Investigating the specific phosphorylation sites of the human mineralocorticoid receptor using phospho-mapping
The purpose of this project was to confirm the specific sites where the G protein- coupled receptor kinase 5 (GRK5)-mediated phosphorylation of the mineralocorticoid receptor (MR) of aldosterone takes place. The significance of the study stems from prior findings indicating that e-cigarette vapors accumulate in the airway epithelium to create adverse respiratory effects, such as the desensitization and downregulation of the beta2- adrenergic receptor (β2AR). Osteopontin (OPN) is known to oppose cardiac β2AR’s cAMP-mediated antifibrotic signaling. Additionally, it is known that aldosterone contributes to OPN upregulation, which could result in the downregulation of β2AR. Aldosterone’s MR is phosphorylated and inhibited by GRK5, potentially at Ser 600 and Ser 843 residues, which provides protection against the adverse effects mediated by aldosterone. A “bottoms up” proteomics approach was applied throughout this study, during which the MR protein was extracted and isolated, digested, purified, and phospho- mapped using co-immunoprecipitation, trypsin protease digestion, liquid chromatography, and mass spectrometry. Phospho-mapping of the MR protein will confirm if GRK5 phosphorylates the MR at the expected residues of Ser 601 and Ser 843.