Copper Transport Protein (Ctr1) Levels in Mice Are Tissue Specific and Dependent on Copper Status

Abstract

Studies were conducted to determine distribution of the copper transporter, Ctr1, a transmembrane protein responsible for cellular copper uptake, in adult mice and in suckling mice nursed by either copper-adequate (Cu+) or copper-deficient (Cu-) dams. Western immunoblot analyses, using immunopurified antibody, detected monomeric (23 kDa) and oligomeric forms of Ctr1 in the membrane fraction of several mouse organs. Immunohistochemical analyses detected abundant Ctr1 protein in liver canaliculi; kidney cortex tubules; small intestinal enterocytes; the choroid plexus and capillaries of brain; intercalated disks of heart; mature spermatozoa; epithelium of mammary ducts; and the pigment epithelium, outer limiting membrane, and outer plexiform layer of the retina. Duodenal Ctr1 distribution was different in the adult compared with suckling mice; adult mice demonstrated strong intracellular staining of the enterocyte, whereas apical staining predominated in suckling mice. In Cu- mice at postnatal d 16 (P16), Ctr1 staining was augmented in kidney, duodenum, and choroid plexus, compared with Cu+ mice. Brain immunoblot data indicated that Ctr1 protein in membrane fractions of Cu- mice was 56% higher than Cu+ mice. Cu- mice had lower hemoglobin (56% of Cu+), and lower copper concentration (% of Cu+) in liver (15%), brain (26%), and kidney (65%). These results suggest that Ctr1 protein is expressed in multiple tissues and found in higher levels in selected organs after perinatal copper deficiency. Enhanced Ctr1 levels and redistribution might compensate in part for the decrease in copper supply. Mechanisms for the enhancement in Ctr1 staining remain to be established.

O-Linked Glycosylation at Threonine 27 Protects the Copper Transporter hCTR1 from Proteolytic Cleavage in Mammalian Cells

“…Although the predicted molecular mass of hCTR1 is 21 kDa, the size of the protein on Western blots using various anti-hCTR1 antibodies or epitope tags has been reported as 24, 28, and 35 kDa and multimeric forms of higher molecular mass (17,18,25,26). The presence of higher mass bands that correspond to dimer, trimer, and higher mass species further complicates the identification of hCTR1 in gels.…”

“…Anti-hCTR1 Antibodies-The identification of hCTR1 on Western blots has been hampered by the tendency of the protein to migrate in SDS-PAGE as a multiplicity of bands of differing apparent molecular mass (17,18,25,26). Several factors give rise to these numerous species, including variable post-translational modifications, multimeric and/or aggregated forms, proteolytic fragments, and irrelevant cross-reacting proteins.…”

“…The predicted molecular mass of hCTR1 is 21 kDa (10), but hCTR1 migrates with a mass of 33-35 kDa, often appearing as a closely spaced smear of bands (17,19). Treatment of (mammalian) cells with tunicamycin or digestion with the N-linked polysaccharide-specific peptide glycosidase PNGase F reduces the apparent mass of hCTR1 to about [24][25][26]20). Two studies identified the site of N-linked glycosylation in hCTR1 at N15, confirming that the amino terminus is extracellular (17,20).…”

Mg, Zn and Cu Transport Proteins: A Brief Overview from Physiological and Molecular Perspectives

“…In intestinal epithelial cells in suckling mice under limited Cu conditions, Ctr1 is predominantly localized on the apical membrane (21). It is likely that Cu induces rapid endocytosis and degradation of CTR1 to prevent excess Cu absorption (22).…”

Posttranslational regulation of copper transporters

“…Copper import in mammals is primarily mediated by the copper transporter (CTR) family of proteins that comprises the homologous CTR1 and CTR2. CTR1 is absolutely required for dietary copper absorption and is essential for cellular copper uptake [1][2][3][4][5][6][7]. Cellular copper export is equally important and is dependent on ATP7A and ATP7B [8][9][10][11][12].…”