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. 1994 Jun;112(2):377–385. doi: 10.1111/j.1476-5381.1994.tb13082.x

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

JW Regan, TJ Bailey, JE Donello, KL Pierce, DJ Pepperl, D Zhang, KM Kedzie, CE Fairbairn, AM Bogardus, DF Woodward, DW Gil
PMCID: PMC1910333  PMID: 8075855

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-1 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.

Keywords: Prostaglandin, small intestine, cDNA, G-protein coupled, adenylyl cyclase, cyclic AMP inhibition, CAT assays

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Selected References

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