Suppression of Prostaglandin E Receptor Signaling by the Variant Form of EP1 Subtype

Abstract

A cDNA clone of prostaglandin (PG) E receptor EP 1 subtype (rEP 1 ) was isolated from a rat uterus cDNA library. It encodes 405 amino acid residues with seven transmembrane-spanning domains and couples to Ca 2؉ mobilization. In addition, three cDNA clones encoding a variant form of rEP 1 were isolated. The open reading frame can code a 366-amino acid protein carrying a specific change of 49 amino acids from the middle of transmembrane segment VI to COOH terminus; it possesses a transmembrane segment VII-like structure lacking an intracellular COOH-terminal tail. Southern blot analysis of rat genomic DNA and genomic polymerase chain reaction demonstrated that these cDNAs were derived from a single copy gene. Northern blot analysis and ribonuclease protection assay revealed that both rEP 1 and rEP 1 -variant receptor mRNAs were highly expressed in the kidney. Immunoblot with an antibody directed toward the specific region of rEP 1 -variant receptor showed that rEP 1 -variant receptor protein was expressed in the membrane of the kidney and Chinese hamster ovary (CHO) cells transfected with rEP 1 -variant cDNA. Thus, the rEP 1 -variant receptor is translated from mRNA which is not spliced at nucleotide position 952 in the segment VI transmembrane region. rEP 1 -variant receptor retained the ligand binding activity with affinity and specificity similar to rEP 1 receptor, but lost the coupling of signal transduction systems by itself. However, when rEP 1 -variant receptor was stably co-expressed with rEP 1 receptor in CHO cells, the Ca 2؉ mobilization mediated by EP 1 receptor was significantly suppressed. Furthermore, when rEP 1 -variant receptor was expressed in CHO cells, cAMP formation by activation of endogenous EP 4 receptor was strongly blocked. These results suggest that the rEP 1 -variant receptor may affect the efficiency of signal coupling of PGE receptors and attenuate the action of PGE 2 on tissues. Prostaglandin (PG)1 E 2 is involved in a broad range of pathophysiological actions in diverse tissues through its binding to cell surface PGE receptor (1, 2). PGE receptors are pharmacologically classified into four subtypes, EP 1 , EP 2 , EP 3 , and EP 4 , and the diversity of PGE 2 actions is ascribed to PGE receptor subtypes coupled to different signal transduction pathways. The existence of PGE receptor subtypes has been given substantial support by cloning of cDNAs encoding these four PGE receptor subtypes from various sources and their characterization as GTP-binding protein-coupled receptors (3-6). In addition, several different isoforms of the PGE receptors have also been identified in the bovine, mouse, rabbit, and human EP 3 subtype (7-12). These isoforms, which are produced by alternative RNA splicing events and differ only in their COOHterminal sequences, couple to different GTP-binding proteins to activate different second messenger systems. The pharmacological actions of these subtypes have been well characterized on the basis of their responses to various agonists and antagonists. It has been co...

Selective Modulation of Wild Type Receptor Functions by Mutants of G-Protein-coupled Receptors

“…A similar effect, that of confining the WT receptor intracellularly, was also reported for a deletion mutant (⌬32) of the CCR5 receptor, conferring a certain level of resistance to human immunodeficiency virus infection and delayed onset of acquired immune deficiency syndrome to heterozygous individuals (11). All the evidence of the dominantnegative effect of these truncated receptors points toward their capacity to form heterocomplexes with their respective WT functional receptors (8,10,11). The ability of an EP1 receptor isoform to inhibit signaling by EP1 as well as EP4 receptors suggests an association between the different receptor subtypes (8).…”

“…This raises the possibility that naturally occurring splice variants, such as CCR2a and CCR2b, with distinct coupling, distribution, and desensitization characteristics could associate in a hetero-oligomeric complex and permit a subtle control of the ligandinduced response by modulating the ratio of the two receptors. Indeed, splice variants of the gonadotropin-releasing hormone receptor and the EP1 subtype of prostaglandin E 2 receptor attenuate the action of the specific ligands (8,10). This is also the case for the carboxyl-terminal deletion mutant of CCR2b whose presence modulates the ligand affinity and responsiveness to MCP-1.…”

“…All the evidence of the dominantnegative effect of these truncated receptors points toward their capacity to form heterocomplexes with their respective WT functional receptors (8,10,11). The ability of an EP1 receptor isoform to inhibit signaling by EP1 as well as EP4 receptors suggests an association between the different receptor subtypes (8). The formation of heterocomplexes between different receptor subtypes may be an efficient mechanism to control the cellular response.…”

“…Alternative splicing can also lead to the formation of nonfunctional receptors or receptors with certain functions that are greatly modified (8,9). Individually, these receptors are not involved in signaling, but some of them can show dominantnegative properties when co-expressed with a functional subtype.…”

The Concise Guide to PHARMACOLOGY 2015/16: G protein‐coupled receptors

“…There is evidence for subtypes of FP [1105], IP [1851,2037] and TP [1005] receptors. mRNA for the EP 1 and EP 3 receptors undergo alternative splicing to produce two [1441] and at least six variants, respectively, which can interfere with signalling [1441] or generate complex patterns of G-protein (G i/o , G q/11 , G s and G 12,13 ) coupling (e.g. [997,1370]).…”