Identification of a prostaglandin E2 receptor splice variant and its expression in rat tissues

Prostaglandins & other Lipid Mediators 63 (2001) 165–173

Identification of a prostaglandin E2 receptor splice variant and its expression in rat tissues Sue Oldfielda, Blair D. Grubbb, Lucy F. Donaldsona,* a

Department of Physiology, University of Bristol, Bristol, UK Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, UK

b

Received 20 October 2000; accepted 28 November 2000

Abstract The intercellular signalling actions of the lipid mediators, the eicosanoids, are transduced by a family of seven transmembrane domain receptors. Members of this receptor family with high affinity for PGE2 are termed EP receptors. There are four known EP receptor genes that are transcribed to generate EP1, EP2, EP3 and EP4 receptors. Two of these receptor transcripts, EP1 and EP3, are further modified by RNA splicing to give multiple receptor isoforms. The EP3 receptor is known to have multiple splice variants in human (9 variants), cow (4 variants), mouse (3 variants) and rat (3 variants). In the rat the three EP3 splice variants differ in the sequence of the intracellular C-terminus. We have identified a fourth splice variant of the rat prostaglandin EP3 receptor that has a greatly truncated intracellular C-terminus when compared to the other EP3 receptor isoforms. Using nested RT-PCR we have shown that this novel splice variant is strongly expressed in rat brain and is also found in spinal cord, kidney and spleen. © 2001 Elsevier Science Inc. All rights reserved. Keywords: prostaglandin receptor; EP3; RT-PCR; rat; kidney; brain, spinal cord

1. Introduction The prostaglandins are metabolites of arachidonic acid, formed by the action of prostaglandin G/H synthase (also known as cyclooxygenase; COX) on free arachidonic acid. The prostaglandins, and other arachidonic acid metabolites are known to exert effects as intracellular signalling molecules [1], but importantly also as intercellular signalling molecules

* Corresponding author. Tel.: ⫹44-117-954-6989; fax: ⫹44-117-928-8923. E-mail address: [email protected] (L.F. Donaldson). 0090-6980/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 0 - 6 9 8 0 ( 0 0 ) 0 0 1 0 4 - 0

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through actions on a family of G-protein coupled receptors [2]. Prostaglandin E2, a prostaglandin important in control of gastric secretion [3], inflammatory processes [4] and nociception [5–7], has high affinity for a family of receptors termed the EP receptors [2]. These receptors were originally characterised on the basis of agonist potency, and three EP receptors defined by functional responses of different tissues [8], for example, ileal constriction is mediated through EP1 receptors, relaxation of tracheal smooth muscle through EP2 receptors and the inhibition of gastric acid secretion through EP3 receptors [2]. With the advent of cloning techniques, four EP receptor genes have been identified in many species, encoding the EP1, EP2, EP3 and EP4 receptors. The EP4 receptor has a similar pharmacological profile to the EP2 receptor [2]. Like many receptors, some of the EP receptor genes have been shown to encode multiple receptors generated through alternative mRNA splicing. The EP1 receptor has two splice variants, one of which has a truncated intracellular tail which does not couple to any signal transduction cascade. This EP1 receptor isoform inhibits signalling at co-expressed EP2 or EP4 receptors, possibly by acting as an endogenous dominant negative receptor [9]. The EP3 receptor gene is also known to generate multiple RNA splice variants in several species. In humans, at least 9 messenger RNA species have been identified, encoding 8 functional EP3 receptor isoforms [10,11], the cow has four known splice variants of the receptor [12], and the rat and mouse have three identified splice variants [13–16]. In the rat two of the splice variants, the EP3A and EP3B receptors, are known to be colocalized in kidney but couple to different signal transduction pathways and subserve different physiological functions [13–16]. The nomenclature used for the three rat EP3 receptor variants in the literature has not yet been formalised; herein we will refer to the known variants as EP3A/␣ [13,17], EP3␤ [17] and EP3B [13] as they are termed in the original publications.

2. Experimental methods RNA extraction Rats were killed by cervical dislocation. Tissues were rapidly removed and frozen on dry ice. Tissue was homogenised in 1ml TriZol (Life Technologies) and total RNA extracted according to the manufacturers instructions. RNA was electrophoresed on agarose gels to confirm integrity and remaining RNA solutions were stored at ⫺80°C until used for reverse transcription. 2.1. Cloning of the novel EP3 receptor splice variant in rat The novel EP3 receptor splice variant was cloned using primers designed against the published rat sequences for the EP3A/␣ and EP3␤ receptors and the polymerase chain reaction (PCR). Primer sequences are given in Table 1. Initially primers were designed against common sequences in the two receptors (EP3-1025 and EP3-1285; Figure 1) to discriminate between the two known splice variants on amplicon size in order to clone these fragments. Rat kidney was dissected and rapidly frozen on dry ice. Total RNA was extracted as described above and reverse transcribed using an oligo dT⫺ primer and MMLV reverse

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Table 1 Primers used in RT-PCR for rat EP3 receptor splice variants Primer

Sequence (5⬘-3⬘)

EP3-840 EP3-1025 EP3-16R2 EP3-1285

5⬘-ATCATGTGTGTACTGTCC-3⬘ 5⬘-GGAATTCTGGATCCCTGGGTTTATCTG-3⬘ 5⬘-CATCATCTGTTAAAACAG-3⬘ 5⬘-GCTCTAGAGTCTCGGTGTGTTTAATGGC-3⬘

Underlined sequences represent restriction enzyme recognition sequences used in the cloning of the EP3A/␣ and EP3␤ PCR fragments (EP3-1025, BamHI; EP3-1285, XbaI).

transcriptase (ABGene) at 37°C for 1 hour. Reverse transcribed RNA was then subject to PCR for the EP3A/␣ and EP3␤ receptors using the primers EP3-1025 and EP3-1285 (Table 1 and Figure 1) under the following amplification conditions; denaturation 94°C, 1 minute, annealing 56°C 1 minutes, extension 72°C 2 minutes for 35 cycles, using Thermoprime DNA polymerase (ABGene). These primers amplified fragments of 227bp and 316bp from reverse transcribed rat kidney RNA as predicted (Figure 2A). The fragments were cloned into pBluescript II KS (Stratagene) and sequenced to confirm identity. One clone isolated in this procedure contained a fragment of DNA which differed in sequence from that expected. This novel sequence is shown in Figure 1 aligned with the other EP3 receptor splice variants and is called the EP3D receptor. Analysis of the sequence of this fragment showed that if translated it could yield a functional EP3 receptor. Further investigation of the tissue expression of this novel splice variant was then performed. 2.2. Reverse transcription polymerase chain reaction (RT-PCR) Tissues were isolated from different rats to that from which the novel splice variant had been cloned, frozen on dry ice and stored at ⫺80°C until used for RNA extraction. Total RNA was extracted and reverse transcribed as described above. A specific EP3D receptor primer, EP3-16R2 was designed against the unique sequence in the EP3D receptor, as shown in Figure 1A and Table 1. Expression of the EP3D receptor was investigated using rat kidney RNA and the EP3-840 and EP3-16R primers. The resulting DNA fragment of the expected size (240bp, Figure 2B) was isolated and sequenced. This primer pair, however, generated multiple competing single primer artefacts (Figure 2B). The approach for the investigation of EP3D receptor expression in multiple tissues was therefore modified. A nested PCR reaction was performed. First round amplification using EP3-840 and EP3-1285 primers was performed under the following conditions; 55°C annealing, 30 seconds; 72°C extension, 90 seconds and 94°C denaturation, 30 seconds for 30 cycles. One twentieth of the reaction was then used as template for the PCR reaction using the nested primers EP3-1025 and EP3-16R2 under the following conditions; 50°C annealing, 60 seconds; 72°C extension, 60 seconds and 94°C denaturation, 60 seconds for 30 cycles. This approach also resulted in non-specific amplification, and was therefore further modified to improve specificity. The remainder of the first amplification reaction was electrophoresed on an agarose gel and the DNA fragment representing the EP3A/␣ receptor (Figure 2C) isolated and gel purified from those lanes where this amplicon was evident. The gel purified EP3A/␣ fragment was then

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Fig. 1. Sequence alignments of the rat EP3 receptor splice variants. Alignments of the nucleotide sequences of the four rat EP3 receptor splice variants showing the annealing positions of the primers used in the RT-PCR analysis of EP3D receptor expression. The areas of black shading show identical nucleotides in two or more sequences and the stop codons used by each receptor splice variant are highlighted in grey. The vertical arrowhead indicates the splice site. Alignments were generated using BOXSHADE software version 3.21 (http://www.ch.embnet.org/software/Box_form.html).

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Fig. 2. Expression of the EP3 receptor isoforms in rat tissues. All the gels are negative images of ethidium bromide stained agarose gels. A) Amplification of the EP3A/␣ (316bp) and EP3␤ (227bp) DNA fragments (lane 2, arrows) from rat kidney with the EP3-1025 and EP3-1285 primers. Lane 1 shows DNA markers of sizes 100 –1000bp at 100bp intervals. B) Amplification of the EP3D splice variant from rat kidney using the EP3-840 and EP3-16R2 primers. The markers in lane 1 are as in A; the arrow indicates the EP3D band of the expected length of 240bp that was isolated and sequenced to confirm expression of this splice variant. C) Expression of EP3D receptor in rat tissues. Gel shows the products of the second round of nested PCR using EP3-1025 and EP3-16R2 primers to give an amplicon of 84bp. The DNA band is only seen in rat brain (arrow, lane 8) and not in any other tissue. (lane 1, markers; lane 2, lung; lane 3, liver; lane 4, kidney; lane 5, testis; lane 6, spinal cord; lane 7, heart; lane 8, brain; lane 9, spleen; lane 10 negative water control; lane 11 DNA markers). D) First round PCR with EP3-840 and EP3-1285 primers prior to gel purification of the EP3A/a amplicon (arrow) from spinal cord, spleen, brain and kidney. The different tissues are run in the same lanes as shown in C. E) EP3D 84bp amplicon amplified from the DNA bands indicated in D, showing expression in all four tissues. Lane 1, DNA markers; lanes 2 and 3; cloned EP3A/␣ and EP3B cDNAs (negative controls); lane 4, spleen; lane 5, kidney; lane 6, spinal cord; lane 7, brain; lane 8 DNA markers.

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used as the template for the nested PCR reaction using the EP3-1025 and EP3-16R2 primers. This strategy was adopted as in previous experiments we had found that the PCR DNA fragments amplified with the EP3-1025 and EP3-1285 primers could consist of heteroduplexes of different DNA strands from the EP3A/␣, EP3␤ or EP3D receptors in any combination. A negative water control was included in all PCR reactions to control for nucleic acid contamination of solutions. As all the primer pairs were designed across a splice site, amplicons of the correct length could not be amplified from potentially contaminating genomic DNA. In the second round of the nested PCR reactions on gel isolated PCR fragments, the cloned EP3A/␣ and EP3B receptors were also included as negative controls.

3. Results and discussion Comparison of the published sequences for the rat EP3 receptors using available databases showed that although the literature terminology suggests there are two EP3 receptor splice variants in the rat (EP3A and EP3B), there are in fact three, EP3A/␣, EP3␤ and EP3B. Alignment of the nucleic acid sequences of these receptor isoforms shows that the sequences are identical except for the region coding for the intracellular C-terminal tail, and the 3⬘ untranslated regions (Figure 1). Takeuchi and colleagues have shown that the two EP3 isoforms first identified, EP3A and EP3B are different in this region due to mRNA splicing at the point indicated by the vertical arrowhead in Figure 1. Alignment of the sequences for the other EP3 splice variant shows that the EP3␤ receptor is also identical to the EP3A and EP3B receptors up to this point but splicing results in the removal of a region of coding region approximately 100bp in length that is present in the EP3A/␣ receptor. The EP3␤ receptor therefore uses a different stop codon (indicated in grey in Figure 1), and thus has a different amino acid sequence to the EP3A/␣ receptor (Figure 3). While subcloning fragments of the rat EP3A/␣ and EP3␤ splice variants for other purposes, we identified a clone that had a completely different sequence to the known EP3 receptor isoforms (shown in Figure 1 as EP3D). Examination of this sequence showed a strong similarity to the EP3A/␣ receptor, but without the same 100bp region that is absent in the EP3␤ receptor. Inclusion of a small amount of additional sequence in the EP3D isoform, not present in either of the other receptors results in the EP3D receptor using the same stop codon as the EP3A/␣ receptor. This 18 base pairs of sequence has high identity with the terminal region of the third intron of the human EP3 receptor [11], at the equivalent splice point. This suggests that that missplicing might result in the inclusion of a small amount of intronic DNA resulting in a novel receptor splice variant. Comparison of the translated sequences in the four EP3 receptor isoforms (Figure 3) shows that the EP3D receptor could be translated and would have a greatly truncated intracellular C-terminus when compared to the other EP3 receptor isoforms. In order to verify that the clone we had identified was expressed in rat tissues we used RT-PCR to investigate the expression of this mRNA in total RNA extracted from rat kidney, as alternative approaches could not distinguish between the different splice variants, due to the extreme similarity of sequence. Using primers EP3-840 and EP3-16R2 the reaction resulted in the amplification of a 250bp product which proved to be the splice variant on

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Fig. 3. Predicted amino acid structures of the four rat EP3 receptor splice variants. The body of the receptor is common to all four splice variants. The difference between the splice variants is in the length and amino acid content of the intracellular tail, as illustrated. Amino acids that are identical in all four splice variant tails are shaded grey. Serine phosphorylation sites are indicated by the solid black circled “P” and a putative site of N-linked glycosylation is indicated by the small circle structure [16].

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sequence analysis (arrow Figure 2B). Thus the EP3D splice variant is expressed in rat kidney. Amplification with this primer pair did not however give reliable results due to the co-amplification of single EP3-16R2 primer products. We therefore used a nested PCR approach to investigate expression of the RNA for this splice variant in multiple rat tissues. Initial nested PCR with first round amplification with EP3-840 and EP3-1285 primers followed by a second round amplification of one twentieth of the first round reaction with EP3-1025 and EP3-16R2 primers showed clear expression of the splice variant amplicon (84bp) in rat brain, but in no other tissues (arrow Figure 2C). As we had previously cloned the EP3D receptor from rat kidney, and confirmed expression in the kidney from another animal we modified the nested PCR approach further to improve sensitivity. We had found during the original cloning that the EP3-1025 and EP3-1285 primer pair amplified a doublet as predicted, but on sequencing these DNA fragments could consist of heteroduplexes of two different strands i.e. EP3A/␣ hybridised to EP3␤. Under certain electrophoresis conditions (low melting agarose gels) a third intermediate amplicon could be seen that on bi-directional sequencing was shown to be such a heteroduplex (not shown). We therefore hypothesized that the EP3D isoform could dimerise with the EP3A/␣ or EP3␤ strands in a similar manner. Gel electrophoresis of the first round amplification with the EP3-840 and EP3-1285 primers showed a clear doublet in brain and spleen, with amplicons also present at much lower levels in spleen and spinal cord (Figure 2D). The EP3 receptor doublet was not evident in the other tissues. Gel purification of the EP3A/␣ amplicon from brain (lane 7), spleen (lane 4), spinal cord (lane 6) and kidney (lane 5) followed by nested PCR on the purified DNA resulted in the clear amplification of the EP3D 84bp fragment (Figure 2E). The cloned EP3A/␣ and EP3B receptor cDNAs were included in the second round PCR reaction (lanes 2 and 3) to show that the EP3-1025 and EP3-16R2 did not amplify the 84bp fragment from cDNAs verified by sequencing as being single cDNA clones. The 84bp fragment was not evident in these reactions, indicating that the first round PCR amplicons did indeed consist of heteroduplexes of the EP3A/␣, EP3␤ and EP3D receptors, possibly explaining why this receptor isoform has not been previously described. In conclusion we have cloned a new EP3 receptor splice variant from rat kidney and shown that it is also expressed in rat brain, spinal cord, and spleen. The EP3D receptor is expressed in the rat central nervous system and other tissues along with the EP3A/␣, and EP3␤ receptor isoforms and may therefore mediate or modulate some of the physiological actions of prostaglandins in these tissues. Other truncated seven transmembrane domain receptor isoforms in the prostaglandin family have been shown to inhibit signalling through endogenous EP receptors; the signal transduction cascade through which the EP3D receptor signals remains to be determined.

Acknowledgments We gratefully acknowledge the support of the Arthritis Research Campaign, Grant No. D0546. The sequence described in this manuscript has been submitted to Genbank with the following accession number: AF302686.

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