Molecular cloning and characterization of the four rat prostaglandin E2 prostanoid receptor subtypes
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, EP3α 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.
Section snippets
Chemicals
Prostaglandin E2, prostaglandin D2, prostaglandin F2α, prostaglandin E1, 17-phenyl-ω-trinor-prostaglandin E2, 11-deoxy-prostaglandin E1, 19(R)-OH-prostaglandin E2, AH23848; [1α(Z),2β,5α]-(±)-7-[5-[[(1,1′-biphenyl)-4-yl]methoxy]-2-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoic acid: AH6809; 6-isopropoxy-9-oxoxanthene-2-carboxylic acid: butaprost; 3-hydroxy-2-[4-hydroxy-4-(1-propylcyclobutyl)-1-butenyl]-5-oxo-methyl ester: enprostil; (dl)-9-keto-11α,15α
Rat prostanoid EP1 receptor cloning
To construct the open reading frame (ORF) of the rat prostanoid EP1 receptor two clones were used, the 5′ clone (clone 8-2) derived from a spleen cDNA library and the 3′ clone (clone 9-1) derived from a kidney cDNA library, with both the 5′ and the 3′ introns removed (see Section 2for details). The ORF is 1215 nt and encodes for a 405 amino acid protein with a predicted relative molecular mass of 43,053 Da. There are 3 potential N-glycosylation sites (Asn 7, 24 and 34), 6 potential protein
Discussion
In the present study all four rat prostanoid EP receptor subtypes have been cloned and characterized with respect to both radioligand binding and functional properties using the same heterologous expression system. Northern blot analysis has been used to examine tissue distribution of prostanoid receptor subtypes EP1, EP2 and EP3α.
Comparison of the deduced amino acid sequences of EP receptor homologues from rat, human and mouse (see Fig. 1) revealed that, in general, rat receptors are more
Acknowledgements
The authors wish to thank Nathalie Ouimet, Helene Juteau, Michel Belley, Yves Gareau, Michel Gallant and Claude Dufresne from the Department of Medicinal Chemistry, Merck Frosst Center for Therapeutic Research for the synthesis of SC19220, butaprost, M&B28767, enprostil and AH23848.
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Both authors contributed equally to this publication and are listed in alphabetical order.