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European Journal of Pharmacology 340 1997 227–241
Molecular cloning and characterization of the four rat prostaglandin
E prostanoid receptor subtypes
2
Yves Boie
a,1
, Rino Stocco
a,1
, Nicole Sawyer
a
, Deborah M. Slipetz
a
, Mark D. Ungrin
a
,
Frank Neuschafer-Rube
b
, Gerhard P. Puschel
b
, Kathleen M. Metters
a
, Mark Abramovitz
a,)
¨¨
a
Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, P.O. Box 1005, Pointe Claire-DorÕal,
Que., Canada H9R 4P8
b
Institut fur Biochemie und Molekulare Zellbiologie, Humboldtallee 23, Georg-August-UniÕersitat, Gottingen, Germany
Received 24 July 1997; revised 26 September 1997; accepted 30 September 1997
Abstract
We have characterized the rat prostanoid EP , EP , EP and EP receptor subtypes cloned from spleen, hepatocyte andror kidney
123
a
4
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
Ž.
performed on EP receptor expressing human embryonic kidney HEK 293 cells. The K values obtained with prostaglandin E for the
D2
prostanoid receptor subtypes EP , EP , EP and EP were approximately 24, 5, 1 and 1 nM, respectively. The rank order of affinities for
123
a
4
various prostanoids at the prostanoid receptor subtypes EP , EP and EP receptor subtypes was prostaglandin E sprostaglandin
23
a
42
E )iloprost )prostaglandin F )prostaglandin D )U46619. The rank order at the prostanoid EP receptor was essentially the same
12
a
21
except that iloprost had the highest affinity of the prostanoids tested. Of the selective ligands, butaprost was selective for prostanoid EP ,
2
M&B28767 and sulprostone were selective for EP and enprostil displayed dual selectivity, interacting with both prostanoid receptor
3
a
subtypes EP and EP . All four receptors coupled to their predominant signal transduction pathways in HEK 293 cells. Notably, using a
13
a
novel aequorin luminescence assay to monitor prostanoid EP mediated increases in intracellular calcium, both iloprost and sulprostone
1
were identified as partial agonists. Finally, by Northern blot analysis EP transcripts were most abundant in liver and kidney whereas
3
prostanoid EP receptor mRNA was expressed in spleen, lung and testis and prostanoid EP receptor mRNA transcripts were
2 1
predominantly expressed in the kidney. The rat prostanoid EP probes also detected additional and abundant transcripts present in all the
1
tissues examined. These were found to be related to the expression of a novel protein kinase gene and not the prostanoid EP gene
1
w
Batshake, B., Sundelin, J., 1996. The mouse genes for the EP prostanoid receptor and the novel protein kinase overlap. Biochem.
1
x
Biophys. Res. Commun. 227, 1329–1333 . q 1997 Elsevier Science B.V.
Keywords: Prostanoid; HEK 293; Aequorin
1. Introduction
Prostaglandin E is thought to be an important mediator
2
of the inflammation and pain responses as demonstrated in
Ž
a number of in vitro and in vivo models Coleman et al.,
.
1989 and references within . In particular, a recent study
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by Mnich et al. 1995 showed that neutralizing mono-
clonal antibodies to prostaglandin E were effective in an
2
)
Corresponding author. Tel.: q1-514-4288525; fax: q1-514-4288615;
e-mail: mark_abramovitz@merck.com.
1
Both authors contributed equally to this publication and are listed in
alphabetical order.
in vivo mouse model of nociception, implicating prosta-
glandin E as the major prostanoid mediating the pain
2
response.
The physiological and pathophysiological actions of
prostaglandin E are mediated through interaction with
2
2
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specific G-protein-coupled EP receptors Coleman et al.,
.
1989; Davies and MacIntyre, 1992 which belong to the
superfamily of G-protein coupled receptors. There are four
2
Prostanoid receptors are designated following the recommendation of
the IUPHAR Commission on Receptor Nomenclature and Classification
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Watson and Girdlestone, 1993 .
0014-2999r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved.
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PII S0014-2999 97 01383-6