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Nitinkumar L. Patel, Stephen G. Schwartz, M. Elizabeth Fini; Characterization of a Novel Orphan G-Protein Coupled Receptor Expressed in Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4628.
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© ARVO (1962-2015); The Authors (2016-present)
GPR158, a novel orphan G-protein coupled receptor of the glutamate clan, has been associated with Alzheimer’s disease and prostate cancer. It is expressed at high levels in the brain and at low levels in other tissues, including the choroid of the eye and the retinal pigment epithelium (NEI website). The function is completely unknown. In this study, we investigated molecular properties of GPR158, as well as its cellular behaviors.
GPR158 was evaluated in the prostate cancer cell line PC-3 (a gift from Dr. Gerhard Coetzee) and the trabecular meshwork cell line TM-1 (a gift from Dr. Donna Peters). GPR158 expression was quantified using qRT-PCR and western blotting. The functional role of glucocorticoid responsive elements (GRE) in the GPR158 promoter (-1053/+25bp, from transcription start site cloned in pGL3 vector) was studied using transient transfection reporter assays. A full-length GPR158 cDNA clone was developed from PC-3 mRNA. This was used to construct a GPR158-Green Fluorescent Protein (GFP) fusion gene to study sub-cellular localization of GPR158 protein.
GPR158 mRNA was expressed in both PC-3 and TM-1. In silico analysis of the 5’-UTR of the GPR158 gene revealed the presence of three glucocorticoid responsive elements (GREs). Reporter assays revealed that GC increased GPR158 mRNA expression through transcriptional activation of its promoter by binding of GRα with GRE. Treatment with 0.5 µM dexamethasone or triamcinolone led to a 4-5-fold increase in GPR158 mRNA expression, with PC-3 cells showing rapid induction in 2-6 hours, and TM-1 cells exhibiting a slow accumulation over 2-3 days. GPR158-GFP fusion protein was predominantly localized in punctuate structures around the perinuclear region of TM-1 cells. We observed a dramatic 4-fold increase in TM-1 cell proliferation upon transient over-expression of GPR158 protein.
We have identified GPR158 as a glucocorticoid-inducible gene expressed in trabecular meshwork cells, suggesting a possible connection with steroid-induced glaucoma. GPR158 appears to shuttle between cytosol and nucleus, and its over-expression stimulates cell proliferation. Further studies will focus on determining whether the protein integrates into the plasma membrane, and on identifying endogenous ligands.
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