Localization of Human Prostaglandin E2 Receptors in Polarized Epithelial Cells

Abstract

The underlying mechanisms of protein sorting in polarized epithelial cells are poorly understood. Several studies have determined membrane targeting of G protein-coupled receptors (GPCRs) using epithelial cells such as Madin-Darby canine kidney (MDCK) cells. Polarized epithelial cells are composed of apical and basolateral plasma membrane domains with specific protein compositions separated by tight junctions. Purinergic, muscarinic, and adrenergic receptors are a few examples of GPCRs that have been shown to localize to specific membranes in MDCK cells. The current work seeks to determine the differences in subcellular localization of the human prostaglandin E2 receptors. The EP receptors are all GPCRs, which differ in their second messenger pathways. The EP3 receptor is unique in that it has eight different isoforms that differ in the lengths of the carboxyl tail. The EP3 isoforms, as well as the EP2 and EP4 receptors, have distinct properties, including different agonist-induced internalization patterns. We have also shown the EP3 isoforms have tissue-specific distribution patterns. To further study the differences among the PGE2 receptors, we examined their subcellular localization patterns in polarized epithelial cells. We have determined the unique subcellular localization patterns for the receptors, as well as three mutants in MDCK cells. The localization patterns for these receptors in human bronchial epithelial (BEAS-2B) cells were quite similar to the MDCK cells, suggesting that receptor localization is not dependent on cell type. Additionally, in an attempt to locate structural motifs responsible for apical or basolateral localization, receptor chimeras between the purinergic P2Y2/P2Y4 receptors and EP3.VI isoform were constructed. Overall, the aim of our work was to define the subcellular localization patterns of various human prostaglandin E2 receptors and our results suggest the differences among them may correlate to the diverse physiological actions of PGE2 throughout the body.