Mastoparan changes the cellular localization of Galphaq/11 and Gbeta through its binding to ganglioside in lipid rafts.
Although it is known that mastoparan, a wasp venom toxin, directly activates Gi/o, mastoparan-induced biological responses are not always explained by this mechanism. For instance, we have demonstrated previously that mastoparan suppressed phosphoinositide hydrolysis induced by carbachol in human astrocytoma cells (FEBS Lett 206:91-94, 1990). In the present study, we examined whether mastoparan affected phosphoinositide hydrolysis by interacting with lipid rafts in PC-12 cells. Mastoparan inhibited UTP-induced increase in [Ca2+]i and phosphoinositide hydrolysis in a concentration-dependent manner. UTP-induced phosphoinositide hydrolysis occurred in lipid rafts, because methyl-beta-cyclodextrin, a disrupting regent of lipid rafts, inhibited the hydrolysis. Mastoparan changed the localization of Galphaq/11 and Gbeta together with cholesterol from lipid rafts to nonraft fractions or cytosol. These changes were inhibited by ganglioside mixtures, suggesting that mastoparan interacts with gangliosides in lipid rafts. In fact, ganglioside mixtures and neuraminidase, but not sialic acid, attenuated the inhibitory effect of mastoparan on phosphoinositide hydrolysis. Furthermore, fluorescence intensity of tyrosine residue of [Tyr3]mastoparan was potentiated by ganglioside mixtures, suggesting the direct binding of mastoparan to gangliosides. Mastoparan caused cytotoxicity of PC-12 cells in a concentration-dependent manner, determined by LDH release. The mastoparan-induced cytotoxicity was significantly inhibited by neuraminidase or gangliosides. The order of inhibitory potency of gangliosides was GT1b approximately GD1b > GD1a > GM1 >> GQ1b, but asialo-GM1 and sialic acid were inactive. These results suggest that mastoparan initially binds to gangliosides in lipid rafts and then it inhibits phosphoinositide hydrolysis by changing the localization of Galphaq/11 and Gbeta in lipid rafts.
Sugama
Jun
J
Department of Cellular Signaling, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba 6-3, Aramaki, Aoba-ku, Sendai, Japan 980-8578.
Ohkubo
Satoko
S
Atsumi
Masanori
M
Nakahata
Norimichi
N
D000818:Animals; D002118:Calcium / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism*; D005732:Gangliosides / Q000378:metabolism* / Q000494:pharmacology; D006868:Hydrolysis; D007295:Inositol Phosphates / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins; D021962:Membrane Microdomains / Q000378:metabolism*; D009439:Neuraminidase / Q000494:pharmacology; D016716:PC12 Cells; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology*; D051381:Rats; D015398:Signal Transduction; D014544:Uridine Triphosphate / Q000494:pharmacology; D014862:Wasp Venoms / Q000378:metabolism / Q000494:pharmacology*
D044387:GTP-Binding Protein beta Subunits; D005732:Gangliosides; D007295:Inositol Phosphates; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D014862:Wasp Venoms; C037500:mastoparan; D009439:Neuraminidase; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D002118:Calcium; D014544:Uridine Triphosphate