Copper-dependent Recycling of hCTR1, the Human High Affinity Copper Transporter

Abstract

Copper is an essential co-factor in many important physiological processes, but at elevated levels it is toxic to cells. Thus at both the organism and cellular level mechanisms have evolved to finely tune copper homeostasis. The protein responsible for copper entry from the circulation in most human cells is hCTR1, a small protein (190 amino acid residues) that functions as a trimer in the plasma membrane. In the present work we employ cell surface biotinylation and isotopic copper uptake studies of overexpressed hCTR1 in HEK293 cells to examine the acute (minutes) response of hCTR1 to changes in extracellular copper. We show that within 10 min of exposure to copper at 2.5 M or higher, plasma membrane hCTR1 levels are reduced (by ϳ40%), with a concomitant reduction in copper uptake rates. We are unable to detect any degradation of internalized hCTR1 in the presence of cycloheximide after up to 2 h of exposure to 0 -100 M copper. Using a reversible biotinylation assay, we quantified internalized hCTR1, which increased upon the addition of copper and corresponded to the hCTR1 lost from the surface. In addition, when extracellular copper is then removed, internalized hCTR1 is promptly (within 30 min) recycled to the plasma membrane. We have shown that in the absence of added extracellular copper, there is a small but detectable amount of internalized hCTR1 that is increased in the presence of copper. Similar studies on endogenous hCTR1 show a cell-specific response to elevated extracellular copper. Copper-dependent internalization and recycling of hCTR1 provides an acute and reversible mechanism for the regulation of cellular copper entry.

Rate and Regulation of Copper Transport by Human Copper Transporter 1 (hCTR1)

“…There was between 35 and 60% decrease in WT cell surface hCTR1 after exposure to 20 -100 M copper (Fig. 8B), in agreement with our previous study (31). However, there was no change in cell surface level of hCTR1 in H139R mutant cells after the addition of extracellular copper (Fig.…”

“…Transferred membranes were removed from the gels and placed on a filter paper wetted with transfer buffer prior to the dotting of 2, 1, and 0.5 ng/l of purified hCTR1 protein, 4 -5 dots for each concentration, along the edge of the membrane. The membranes were then dried at room temperature for Ͼ2 h, after which Western blot analysis was performed with rabbit anti-hCTR1 C-terminal antibody as described (31).…”

“…PAGE and Western Blots-PAGE and subsequent Western blotting was performed as described previously (31). The following primary antibodies were used: rabbit anti-hCTR1 antibody against hCTR1 C-terminal tail (10), mouse anti-FLAG (GenScript, Piscataway, NJ), rabbit anti-FLAG (Sigma-Aldrich), mouse anti-Na,K-ATPase ␤1 (Affinity Bioreagents, Golden, CO), mouse anti-␤-catenin (BD Biosciences), and mouse anti-␣1 subunit of Na,K-ATPase (Affinity Bioreagents).…”

“…All of the biotinylation procedures were performed at 4°C. Caco-2 cells were grown in Transwell culture plates until polarized, and either the apical or the basolateral membrane was biotinylated (30,31).…”

“…The model is based on the observation that preincubation of cells in high copper conditions stimulates endocytosis of hCTR1 (26,27,31). If endocytosis is required for copper transport, then the process should occur at an elevated rate in cells expressing H139R when compared with cells expressing WT, reflecting the relative rates of copper uptake.…”