Studies on regulatory mechanism of human Copper Transporter CTR1

Kar, Sumanta (2023) Studies on regulatory mechanism of human Copper Transporter CTR1. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Sumanta Kar (17RS007)) 17RS007.pdf - Submitted Version Restricted to Repository staff only Download (7MB)

Abstract

Copper (Cu) is an essential micronutrient for all forms. The reduced form of Cu, Cu (I) is imported in cells and is bio-utilizable but not Cu (II). Copper transporter-1 (CTR1) is the primary high affinity copper importer in eukaryotes; however, its regulatory mechanism and role in Cu (II) reduction remain unknown. CTR1 has an extracellular amino-terminal domain, three transmembrane domains and a cytoplasmic carboxy terminus. Here, we demonstrate that the extracellular amino-terminus (1-67 amino acids) of human CTR1 contains two methionine-histidine clusters and neighboring aspartates that bind Cu (II) and Cu (I) in a distinct manner prior to its import. We have determined that hCTR1 localizes at the basolateral membrane of polarized MDCK-II cells and in excess copper conditions, it gets endocytosed to Common Recycling Endosomes (CRE). We observed that the aspartate residues, D13 and D37 facilitate the transient binding of both Cu (II) and Cu (I) during reduction process. Using mutational studies, we deduced that the first Methionine cluster (7Met-Gly-Met9) and Asp13 facilitate copper uptake and endocytosis of hCTR1 in a copper-responsive fashion. We demonstrate that histidine clusters bind Cu (II) and are essential in copper-limiting conditions. Further, we also demonstrate that the Methionines of the N-terminus are essential in stabilizing the reduced form of Cu, i.e., Cu (I) which is bio-utilizable. We discovered that two amino-terminal His-Met-Asp clusters exhibit functional complementarity. We propose a novel mechanism by which the two His-Met residue clusters of the amino-terminus of hCTR1 not only bind copper but also maintain its reduced state, which is essential for intracellular copper uptake. In mammalian systems, CTR1 imports copper in its trimeric form. At higher extracellular Cu, hCTR1 endocytoses as a self-regulatory mechanism to limit copper uptake. We tried to decipher the Cu-induced regulatory mechanism that triggers its endocytosis. Using in vivo crosslinkers, we found that CTR1 exists in two conformational states - post-synthesis and during plasma membrane targeting, hCTR1 remains primarily as a ‘loose-grip’ trimer, whereas, extracellular Cu induces ‘tight-grip’ functional trimer formation that is capable of copper import. Under TIRF microscope in HEK293T cells, we observed that CTR1 undergoes copper-induced clustering at the plasma membrane before its onset of endocytosis. We found that this pre-endocytic clustering of hCTR1 causes a change in membrane tension and decrease in membrane fluctuation, that favors initiation of endocytosis. Finally, we also found that the Adapter Protein, AP-2 is recruited to CTR1 as it enters a pre-endocytic state in response to copper. To summarize, my study identifies the mechanistic underpinnings of copper reduction and import by CTR1. Further, we decipher the regulatory mechanism of CTR1 endocytosis, a phenomenon that is instrumental in limiting excess copper uptake in cells.

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