IDENTIFICATION OF A NOVEL ALTERNATIVE SPLICING OF RGS5 mRNA IN HUMAN OCULAR TISSUES

Abstract

Abstract: : Purpose: RGS proteins (regulator of G protein signaling) act as GTPase–activating proteins (GAPs) for Gα subunits and negatively regulate G protein–coupled receptor signaling. These studies were performed to investigate the molecular mechanisms of RGS5 and its alternative splicing variant (RGS5s) in the regulation of G protein and G protein coupled receptors. Methods:Total RNA was isolated from human ocular tissues, primary human ciliary smooth muscle and trabecular meshwork cells. RT–PCR was performed to detect alternatively spliced variants of RGS5 mRNA. Full length of wild type RGS5 and RGS5 variants were cloned into pcDNA3 expression vector, the functional studies were performed using FLIPR and MAP kinase assays Results:This study is the first to document alternative splicing of an RGS5 gene. We therefore studied RGS5 and RGS5s mRNA distribution in human tissues. In the eye, RGS5s was found to be highly expressed in the ciliary body and trabecular meshwork (TM). It was also expressed in the kidney, brain, spleen, skeletal muscle and small intestine, but was not detectable in the liver, lung, heart. RGS5s was not found in monkey and rat ocular tissues, indicating species specificity for the eye. Comparing the recombinant RGS5 and RGS5s expression in HEK 293/EBNA cells, RGS5s was present almost exclusively in the cytosolic fraction, whereas RGS5 was present in both membrane and cytosolic fractions. The data suggest that the N–terminal of RGS5 may be important for protein translocation to the cell membrane. Both RGS5 and RGS5s antagonized the rapid phosphorylation of p44/42 MAP kinase induced by Gαi coupled cannibinoid receptor–1 (CB–1) activation. RGS5, but not RGS5s, inhibited the Ca²⁺ signaling initiated by activation of Gαq coupled angiotensin II receptors (AT1) and prostaglandin FP receptors. Cotransfection of RGS5s with RGS5 resulted in the blockade of RGS5 actions with respect to inhibition of the signal transduction initiated by activation of both AT1 and FP receptor, suggesting that RGS5s may contain functional domains that compete with RGS5 in the regulation of the Gαq coupled AT1 and FP receptors. Conclusions:The unique expression pattern, cellular localization and functions of RGS5s suggest that RGS5s may play a critical role in the regulation of intracellular signaling pathways.

Keywords: gene/expression • pharmacology • intraocular pressure