RT Journal Article SR Electronic T1 Differential Internalization of the Prostaglandin F2α Receptor Isoforms: Role of Protein Kinase C and Clathrin JF Journal of Pharmacology and Experimental Therapeutics JO J Pharmacol Exp Ther FD American Society for Pharmacology and Experimental Therapeutics SP 219 OP 224 DO 10.1124/jpet.302.1.219 VO 302 IS 1 A1 Srinivasan, Dinesh A1 Fujino, Hiromichi A1 Regan, John W. YR 2002 UL http://jpet.aspetjournals.org/content/302/1/219.abstract AB FP prostanoid receptors are G-protein-coupled receptors that mediate the actions of prostaglandin F2α(PGF2α). Alternative mRNA splicing gives rise to two isoforms, FPA and FPB, which are identical except for their intracellular carboxyl termini. In this study, we examined the internalization of recombinant FLAG-epitope-tagged FPA and FPB 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 PGF2α and were examined by immunofluorescence microscopy. In the absence of PGF2α, FPA-expressing cells were labeled predominantly on the cell surface; however, FPB-expressing cells were labeled on both the cell surface and intracellularly, indicating constitutive internalization of the FPB isoform. After treatment with PGF2α, FPA-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 FPA-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 PGF2α- and PMA-induced receptor internalization. However, Gö 6976 did not block constitutive internalization of the FPB isoform, suggesting that the mechanisms of receptor internalization differ between the FPA and FPB isoforms. Furthermore, pretreatment with sucrose, an inhibitor of clathrin-dependent internalization, blocked PGF2α-induced internalization of the FPA isoform but did not block constitutive internalization of the FPB isoform. In conclusion, the FPAreceptor isoform shows an agonist-induced internalization involving PKC and clathrin, whereas the FPB isoform undergoes agonist-independent internalization that does not involve PKC or clathrin. The American Society for Pharmacology and Experimental Therapeutics