EP3 Receptor Isoforms Differentially Expressed in Subpopulations of Primate Granulosa cells Couple to Unique G Proteins.

Abstract

Prostaglandin (PG) E2 is necessary for ovulation of mammalian follicles. PGE2 acts through four distinct G protein coupled receptors (PTGER1-PTGER4, also called EP1-EP4). Among them, EP3 is unique in having multiple isoforms generated by alternative mRNA splicing. Each isoform has a distinct amino acid composition in the C-terminal region which couples to G proteins. Therefore, we hypothesized that each EP3 isoform couples to different G proteins to modulate intracellular signaling in primate granulosa cells. Using 3' rapid amplification of cDNA ends (3' RACE), six EP3 mRNAs were identified in cynomolgus monkey granulosa cells. Three of these isoforms shared significant sequence homology with EP3 isoform 5 (EP3-5), EP3-7, and EP3-9 as described previously in human tissues. In addition, three isoforms were identified which have not been previously reported. EP3-5, EP3-7, and EP3-9 were transiently transfected into Chinese hamster ovary (CHO) cells and were detected in a punctate pattern in the cytoplasm as well as in the plasma membrane. Stimulation of EP3-5 by sulprostone, an EP3 agonist, reduced cAMP compared to vehicle-treated cells. Pretreatment with pertussis toxin (PTX) increased sulprostone-stimulated cAMP compared to sulprostone alone, indicating that EP3-5 couples to the Gi protein. Addition of sulprostone to EP3-9 transfected cells increased cAMP, which was blocked by the general G protein inhibitor GDP-β-S. Sulprostone also increased intracellular Ca2+ in EP3-9 transfected cells, which was blocked by PTX or GDP-β-S. Therefore, EP3-9 likely couples to both Gs to increase cAMP and a PTX-sensitive G protein to regulate intracellular Ca2+. No G protein associated function was identified for EP3-7. Laser capture microdissection and qPCR were used to identify subpopulations of granulosa cells which preferentially expressed these EP3 isoforms 36 hours after an ovulatory dose of hCG, or just before ovulation. EP3-5 mRNA was significantly lower in apex granulosa cells compared to non-apex granulosa cells; EP3-9 mRNA was higher in cumulus cells when compared to mural granulosa cells. In summary, each EP3 receptor isoform may function through different G proteins. Differential expression of EP3 isoforms in subpopulations of granulosa cells may contribute to the different roles that these subpopulations play in the process of ovulation. Supported by NIH/NICHD HD054691.