The utilization of recombinant prostanoid receptors to determine the affinities and selectivities of prostaglandins and related analogs

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Abstract

Stable cell lines that individually express the eight known human prostanoid receptors (EP1, EP2, EP3, EP4, DP, FP, IP and TP) have been established using human embryonic kidney (HEK) 293(EBNA) cells. These recombinant cell lines have been employed in radioligand binding assays to determine the equilibrium inhibitor constants of known prostanoid receptor ligands at these eight receptors. This has allowed, for the first time, an assessment of the affinity and selectivity of several novel compounds at the individual human prostanoid receptors. This information should facilitate interpretation of pharmacological studies that employ these ligands as tools to study human tissues and cell lines and should, therefore, result in a greater understanding of prostanoid receptor biology.

Introduction

Prostanoids provoke diverse biological actions in many tissues and cell types through interaction with G-protein-coupled prostanoid receptors. The cloning of eight human prostanoid receptors has now been described: EP1 [1], EP2 [2], EP3 [3], EP4 [4], DP [5], FP [6], IP [7] and TP [8]. In addition, isoforms have been identified in human for EP3 (six isoforms) [9] and TP (two isoforms) [10] which are generated by alternative splicing at the mRNA level and vary in the length and amino acid composition of the carboxyl-terminal tails. These represent all of the prostanoid receptors for which there is compelling pharmacological evidence.

Elucidating the particular receptor(s) involved in prostanoid-mediated events has been complicated by several factors, which have made simple ligand:receptor structure–activity relationships difficult to determine. (1) Although prostanoids exhibit a degree of receptor selectivity, i.e. the EP subtypes interact preferentially with prostaglandin (PG)E2, DP with PGD2, FP with PGF, IP with prostacyclin (PGI2) and TP with thromboxane (TX)A2, these ligands are promiscuous and cross-react to an extent with each of the receptors. (2) This cross-reactivity extends to the second messenger pathways, i.e. EP1, FP and TP signal through mobilization of calcium, EP2, EP4, DP, and IP through stimulation of adenylyl cyclase and EP3 through inhibition of adenylyl cyclase. (3) Many tissues and cell types express heterogeneous receptor populations. (4) There is a limited range of selective agonists and, in particular, antagonists available for most of the prostanoid receptors. (5) Although the ligand binding affinities of the eight receptors have recently been determined for a variety of ligands in mouse [11], accurate affinities for the ligands are, as yet, not available for other species, including, most importantly, human.

In order to resolve some of these problems we have established stable human embryonic kidney (HEK) 293(EBNA) cell lines (HEK cells) individually expressing recombinant human prostanoid receptors. We have used EP3-III [3] and the placental form of the TP receptor [8]. These cell lines have been used to determine the affinities, and therefore the selectivities, of known prostanoid-receptor ligands.

Section snippets

Stable expression of prostanoid receptors in the human embryonic kidney (HEK) 293(EBNA) cell line

Prostanoid receptor (PG) cDNAs corresponding to full-length coding sequences were subcloned into the appropriate sites of the mammalian expression vector pCEP4 (Invitrogen). pCEP4PG plasmid DNA was prepared using the Qiagen plasmid preparation kit (Qiagen) and transfected into HEK cells using Lipofectamine (Gibco-BRL) according to the manufacturer’s instructions. HEK cells expressing the cDNA together with the hygromycin resistance gene were selected in Dulbecco’s modified Eagle’s medium (DMEM)

Radioligand binding assay conditions

The radioligand binding assay conditions used for each prostanoid receptor were optimized with respect to cation (Ca2+, Mg2+ or Mn2+), cation concentration (0.3–100 mM), pH (5–8) and GTPγS concentration (1–1000 μM). For each receptor, the appropriate conditions were chosen to maximize the affinity of the specific binding of the radioligand. For equilibrium competition assays, the radioligand concentration was chosen to give 20 000 cpm per incubation and the membrane protein concentrations were

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