Abstract
Prostaglandin F2α receptors (FP) are G protein-coupled receptors that bind prostaglandin F2α(PGF2α), resulting in the activation of an inositol phosphate (IP) second messenger pathway. Alternative mRNA splicing generates two FP receptor isoforms. These isoforms, designated FPA and FPB, are otherwise identical except for their carboxyl termini. FPB is essentially a truncated version of FPA that lacks the 46 carboxyl-terminal amino acids, including four putative protein kinase C (PKC) phosphorylation sites. Until now, functional differences between these FP receptor isoforms have not been identified. We now report that pretreatment with the PKC inhibitor bisindolylmaleimide I enhanced PGF2α-stimulated IP accumulation in transfected cells stably expressing the FPA isoform but not in cells stably expressing the FPB isoform. Whole-cell phosphorylation experiments showed a strong agonist-dependent phosphorylation of the FPA isoform but little or no phosphorylation of the FPB. Pretreatment of cells with bisindolylmaleimide I decreased PGF2α-stimulated phosphorylation of the FPA isoform consistent with a PKC-dependent phosphorylation. In vitro phosphorylation of an FPAcarboxyl-terminal fusion protein by recombinant PKCα showed that the carboxyl terminus of the FPA is a substrate for PKC. These results suggest that PKC-dependent phosphorylation is responsible for differential regulation of second messenger signaling by FP prostanoid receptor isoforms.
Footnotes
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Send reprint requests to: John W. Regan, Ph.D., Department of Pharmacology & Toxicology, College of Pharmacy, University of Arizona, 1703 E. Mabel St., Box 210207, Tucson, AZ 85721-0207. E-mail: regan{at}pharmacy.arizona.edu
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Supported in part by National Institutes of Health Grant EY11291 and grants from Allergan Inc. K.L.P. was supported by an individual predoctoral fellowship from the National Science Foundation.
- Abbreviations:
- GPCR
- G protein-coupled receptor
- PKC
- protein kinase C
- GRK
- G protein-coupled receptor kinase
- PGF2α
- prostaglandin F2α
- IP
- inositol phosphate
- HEK
- human embryonic kidney
- TMX
- thymeleatoxin
- BIM
- bisindolylmaleimide I
- DMEM
- Dulbecco's modified Eagle's medium
- GST
- glutathione-S-transferase
- PCR
- polymerase chain reaction
- HA
- hemagglutinin
- RIPA
- radioimmunoprecipitation assay
- PAGE
- polyacrylamide gel electrophoresis
- PLC
- phospholipase C
- PMA
- phorbol 12-myristate 13-acetate
- Received July 14, 1999.
- Accepted October 18, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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