Involvement of lipid rafts in multiple signal transductions mediated by two isoforms of thromboxane A₂ receptor: Dependency on receptor isoforms and downstream signaling types

Lipid rafts, microdomains in the plasma membrane, are known to be involved in G protein-coupled receptor signal transduction; however, their involvement in thromboxane A₂ receptor (TP) signaling remains to be clarified. We examined whether two isoforms of TP, TPα and TPβ, utilize lipid rafts for multiple G protein signal transduction. Sucrose density gradient centrifugation followed by western blotting of HEK cells expressing TPα or TPβ revealed the localization of both TPα and TPβ in lipid rafts. Furthermore, methyl-β-cyclodextrin, which destroys lipid raft structure by depleting cholesterol, influenced G protein signaling elicited by TPα and TPβ to varying degrees. Phosphatidylinositol hydrolysis and cAMP accumulation induced by TPα or TPβ stimulation was markedly inhibited by methyl-β-cyclodextrin. In contrast, treatment with methyl-β-cyclodextrin partially inhibited RhoA activation induced by TPα stimulation, but failed to affect TPβ stimulation. Furthermore, the inhibitory action of methyl-β-cyclodextrin on cAMP accumulation was specific to TPα and TPβ, because methyl-β-cyclodextrin enhanced forskolin and β-adrenergic stimulation-induced cAMP accumulation. These results indicate that TP isoforms depend on lipid rafts during G_q and G_s signaling, while G₁₃ signaling mediated by TP isoforms does not. Moreover, TPα seems to be more lipid raft-dependent with respect to RhoA activation than TPβ. These results indicate that the two isoforms of the TP mediate multiple signal transductions with varying degrees of lipid raft dependency. Moreover, our results provide a deeper understanding of the function of lipid rafts in G protein signaling and the physiological meaning of TP isoforms.