP7A protein, human; C486202:Atp7a protein, rat
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