Determining the intracellular trafficking pathway of tachykinin-copper(II)-receptor complexes and elucidating the intracellular fate of copper

Abstract

Neurokinin B (NKB) is a member of the tachykinin family of peptides. In mammals the tachykinins are found throughout the central and peripheral nervous system and play diverse roles including in neuronal transmission, pain, inflammation and hormone regulation. NKB is part of this family due to its highly conserved FxGLM-NH2 carboxy terminus that is crucial for its cognate NK3R receptor binding and stimulation. In humans NKB is predominant in the hypothalamus and spinal cord and has a key role in reproduction, but has also been linked to disorders such as Alzheimer's Disease. NKB binds copper at its N-terminus and undergoes conformational changes, however this does not appear to disrupt receptor activation and endocytosis. It is not well understood why NKB binds copper, but this feature may be key to understanding the function and misfunction of NKB in the normal brain and in disease states. This is because copper is important for the normal physiology of the brain, but can be toxic if not well controlled. Dysregulated copper is a feature of several neurological disorders, including Menkes and Wilson's diseases and Alzheimer's disease. This thesis elucidates the cellular pathways taken by copper, NKB and the NK3 receptor and establishes how NKB can mitigate against the potentially toxic effects of copper. This work shows that NKB in apo- and copper-bound forms undergoes NK3R mediated endocytosis. Copper-bound NKB triggers more rapid endocytosis that apo-NKB. The metal-peptide-receptor complex enters into early endosomes from the cell surface via clathrin dependent and independent pathways. Copper dissociates from the complex, but, unexpectedly, remains enclosed in a vesicle and undergoes recycling out of cell thereby preventing cellular metal accumulation. The work shows that NKB undergoes further downstream endo-lysosomal degradation while NK3R receptor enters into novel nuclear envelope associated endosomal pathways (NAE) and shuttles to the nuclear envelope and nucleoplasm. This work has revealed the intricate cellular trafficking pathways taken by NKB and the NK3 receptor and the effect that copper has on the processes. The receptor mediated endocytosis of the copper-NKB complex is the first time the process has been described for a metal other than iron. The unexpected finding that copper does not accumulate, but is recycled out of the cell is thought to be a previously unrecognised protective mechanism that avoids having copper retained in the cell. This suggests that NKB binds copper for reasons other than as being part of the cellular copper homeostasis mechanisms.

Date of Award	2021
Original language	English