title: Involvement of lipid rafts in multiple signal transductions mediated by two isoforms of thromboxane A₂ receptor: dependency on receptor isoforms and downstream signaling types.
abstract: Lipid rafts, microdomains in the plasma membrane, are known to be involved in G protein-coupled receptor signal transduction; however, their involvement in thromboxane A(2) 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 G(13) 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.
authors.affiliation: Department of Cellular Signaling, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba 6-3, Aramaki, Aoba-ku, Sendai 980-8578, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Goto
authors.forename: Masaki
authors.initials: M
authors.lastname: Saito
authors.forename: Yutaro
authors.initials: Y
authors.lastname: Obara
authors.forename: Takahiro
authors.initials: T
authors.lastname: Moriya
authors.forename: Norimichi
authors.initials: N
authors.lastname: Nakahata
mesh_terms: D000242:Cyclic AMP / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism*; D020033:Protein Isoforms; D044244:Receptors, Thromboxane A2, Prostaglandin H2 / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D047392:beta-Cyclodextrins / Q000494:pharmacology; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D020033:Protein Isoforms; D044244:Receptors, Thromboxane A2, Prostaglandin H2; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C066737:RHOA protein, human; D000242:Cyclic AMP; D007770:L-Lactate Dehydrogenase; D020742:rhoA GTP-Binding Protein