The C-terminus of the thromboxane receptor contributes to coupling and desensitization in a mouse mesangial cell line

To investigate regulatory domains of the thromboxane A2 (TxA2) receptor, we constructed a truncated form of the mouse TxA2 receptor and expressed it in a mesangial cell line. The mutant receptor lacked 22 amino acids in the C-terminus including four potential phosphorylation sites. Ligand binding of mutant receptors was identical with the wild type. Stimulation with TxA2 agonist induced increases in inositol trisphosphate (IP3) generation and [Ca++]i by both wild-type and mutant receptors. However, the initial increase in IP3 generation by the mutant receptor was only approximately 50% of that seen in the wild type. Exposure of wild-type receptors to TxA2 agonist caused desensitization of IP3 and calcium responses. Pretreatment with TxA2 agonist caused some desensitization of mutant receptors, but the extent of desensitization was reduced compared with the wild type. The protein kinase C inhibitor staurosporine attenuated TxA2-induced desensitization of wild-type receptors, but had little effect on TxA2-induced desensitization of mutant receptors. Pretreatment with low concentrations of the phorbol ester, phorbol 12,13-dibutyrate (100 nM), reduced subsequent responsiveness of wild-type but not mutant TxA2 receptors. In contrast, high-dose phorbol 12,13-dibutyrate (1 microM) produced a similar degree of desensitization of both receptor types. These data suggest that: 1) the C-terminus participates in coupling of the TxA2 receptor to its effector systems; 2) the C-terminus contributes to agonist-specific desensitization of the TxA2 receptor; and 3) protein kinase C-induced desensitization of the TxA2 receptor is complex and depends, in part, on C-terminal domains of the TxA2 receptor.