Differential Desensitization of Thromboxane A 2 Receptor Subtypes

Abstract

Abstract Two subtypes of the thromboxane A 2 (TxA 2 ) receptor (TxA 2 R-E and TxA 2 R-P), which differ in their alternatively spliced cytoplasmic tails, have been identified. The initial concentration of the TxA 2 mimetic IBOP required to reduce peak intracellular Ca 2+ concentration ([Ca 2+ ] i ) induced by a second addition of IBOP (100 nmol/L) was similar (IC 50 for TxA 2 R-E and TxA 2 R-P, 0.46±0.16 and 0.40±0.07 nmol/L) in fibroblasts overexpressing either the TxA 2 R-E or -P subtype. Although the number of TxA 2 binding sites decreased in TxA 2 R-P cells after prolonged stimulation with a TxA 2 mimetic, those in the TxA 2 R-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 TxA 2 -induced [Ca 2+ ] i mobilization was measured. Forskolin reduced the IBOP-induced peak [Ca 2+ ] i in neither TxA 2 R-E nor TxA 2 R-P cells; however, treatment with phorbol esters (IC 50 , 0.57±0.70 nmol/L) strongly prevented IBOP-mediated [Ca 2+ ] i rise in TxA 2 R-E but not in TxA 2 R-P cells. Desensitization of TxA 2 R-E by phorbol esters was prevented by the PKC inhibitor calphostin C or by downregulation of PKC-α. Thus, the response of TxA 2 R-E to prolonged stimulation differs from that of TxA 2 R-P in both the regulation of the number of binding sites and the mechanism for desensitization; agonists that activate PKC-α might interfere with TxA 2 R-E–mediated signaling.