Abstract
Although most heterotrimeric G proteins are thought to dissociate into Gα and Gβγ subunits upon activation, the evidence in the Gi/o family has long been inconsistent and contradictory. The Gi/o protein family mediates inhibition of cAMP production and regulates the activity of ion channels. On the basis of experimental evidence, both heterotrimer dissociation and rearrangement have been postulated as crucial steps of Gi/o protein activation and signal transduction. We have now investigated the process of Gi/o activation in living cells directly by two-photon polarization microscopy and indirectly by observations of G protein-coupled receptor kinase-derived polypeptides. Our observations of existing fluorescently labeled and non-modified Gαi/o constructs indicate that the molecular mechanism of Gαi/o activation is affected by the presence and localization of the fluorescent label. All investigated non-labeled, non-modified Gi/o complexes dissociate extensively upon activation. The dissociated subunits can activate downstream effectors and are thus likely to be the major activated Gi/o form. Constructs of Gαi/o subunits fluorescently labeled at the N terminus (GAP43-CFP-Gαi/o) seem to faithfully reproduce the behavior of the non-modified Gαi/o subunits. Gαi constructs labeled within the helical domain (Gαi-L91-YFP) largely do not dissociate upon activation, yet still activate downstream effectors, suggesting that the dissociation seen in non-modified Gαi/o proteins is not required for downstream signaling. Our results appear to reconcile disparate published data and settle a long running dispute.
Highlights
The nature of the activated form of heterotrimeric Gi/o proteins is unclear
We have shown recently that, using 2PPM, linear dichroism (LD) can be detected in ϳ80% of fluorescent proteins (FPs)-labeled membrane proteins expressed in mammalian cells, including several G proteins [18]
Dissociation of G Protein Heterotrimers Is Not Required for Activation of Downstream Effectors—To determine how dissociation of G protein heterotrimers relates to their functional activity, we investigated the ability of constitutively active G␣-FP constructs to regulate the activity of GIRK channels (Fig. 8)
Summary
Results: Non-modified Gi/o heterotrimers dissociate upon activation, whereas fluorescently labeled Gi/o heterotrimers dissociate or rearrange depending on fluorescent protein localization. Conclusion: Dissociated G␣GTP and G␥ subunits represent the major activated form of Gi/o proteins. Most heterotrimeric G proteins are thought to dissociate into G␣ and G␥ subunits upon activation, the evidence in the Gi/o family has long been inconsistent and contradictory. On the basis of experimental evidence, both heterotrimer dissociation and rearrangement have been postulated as crucial steps of Gi/o protein activation and signal transduction. G␣i constructs labeled within the helical domain (G␣i-L91-YFP) largely do not dissociate upon activation, yet still activate downstream effectors, suggesting that the dissociation seen in non-modified G␣i/o proteins is not required for downstream signaling. Our results appear to reconcile disparate published data and settle a long running dispute
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