Molecular cloning and characterization of the four rat prostaglandin E₂ prostanoid receptor subtypes

Abstract

We have characterized the rat prostanoid EP₁, EP₂, EP_3α and EP₄ receptor subtypes cloned from spleen, hepatocyte and/or kidney cDNA libraries. Comparison of the deduced amino acid sequences of the rat EP receptors with their respective homologues from mouse and human showed 91% to 98% and 82% to 89% identity, respectively. Radioreceptor binding assays and functional assays were performed on EP receptor expressing human embryonic kidney (HEK) 293 cells. The K_D values obtained with prostaglandin E₂ for the prostanoid receptor subtypes EP₁, EP₂, EP_3α and EP₄ were approximately 24, 5, 1 and 1 nM, respectively. The rank order of affinities for various prostanoids at the prostanoid receptor subtypes EP₂, EP_3α and EP₄ receptor subtypes was prostaglandin E₂=prostaglandin E₁>iloprost>prostaglandin F_2α>prostaglandin D₂>U46619. The rank order at the prostanoid EP₁ receptor was essentially the same except that iloprost had the highest affinity of the prostanoids tested. Of the selective ligands, butaprost was selective for prostanoid EP₂, M&B28767 and sulprostone were selective for EP_3α and enprostil displayed dual selectivity, interacting with both prostanoid receptor subtypes EP₁ and EP_3α. All four receptors coupled to their predominant signal transduction pathways in HEK 293 cells. Notably, using a novel aequorin luminescence assay to monitor prostanoid EP₁ mediated increases in intracellular calcium, both iloprost and sulprostone were identified as partial agonists. Finally, by Northern blot analysis EP₃ transcripts were most abundant in liver and kidney whereas prostanoid EP₂ receptor mRNA was expressed in spleen, lung and testis and prostanoid EP₁ receptor mRNA transcripts were predominantly expressed in the kidney. The rat prostanoid EP₁ probes also detected additional and abundant transcripts present in all the tissues examined. These were found to be related to the expression of a novel protein kinase gene and not the prostanoid EP₁ gene [Batshake, B., Sundelin, J., 1996. The mouse genes for the EP₁ prostanoid receptor and the novel protein kinase overlap. Biochem. Biophys. Res. Commun. 227, 1329–1333].

Introduction

Prostaglandin E₂ is thought to be an important mediator of the inflammation and pain responses as demonstrated in a number of in vitro and in vivo models (Coleman et al., 1989and references within). In particular, a recent study by Mnich et al. (1995)showed that neutralizing monoclonal antibodies to prostaglandin E₂ were effective in an in vivo mouse model of nociception, implicating prostaglandin E₂ as the major prostanoid mediating the pain response.

The physiological and pathophysiological actions of prostaglandin E₂ are mediated through interaction with specific G-protein-coupled EP² receptors (Coleman et al., 1989; Davies and MacIntyre, 1992) which belong to the superfamily of G-protein coupled receptors. There are four subtypes of the prostaglandin E₂ receptor, EP₁, EP₂, EP₃ and EP₄, which have been cloned from both human (Funk et al., 1993; Adam et al., 1994; Regan et al., 1994; Bastien et al., 1994) and mouse (Sugimoto et al., 1992; Watabe et al., 1993; Honda et al., 1993; Katsuyama et al., 1995) and which couple to different major signal transduction pathways; namely elevation of intracellular Ca²⁺ (EP₁) and stimulation (EP₂, EP₄) or inhibition of adenylyl cyclase (EP₃). Rat prostanoid receptors cloned include the EP₁ (Okuda-Ashitaka et al., 1996), EP₃ (Takeuchi et al., 1993, Takeuchi et al., 1994; Neuschäfer-Rube et al., 1994) and EP₄ (Sando et al., 1994). In addition, several different isoforms of the EP₃ subtype have also been identified (Breyer et al., 1994a, Breyer et al., 1994b; Schmid et al., 1995), which are produced by alternative splicing and differ only in the length and amino acid composition of their carboxyl-terminal regions.

Rat in vivo models of inflammation and pain are widely used in pharmacology (Chau, 1989). In order to evaluate the true therapeutic utility of prostanoid EP receptor ligands in these models it is necessary to establish the relative potencies of these compounds at the four rat prostanoid receptor EP subtypes. This information can then be correlated with similar results using the human homologues.

We report here, therefore, the molecular cloning of the four rat prostaglandin E₂ receptor subtypes, EP₁, EP₂, EP_3α and EP₄. As a result the prostanoid receptor EP subtypes have been characterized in terms of radioligand binding and functional activities in assays performed under comparable experimental conditions. In addition, expression of prostanoid receptor subtypes, EP₁, EP₂ and EP₃, has been delineated by Northern blot analysis.