Abstract: SA-PO355
G Protein-Coupled Receptor 37L1 Is Expressed on the Nuclear Envelope
Session Information
- Hypertension and CVD: Mechanisms
November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Hypertension and CVD
- 1403 Hypertension and CVD: Mechanisms
Authors
- Konkalmatt, Prasad, George Washington University, Washington, District of Columbia, United States
- Armando, Ines, George Washington University, Washington, District of Columbia, United States
- Jose, Pedro A., George Washington University School of Medicine, Washington, District of Columbia, United States
Background
G protein-coupled receptors (GPCRs), expressed on the plasma membrane, interact with various types of ligands, which trigger a cascade of signal transduction events leading to different intracellular responses that in turn manifest in physiological changes. Some GPCRs also reside and exert signals from intracellular organelles such as the nucleus, endoplasmic reticulum, and Golgi apparatus. Recently, we reported that G protein-coupled receptor 37L1 (GPR37L1) is expressed in the apical membrane of renal proximal tubule cells (RPTCs) and participates in luminal sodium transport and blood pressure regulation by regulating the renal expression of NHE3. However, the mechanism by which GPR37L1 regulates NHE3 expression and function in the RPTC has not been studied.
Methods
We employed Tandem affinity purification using GPR37L1 tagged with streptavidin and calmodulin binding peptides, followed by mass spectrophotometry (MS) analyses to identify the proteins interacts with GPR37L1. Subcellular location of GPR37L1 was determined by confocal fluorescence imaging and immunoblotting on the cells or the nucleus prepared from the RPTCs expressing GPR37L1 tagged with green fluorescence protein (GPR37L1-GFP).
Results
Tandem affinity purification of GPR37L1, combined with MS analyses, revealed the association of GPR37L1 with mediators of nuclear importing proteins, such as RAN-GTPase, importin-5, and importin-7. In silico analyses of GPR37L1 amino acid sequence revealed the presence of a potential nuclear localization signal at the N-terminus. Confocal fluorescence imaging of RPTCs expressing GPR37L1-GFP showed distinct nuclear membrane expression of GPR37L1. In addition, fluorescence imaging of nuclei isolated from the RPTCs expressing GPR37L1-GFP showed the expression on the nuclear envelope. Immunoblot analyses confirmed the presence of GPR37L1 in the nuclear protein prepared from the RPTCs expressing GPR37L1-GFP. The purity of the nuclear protein preparations was confirmed by the presence of histone deacetylase 2, a marker for nuclear protein, the absence of Na,K-ATPase, a marker for plasma membrane, and the absence of calnexin, a marker for endoplasmic reticulum.
Conclusion
Our results show that GPR37L1 also resides on the nuclear envelope and may play a critical role in the regulation of the expression of genes responsible for maintaining normal electrolyte balance and blood pressure.
Funding
- NIDDK Support