Investigating the Specific Phosphorylation Sites of the Human Mineralocorticoid Receptor Using Phospho-mapping
Abstract
The purpose of this project is to confirm the specific sites where G protein receptor kinase 5 (GRK-5)- mediated phosphorylation of the mineralocorticoid receptor (MR) of aldosterone occurs. The significance of the study stems from prior findings indicating that e-cigarette vapors induce lung inflammation and tissue destruction. While research has typically focused on the effects of tobacco use, the detrimental influence of e-cigarette vapors, which accumulate in the airway epithelium, must also be identified. This may include desensitization of the beta2-adrenergic receptor (β2AR), a protective receptor against airway inflammation and fibrosis and the only AR subtype expressed in human bronchial cells. Osteopontin (OPN), a pro-inflammatory and pro-fibrotic cytokine, is known to oppose cardiac β2AR’s antifibrotic signaling, and therefore may provoke β2AR desensitization in bronchial cells.
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 GRK-5, potentially at Ser 601 and Ser 843 residues, which provides protection against aldosterone-induced adverse effects. Hence, the phosphorylation by GRK-5 on the MR is a primary focus of our study, as this mechanism suppresses MR function.
The study involves isolating the MR from a human tissue sample by co-immunoprecipitation, digesting the MR with trypsin protease, purifying the resulting peptide fragments through liquid chromatography, and characterizing the peptide fragments by MALDI-TOF mass spectrometry to determine the protein’s phosphorylation sites. Phospho-mapping of the fragments will confirm if GRK-5 phosphorylates the MR at the expected Ser 601 and Ser 843 residues.
Faculty Sponsors
Dr. Beatrix Aukszi
Project Type
Event
Location
Alvin Sherman Library
Start Date
4-6-2022 12:00 PM
End Date
4-7-2022 5:00 PM
Investigating the Specific Phosphorylation Sites of the Human Mineralocorticoid Receptor Using Phospho-mapping
Alvin Sherman Library
The purpose of this project is to confirm the specific sites where G protein receptor kinase 5 (GRK-5)- mediated phosphorylation of the mineralocorticoid receptor (MR) of aldosterone occurs. The significance of the study stems from prior findings indicating that e-cigarette vapors induce lung inflammation and tissue destruction. While research has typically focused on the effects of tobacco use, the detrimental influence of e-cigarette vapors, which accumulate in the airway epithelium, must also be identified. This may include desensitization of the beta2-adrenergic receptor (β2AR), a protective receptor against airway inflammation and fibrosis and the only AR subtype expressed in human bronchial cells. Osteopontin (OPN), a pro-inflammatory and pro-fibrotic cytokine, is known to oppose cardiac β2AR’s antifibrotic signaling, and therefore may provoke β2AR desensitization in bronchial cells.
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 GRK-5, potentially at Ser 601 and Ser 843 residues, which provides protection against aldosterone-induced adverse effects. Hence, the phosphorylation by GRK-5 on the MR is a primary focus of our study, as this mechanism suppresses MR function.
The study involves isolating the MR from a human tissue sample by co-immunoprecipitation, digesting the MR with trypsin protease, purifying the resulting peptide fragments through liquid chromatography, and characterizing the peptide fragments by MALDI-TOF mass spectrometry to determine the protein’s phosphorylation sites. Phospho-mapping of the fragments will confirm if GRK-5 phosphorylates the MR at the expected Ser 601 and Ser 843 residues.
