Gi protein activation in intact cells involves subunit rearrangement rather than dissociation

Abstract

G protein-coupled receptors transduce diverse extracellular signals, such as neurotransmitters, hormones, chemokines, and sensory stimuli, into intracellular responses through activation of heterotrimeric G proteins. G proteins play critical roles in determining specificity and kinetics of subsequent biological responses by modulation of effector proteins. We have developed a fluorescence resonance energy transfer (FRET)-based assay to directly measure mammalian G protein activation in intact cells and found that Gi proteins activate within 1-2 s, which is considerably slower than activation kinetics of the receptors themselves. More importantly, FRET measurements demonstrated that G␣i-and G␤␥-subunits do not dissociate during activation, as has been previously postulated. Based on FRET measurements between G␣i-yellow fluorescent protein and G␤␥-subunits that were fused to cyan fluorescent protein at various positions, we conclude that, instead, G protein subunits undergo a molecular rearrangement during activation. The detection of a persistent heterotrimeric composition during G protein activation will impact the understanding of how G proteins achieve subtype-selective coupling to effectors. This finding will be of particular interest for unraveling G␤␥-induced signaling pathways. Avariety of physiological signals such as neurotransmitters, hormones, and light are detected by members of the seven transmembrane domain receptor family. These G protein-coupled receptors (GPCRs) activate G proteins by promoting binding of GTP in exchange for GDP. Both, G␣ and G␤␥-subunits of activated G proteins can regulate downstream effectors such as adenylyl cyclases, phospholipases, or ion channels. Based on biochemical experiments and structural studies, it is known that conformational rearrangements in the ''switch regions'' of the ␣-subunits on GTP binding weaken the interaction with G␤␥-subunits (1-3). It is generally assumed that the reduced affinity of G␣-GTP for G␤␥ causes G proteins to dissociate into G␣-GTP and a G␤␥ complex. Reassociation is then assumed to occur on hydrolysis of the GTP bound to G␣, which can be accelerated by RGS proteins (4-6). However, this model fails to explain well established phenomena in G protein-mediated signaling. Most importantly, a major open question is how G␤␥ effectors, such as G proteinactivated inwardly rectifying K ϩ (GIRK) channel, are selectively regulated through specific G␣ subtypes despite the lack of subtype selectivity for G␤␥ subtypes (7-10). In attempts to answer this central question, it has been hypothesized that selectivity may be caused by scaffolding of G proteins and effectors, either by direct binding of G␣ to its effector (11), or by temporally and spatially restricting G protein activity (12, 13).The possibility to investigate protein-protein interactions in living cells by using fluorescence resonance energy transfer (FRET) between recombinant fluorescent proteins (14), has recently led to new insights in temporal signaling properties of G protein effectors (1...

Rapid-mix flow cytometry measurements of subsecond regulation of G protein-coupled receptor ternary complex dynamics by guanine nucleotides

“…GTPγS induced separation of the receptor and G protein as did GDP (at half the rate), making it unlikely that RG separation is sufficient for activation. These results are compatible with a cell activation model regulated by the G protein conformational changes rather than disassembly of the Gαβγ heterotrimer [7,9,19].…”

“…Although there is much recent evidence that concurs with our results [6][7][8][9], it is difficult to arrive at a definitive conclusion because of the model nature of our experiments. One might infer from structural data that the interface domains of the components of the ternary complex involves an intricate system of ON and OFF allosteric switches that trigger their stepwise disassembly from erstwhile partners in exchange for new ones [50].…”

“…Furthermore, other experimental evidence has led to questions about the absolute necessity of including subunit dissociation as an element of activation [5][6][7][8][9]19].…”

“…For example, Klein, Levitzki and coworkers [5,6] used a Gα subunit fused to its cognate Gβγ subunit, to show that both the fusion of subunits and therefore absence of dissociation of subunits did not impair signaling function. Other evidence has come from FRET assays using fluorescently tagged Gα and Gβγ subunits (vide infra) [7][8][9].…”

Interaction of nucleoside diphosphate kinase B with heterotrimeric G protein βγ dimers: consequences on G protein activation and stability

“…Both can directly interact with specific effector proteins. Note, however, that the concept of heterotrimer dissociation has recently been challenged (Bünemann et al 2003). After the hydrolysis of GTP to GDP and inorganic phosphate by the intrinsic GTPase activity present in Gα, the G protein returns to its inactive heterotrimeric state.…”