Wilber Romero Fernández
The human M3 muscarinic acetylcholine receptor (M3R) regulates many important physiological roles in the central and peripheral nervous systems, and it is involved in the pathophysiology of several neurodegenerative and autoimmune diseases, representing attractive potential pharmacological target for intervention. However, the lack of structural information on this receptor hampered the development of new potent antagonist with increased selectivity and lower side effects. Such structural information can be only achieved by means of experimental biophysical techniques, which require large quantities of pure receptor. Considering that under physiological conditions the expression of G-protein coupled receptors (GPCRs) is relatively low, optimization of the receptor overexpression is a pre-requisite for structural studies efforts to be performed. In addition, although is has well established that GPCR undergo post-translational and increase evidences support that these are tight links to receptor roles, little progress has been made in the post-translational modifications field in some GPCRs, such as the case of M3R. In this study, we provide some strategies to improve muscarinic receptor heterologous expression in mammalian cells guaranteeing proper post-translational modifications. In addition, we have been able to extract high levels of functional receptor from COS-7 cells using a detergent combination tested, and to purify the receptor to near homogeneity-keeping the full wild type receptor properties- by means of different affinity purification methods. Regarding the post-translational modifications studied, our findings provide the first evidence of the critical role that N-glycan chains play in determining muscarinic receptor distribution and localization, as well as in cell integrity. Furthermore, our data reveal a role for palmitoylation in determining M3R residence within lipid raft, as well as in receptor internalization and down-regulation
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