Hsp90 interactions and acylation target the G protein Galpha 12 but not Galpha 13 to lipid rafts.
The heterotrimeric G proteins, G(12) and G(13), are closely related in their sequences, signaling partners, and cellular effects such as oncogenic transformation and cytoskeletal reorganization. Yet G(12) and G(13) can act through different pathways, bind different proteins, and show opposing actions on some effectors. We investigated the compartmentalization of G(12) and G(13) at the membrane because other G proteins reside in lipid rafts, membrane microdomains enriched in cholesterol and sphingolipids. Lipid rafts were isolated after cold, nonionic detergent extraction of cells and gradient centrifugation. Galpha(12) was in the lipid raft fractions, whereas Galpha(13) was not associated with lipid rafts. Mutation of Cys-11 on Galpha(12), which prevents its palmitoylation, partially shifted Galpha(12) from the lipid rafts. Geldanamycin treatment, which specifically inhibits Hsp90, caused a partial loss of wild-type Galpha(12) and a complete loss of the Cys-11 mutant from the lipid rafts and the appearance of a higher molecular weight form of Galpha(12) in the soluble fractions. These results indicate that acylation and Hsp90 interactions localized Galpha(12) to lipid rafts. Hsp90 may act as both a scaffold and chaperone to maintain a functional Galpha(12) only in discrete membrane domains and thereby explain some of the nonoverlapping functions of G(12) and G(13) and control of these potent cell regulators.
Metabolic Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
Abdul A
AA
Waheed
Teresa L Z
TL
Jones
D016475:3T3 Cells; D000215:Acylation; D000818:Animals; D016227:Benzoquinones; D019556:COS Cells; D002460:Cell Line; D003505:Cyclodextrins / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D047029:Lactams, Macrocyclic; D021962:Membrane Microdomains / Q000378:metabolism*; D051379:Mice; D017830:Octoxynol / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011809:Quinones / Q000494:pharmacology; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection; D047392:beta-Cyclodextrins
D016227:Benzoquinones; D003505:Cyclodextrins; D004268:DNA-Binding Proteins; D003902:Detergents; D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D011809:Quinones; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D017830:Octoxynol; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D020962:Heterotrimeric GTP-Binding Proteins; D003545:Cysteine; C001277:geldanamycin