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Title: The role of ubiquitination in the post-endocytic trafficking of M2 muscarinic cholinergic receptor
Author: Zenko, Dmitry
ISNI:       0000 0004 7961 2914
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 2019
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Prolonged agonist stimulation of mAChRs promotes their down-regulation which has been implicated in a number of disease states such as bipolar disorder and cognitive impairment seen in Alzheimer's disease. Despite the fundamental importance of receptor down-regulation in the regulation of mAChR signalling it remains unknown which molecular mechanisms regulate the internalisation and post-endocytic trafficking of these receptors. As a first step in understanding of this process, the mechanisms of M2 mAChR internalisation and trafficking were investigated. Using a number of cutting-edge techniques, it was demonstrated that M2 mAChR undergoes rapid and extensive internalisation though CME in β-arrestin, clathrin and dynamin-dependent manner. Subsequently, the receptor is delivered to lysosomes for proteolysis. In the current study, the role of ubiquitination in lysosomal degradation of M2 mAChR was investigated. Ubiquitination has been shown to regulate the lysosomal trafficking of a number of GPCRs, however, is not always the major prerequisite for down-regulation of these receptors. Upon carbachol stimulation, the activated M2 mAChR expressed in HEK293 cells, undergoes ubiquitination, which serves as a sorting signal regulating transfer to intraluminal vesicles and subsequent trafficking to the lysosome. Substitution of 28 intracellular lysine residues with arginine prevented ubiquitin modification of the M2 mAChR and trafficking to the lysosome and promoted recycling via a Rab11 positive endosomal compartment. Biochemical disruption of ubiquitin interacting proteins prevents efficient proteolysis, suggesting that lysosomal trafficking of ubiqutinated M2 mAChR require functional ESCRT to promote transfer of M2 mAChR from the limiting endosome membrane to lumen. Finally, the importance of this post-translational modification was demonstrated to be conserved in primary cultures of rat dorsal root ganglia. Together, these results show for the first time that M2 mAChR requires ubiquitination and interaction with the ESCRT complex for efficient lysosomal sorting, and this process is critical for the efficient sorting of synaptic receptors for neuronal transport.
Supervisor: Hislop, James Sponsor: University of Aberdeen
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Muscarinic receptors ; Acetylcholine ; Post-translational modification ; Ubiquitin