Differential desensitization of thromboxane A2 receptor subtypes

Two subtypes of the thromboxane A2 (TxA2) receptor (TxA2R-E and TxA2R-P), which differ in their alternatively spliced cytoplasmic tails, have been identified. The initial concentration of the TxA2 mimetic IBOP required to reduce peak intracellular Ca2+ concentration ([Ca2+]i) induced by a second addition of IBOP (100 nmol/L) was similar (IC50 for TxA2R-E and TxA2R-P, 0.46 +/- 0.16 and 0.40 +/- 0.07 nmol/L) in fibroblasts overexpressing either the TxA2R-E or -P subtype. Although the number of TxA2 binding sites decreased in TxA2R-P cells after prolonged stimulation with a TxA2 mimetic, those in the TxA2R-E cells increased markedly. To determine whether the mechanism for desensitization differs between subtypes, the effect of activation of protein kinase C (PKC) or cAMP-dependent kinase on TxA2-induced [Ca2+]i mobilization was measured. Forskolin reduced the IBOP-induced peak [Ca2+]i in neither TxA2R-E nor TxA2R-P cells; however, treatment with phorbol esters (IC50, 0.57 +/- 0.70 nmol/L) strongly prevented IBOP-mediated [Ca2+]i rise in TxA2R-E but not in TxA2R-P cells. Desensitization of TxA2R-E by phorbol esters was prevented by the PKC inhibitor calphostin C or by downregulation of PKC-alpha. Thus, the response of TxA2R-E to prolonged stimulation differs from that of TxA2R-P in both the regulation of the number of binding sites and the mechanism for desensitization; agonists that activate PKC-alpha might interfere with TxA2R-E-mediated signaling.