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Role of C-terminal serines in desensitization and phosphorylation of the mouse thromboxane receptor.

Publication ,  Journal Article
Spurney, RF
Published in: J Biol Chem
October 23, 1998

To investigate the role of C-terminal hydroxyamino acids in desensitization of the receptor for thromboxane A2 (TxA2), we created a mutant TxA2 receptor (TP receptor) in which serines at positions 321, 322, and 328 were replaced with either alanine or glycine. Mutant and wild type receptors were expressed in a mesangial cell line, and clones expressing similar numbers of receptors were studied. Affinity and specificity of TxA2 binding to the mutant receptor were identical to wild type receptors. In contrast, TxA2-induced inositol trisphosphate generation by the mutant receptor was enhanced compared with the wild type. Prior treatment with the TxA2 agonist U46619 reduced subsequent U46619-induced increases in inositol trisphosphate generation by both receptors; however, the extent of desensitization was significantly reduced in the receptor mutant. Protein kinase C (PKC) inhibitors attenuated TxA2-induced desensitization of wild type receptors, but had little effect on TxA2-induced desensitization of mutant receptors. Pretreatment with the phorbol ester phorbol 12, 13-dybutyrate (PDBu) (100 nM) decreased subsequent responsiveness of wild type but not mutant TP receptors. -induced desensitization of wild type receptors was associated with enhanced phosphorylation of receptor proteins. This agonist-specific phosphorylation of the TP receptor was largely prevented by inhibitors of PKC. Treatment with 100 nM PDBu increased phosphorylation of both wild type and mutant TP receptors, but the extent of phosphorylation of the receptor mutant was reduced compared with the wild type. Increasing the concentration of PDBu from 100 nM to 1 microM PDBu reduced responsiveness of both mutant and wild type receptors without enhancing phosphorylation of either of the receptor proteins. These data suggest that 1) phosphorylation of C-terminal serines contributes to agonist-specific desensitization of the TP receptor, 2) PKC-induced desensitization of TP receptors is caused, in part, by phosphorylation of C-terminal serines, and 3) desensitization of TP receptors by PKC is complex and involves mechanisms that may not require direct phosphorylation of receptor proteins.

Duke Scholars

Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

October 23, 1998

Volume

273

Issue

43

Start / End Page

28496 / 28503

Location

United States

Related Subject Headings

  • Thromboxane A2
  • Staurosporine
  • Serine
  • Recombinant Proteins
  • Receptors, Thromboxane
  • Protein Kinase C
  • Precipitin Tests
  • Phosphorylation
  • Phorbol 12,13-Dibutyrate
  • Mutagenesis, Site-Directed
 

Citation

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Spurney, R. F. (1998). Role of C-terminal serines in desensitization and phosphorylation of the mouse thromboxane receptor. J Biol Chem, 273(43), 28496–28503. https://doi.org/10.1074/jbc.273.43.28496
Spurney, R. F. “Role of C-terminal serines in desensitization and phosphorylation of the mouse thromboxane receptor.J Biol Chem 273, no. 43 (October 23, 1998): 28496–503. https://doi.org/10.1074/jbc.273.43.28496.
Spurney, R. F. “Role of C-terminal serines in desensitization and phosphorylation of the mouse thromboxane receptor.J Biol Chem, vol. 273, no. 43, Oct. 1998, pp. 28496–503. Pubmed, doi:10.1074/jbc.273.43.28496.

Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

October 23, 1998

Volume

273

Issue

43

Start / End Page

28496 / 28503

Location

United States

Related Subject Headings

  • Thromboxane A2
  • Staurosporine
  • Serine
  • Recombinant Proteins
  • Receptors, Thromboxane
  • Protein Kinase C
  • Precipitin Tests
  • Phosphorylation
  • Phorbol 12,13-Dibutyrate
  • Mutagenesis, Site-Directed