The purpose of this project is to confirm the specific sites where the G protein receptor kinase 5 (GRK-5) 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 could induce lung inflammation and tissue destruction. While research has typically focused on the effects of traditional tobacco use, the detrimental influence of e-cigarette vapors, which accumulate in the airway epithelium to create adverse respiratory effects, must also be identified.
Such effects include desensitization and downregulation of the beta 2-adrenergic receptor (β2AR), a cAMP-mediated receptor that is protective against airway inflammation and fibrosis, and the only AR subtype that expressed in human bronchial cells. Osteopontin (OPN), a pro-inflammatory and pro-fibrotic cytokine, is known to oppose cardiac β2AR’s cAMP-mediated antifibrotic signaling. Thus, it is possible that OPN upregulation causes β2AR desensitization in human bronchial cells.
Additionally, it is known that aldosterone contributes to OPN upregulation, which could result in the downregulation of β2AR. Aldosterone’s mineralocorticoid receptor (MR) is phosphorylated and thus inhibited by G-protein receptor kinase 5 (GRK-5), potentially at Ser 601 and Ser 843 residues, which provides protection against the adverse effects mediated by aldosterone.
Hence, confirmation of the GRK-5-mediated phosphorylation sites of the MR is a primary focus of our study, as this mechanism suppresses MR function.
The first course of action for the study is to isolate the MR from a sample of human tissue by co-immunoprecipitation (co-IP). Then, the presence and concentration of MR is tested using standard electrophoretic techniques to ensure the protein is suitable for further analysis. The MR sample is then digested with a trypsin protease and the peptide fragments are purified through liquid chromatography. Characterization of the peptide fragments are carried out by MALDI-TOF mass spectrometry, relying on database information as well as standards created from human recombinant MR protein, to determine the protein’s phosphorylation sites. Phospho-mapping of the fragments will confirm if GRK-5 phosphorylates the MR at the expected residues of Ser 601 and Ser 843.