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

To investigate the role of C-terminal hydroxyamino acids in desensitization of the receptor for thromboxane A(2) (TxA(2)), we created a mutant TxA(2) 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 TxA(2) binding to the mutant receptor were identical to wild type receptors. In contrast, TxA(2)-induced inositol trisphosphate generation by the mutant receptor was enhanced compared with the wild type. Prior treatment with the TxA(2) 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 TxA(2)-induced desensitization of wild type receptors, but had little effect on TxA(2)-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, U46619-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 mu M 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.