Abstract

FP prostanoid receptors are G-protein-coupled receptors that mediate the actions of prostaglandin F_2α(PGF_2α). Alternative mRNA splicing gives rise to two isoforms, FP_A and FP_B, which are identical except for their intracellular carboxyl termini. In this study, we examined the internalization of recombinant FLAG-epitope-tagged FP_A and FP_B receptors that were stably expressed in human embryonic kidney-293 cells. Cell surface receptors on live cells were labeled with anti-FLAG antibodies either in the presence or absence of PGF_2α and were examined by immunofluorescence microscopy. In the absence of PGF_2α, FP_A-expressing cells were labeled predominantly on the cell surface; however, FP_B-expressing cells were labeled on both the cell surface and intracellularly, indicating constitutive internalization of the FP_B isoform. After treatment with PGF_2α, FP_A-expressing cells were labeled intracellularly, reflecting receptor internalization, which could be mimicked with phorbol 12-myristyl 13-acetate (PMA), an activator of protein kinase C (PKC). Pretreatment of FP_A-expressing cells with Gö 6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo[3,4-c]carbozole], an inhibitor of PKC, blocked both PGF_2α- and PMA-induced receptor internalization. However, Gö 6976 did not block constitutive internalization of the FP_B isoform, suggesting that the mechanisms of receptor internalization differ between the FP_A and FP_B isoforms. Furthermore, pretreatment with sucrose, an inhibitor of clathrin-dependent internalization, blocked PGF_2α-induced internalization of the FP_A isoform but did not block constitutive internalization of the FP_B isoform. In conclusion, the FP_Areceptor isoform shows an agonist-induced internalization involving PKC and clathrin, whereas the FP_B isoform undergoes agonist-independent internalization that does not involve PKC or clathrin.