Molecular cloning and expression of human EP₃ receptors: evidence of three variants with differing carboxyl termini

Abstract

1 The polymerase chain reaction (PCR) was used in combination with plaque hybridization analysis to clone four variants of the EP3 prostaglandin receptor from a human small intestine cDNA library. 2 Three of these variants, i.e. the EP3A, EP3E and EP3D, share the same primary amino acid sequence except for their carboxyl termini, which diverge from one another at the same point, approximately 10 amino acids away from the end of the seventh membrane spanning domain of the receptor. The fourth variant (EP3A1) has a nucleotide coding sequence identical to EP3A but has a completely different 3' untranslated sequence. 3 The carboxyl termini of the three isoforms differ most obviously in length with the EP3A being the longest (41 amino acids) and the EP3E being the shortest (16 amino acids). They also differ in content with the EP3A containing 9 serine and threonines in its carboxyl terminus and the EP3E none. 4 Transient expression in eukaryotic cells showed that the human EP3 receptor variants had similar but not identical radioligand binding properties and differed in their functional coupling to second messenger pathways. Up to 3 pmol mg-' protein of [3H]-prostaglandin E2 binding could be obtained with more than 95% specific binding. Using a reporter gene assay, as a measure of intracellular cyclic AMP levels, the EP3A coupled more efficiently to the inhibition of adenylyl cyclase than did the EP3E. 5 PCR was used to confirm the presence of mRNAs encoding the four human EP3 receptor variants in tissues of the human small intestine, heart and pancreas. These findings indicate that the EP3 receptor variants identified here are likely to be expressed in tissues. The differences in the carboxyl termini at the protein level, and in the 3' untranslated regions at the mRNA level, could be profound in terms of the regulation and functional coupling of these receptor isoforms.

The mouse prostaglandin E receptor EP2 subtype: cloning, expression, and Northern blot analysis

“…The ATG assigned as the initiation codon matched well to the Kozak consensus sequence for translation initiation [17]. Analysis of the predicted amino acid sequence indicated that it possesses seven hydrophobic segments typical of guanine nucleotide-binding protein-coupled rhodopsin-type receptors, and is 86% identical to that of the human EP2 receptor [11]. Two potential N-glycosylation sites [18] were found in the N-terminal and the first extracellular loop regions.…”

“…Degenerate PCR primers, 5'-CTGGGATCCATGCTCATGCT(CG)TT(CT)GC(ACGT)ATGG-C-3' and 5'-GCAGAATTCAGACGGCGAA(CG)GT(AG)AT(GTF GTCAT-3', corresponding to the putative third and sixth transmembrane domains of the human EP 2 receptor [11] were synthesized. The thermal cycle program was 94°C for 0.8 min, 48°C for 1 min, 72°C for 3 min for the first 2 cycles, 94°C for 0.8 min, 50°C tbr 1 min, 72°C for 0014-5793/95/$9.50 ~ 1995 Federation of European Biochemical Societies.…”

“…Using these mouse clones, we characterized their structural, binding and signaling properties and tissue distribution [6,8 10]. Recently, Regan et al cloned a human PGE receptor, which is similar in its ligand binding properties to the pharmacologically defined EP 2 subtype [11]. However, tissue distribution study using the human clone has not been well established because of the restriction of available human tissues.…”

Exclusive expression of the Gs‐linked prostaglandin E2 receptor subtype 4 mRNA in human mononuclear Jurkat and KM‐3 cells and coexpression of subtype 4 and 2 mRNA in U‐937 cells

“…receptors A 930 bp RT-PCR fragment of the EP4 receptor [18] (primers EP4-3F and -2R) and a 1149 bp fragment of the EP2 receptor [19] (primers EP2-3F and -2R) were cloned into the Smal restriction site of pBlueScript. From the construct containing the EP4R insert, a 171 bp fragment was removed by restriction enzyme digestion with BssHlI (at positions 1037 and 1208, GenBank accession number L25124), followed by gel electrophoresis, extraction (Genomed, Bad Oeynhausen, Germany) and religation of the new construct.…”

“…Since the Gs-linked EP2 receptor has a lower affinity for PGE2 than the Gs-linked EP4 receptor, this would implicate a high number of the high affinity EP4 receptor (K,l 1-2 nM [18,23]) and a small number of the low affinity EP2 receptor (Kd 100 nM [19]). This contrasts with findings of a highly abundant low affinity (Kd 137 nM) and a rare high affinity (Kd 3.1 nM) PGEz receptor on U-937 cells [24].…”