Vol. 302, Issue 1, 219-224, July 2002

Differential Internalization of the Prostaglandin F₂ Receptor Isoforms: Role of Protein Kinase C and Clathrin

Dinesh Srinivasan, Hiromichi Fujino and John W. Regan

Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona

FP prostanoid receptors are G-protein-coupled receptors that mediate the actions of prostaglandin F₂ (PGF₂). 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 PGF₂ and were examined by immunofluorescence microscopy. In the absence of PGF₂, 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 PGF₂, 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 PGF₂- 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 PGF₂-induced internalization of the FP_A isoform but did not block constitutive internalization of the FP_B isoform. In conclusion, the FP_A receptor 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.