Differential internalization of the prostaglandin F receptor isoforms: Role of protein kinase C and clathrin

Dinesh Srinivasan, Hiromichi Fujino, John W. Regan

Research output: Contribution to journalArticle

23 Scopus citations

Abstract

FP prostanoid receptors are G-protein-coupled receptors that mediate the actions of prostaglandin F (PGF). 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 PGF and were examined by immunofluorescence microscopy. In the absence of PGF, 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 PGF, 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-oxo5H-indolo[2,3-a] pyrrolo[3,4-c]carbozole], an inhibitor of PKC, blocked both PGF- 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 PGF-induced internalization of the FPA isoform but did not block constitutive internalization of the FPB isoform. In conclusion, the FPA receptor 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.

Original languageEnglish (US)
Pages (from-to)219-224
Number of pages6
JournalJournal of Pharmacology and Experimental Therapeutics
Volume302
Issue number1
DOIs
StatePublished - Jul 4 2002

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

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