A FRET map of membrane anchors suggests distinct microdomains of heterotrimeric G proteins.
The standard model of heterotrimeric G protein signaling postulates a dissociation of Galpha and Gbetagamma subunits after activation. We hypothesized that the different combination of lipid-modifications on Galpha and Galphabetagamma subunits directs them into different microdomains. By characterizing rapidly and at high sensitivity 38 fluorescence resonance energy transfer (FRET) pairs of heterotrimeric-G-protein constructs, we defined their microdomains in relation to each other, free from the constraints of the raft/non-raft dualism. We estimated that in a cell approximately 30% of these membrane-anchored proteins are mostly clustered in 3400-16,200 copies of 30-nm microdomains. We found that the membrane anchors of Galpha and Galphabetagamma subunits of both the G(i/o) and G(q) family co-cluster differently with microdomain markers. Moreover, anchors of the Galpha(i/o) and Galpha(q) subunits co-clustered only weakly, whereas constructs that contained the anchors of the corresponding heterotrimers co-clustered considerably, suggesting the existence of at least three types of microdomain. Finally, FRET experiments with full-length heterotrimeric G proteins confirmed that the inactive, heterotrimerized Galpha subunit is in microdomains shared by heterotrimers from different subclasses, from where it displaces upon activation into a membrane-anchor- and subclass-specific microdomain.
Abankwa
Daniel
D
Ecole Polytechnique Fédérale de Lausanne (EPFL), Institut des Sciences et Ingénierie Chimiques, CH-1015 Lausanne, Switzerland. d.abankwa@imb.uq.edu.au
Vogel
Horst
H
D000595:Amino Acid Sequence; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits / Q000737:chemistry / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000737:chemistry / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D021962:Membrane Microdomains / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D021381:Protein Transport; D015203:Reproducibility of Results
D015415:Biomarkers; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D017261:Glycosylphosphatidylinositols; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins