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Title: The trafficking of melanocortin 4 receptor mutants
Author: Ward, Natalie Anne
ISNI:       0000 0004 2742 767X
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2011
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MUTATED versions of membrane proteins often fail to express at the plasma membrane, but instead are trapped in the secretory pathway, resulting in disease. The retention of these mutant proteins is thought to result from local misfolding, which prevents export from the endoplasmic reticulum (ER), targeting the receptor for degradation via the Ek-associated quality control system. The rhodopsin-like G protein- coupled melanocortin 4 receptor (MC4R) is an example of such a membrane protein. Over a hundred natural MC4R mutations are linked with an obese phenotype and to date represent the most common monogenic cause of severe early-onset obesity. More than 80% of these mutations result in a substantial proportion of MC4R being retained intracellularly. If these receptors were expressed at the plasma membrane, they are likely to be functional, as many mutations occur in domains distinct from those associated with ligand or G-protein binding. The objective of this thesis was to develop high throughput cell culture based assays with which to monitor MC4R cell surface expression, with the view to screening potential pharmacological chaperones for their ability to promote trafficking to the plasma membrane. Subsequently, the rescued receptors were assessed for signalling capability, to investigate whether their cell surface rescue restored functionality. The work presented herein confirms that clinically occurring MC4R mutants V50M, S58C and I137T are intracellularly retained in HEK-293 cells to differing degrees. Whole cell ELISA and 96-well fluorescence-based assays with N-terminally HA-tagged and C-terminally mCherry-tagged mutant MC4R were used to screen a number of MC4R-selective compounds. Compound A had previously been shown to be a potent antagonist of MC4R, and in addition to this compound, three related compounds B, C and D increased the cell surface expression of wild type and mutant MC4R, thus acting as pharmacological chaperones. They appeared to be receptor specific, as ligands for other proteins failed to alter the cell surface expression of wild type or mutant MC4R, and also had an intracellular site of action. There appears to be a unique rescue profile for each drug which did not correlate with potency, suggesting distinct receptor conformations induced by the different mutations. Functionality of V50M and S58C was rescued following their relocation to the cell surface. IV From the computational analysis using an MC4R model, it is evident that such point mutations can result in markedly changed interactions between amino acidside chains, which may alter helical-helical packing and affect receptor stability and/or activation steps, without directly affecting the ligand binding pocket. Following rescue to the plasma membrane, the ECso and Emax values of V50M and S58C increased significantly, indicating that the misfold induced by these mutations have a lesser impact on the overall structure and stability of the receptor than the one induced by I137T. Following cell surface rescue, novel derivatives of Compound A were also shown to act as competitive antagonists of varying potencies. For the most part, there appears to be a clear separation in potency between Compound A and D, and Compound Band C, indicating that exchange of the centre linker azetidine ring for the piperazine ring results in a marked decrease in potency. Investigation into binding modes of Compounds A and B, using our MC4R model, suggests differential binding of the two compounds when the linker ring is exchanged. The rescue of intracellularly retained MC4R mutants using pharmacological chaperones is a promising and exciting concept that could prove to be a novel therapeutic avenue in the treatment of early-onset obesity.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available