Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates heterotrimeric G protein alpha subunits (Gα) and serves as an essential Gα chaperone. Mechanisms by which Ric-8A catalyzes these activities, which are stimulated by Casein Kinase II phosphorylation, are unknown. We report the structure of the nanobody-stabilized complex of nucleotide-free Gα bound to phosphorylated Ric-8A at near atomic resolution by cryo-electron microscopy and X-ray crystallography. The mechanism of Ric-8A GEF activity differs considerably from that employed by G protein-coupled receptors at the plasma membrane. Ric-8A engages a specific conformation of Gα at multiple interfaces to form a complex that is stabilized by phosphorylation within a Ric-8A segment that connects two Gα binding sites. The C-terminus of Gα is ejected from its beta sheet core, thereby dismantling the GDP binding site. Ric-8A binds to the exposed Gα beta sheet and switch II to stabilize the nucleotide-free state of Gα.
The G‐protein complex is a cytoplasmic on–off molecular switch that is set by plasma membrane receptors that activate upon binding of its cognate extracellular agonist. In animals, the default setting is the “off” resting state, while in plants, the default state is constitutively “on” but repressed by a plasma membrane receptor‐like protein. De‐repression appears to involve specific phosphorylation of key elements of the G‐protein complex and possibly target proteins that are positioned downstream of this complex. To address this possibility, protein abundance and phosphorylation state are quantified in wild type and G‐protein deficient Arabidopsis roots in the unstimulated resting state. A total of 3246 phosphorylated and 8141 non‐modified protein groups are identified. It has been found that 428 phosphorylation sites decrease and 509 sites increase in abundance in the G‐protein quadrupole mutant lacking an operable G‐protein‐complex. Kinases with known roles in G‐protein signaling including MAP KINASE 6 and FERONIA are differentially phosphorylated along with many other proteins now implicated in the control of G‐protein signaling. Taken together, these datasets will enable the discovery of novel proteins and biological processes dependent on G‐protein signaling.
more » « less- Award ID(s):
- 1759023
- NSF-PAR ID:
- 10463105
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- PROTEOMICS
- Volume:
- 18
- Issue:
- 24
- ISSN:
- 1615-9853
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Protein intrinsically disordered regions (IDRs) are often targets of combinatorial post-translational modifications (PTMs) that serve to regulate protein structure and/or function. Emerging evidence suggests that the N-terminal tails of G protein γ subunits – essential components of heterotrimeric G protein complexes – are intrinsically disordered, highly phosphorylated governors of G protein signaling. Here, we demonstrate that the yeast Gγ Ste18 undergoes combinatorial, multi-site phosphorylation within its N-terminal IDR. Phosphorylation at S7 is responsive to GPCR activation and osmotic stress while phosphorylation at S3 is responsive to glucose stress and is a quantitative indicator of intracellular pH. Each site is phosphorylated by a distinct set of kinases and both are also interactive, such that phosphomimicry at one site affects phosphorylation on the other. Lastly, we show that phosphorylation produces subtle yet clear changes in IDR structure and that different combinations of phosphorylation modulate the activation rate and amplitude of the scaffolded MAPK Fus3. These data place Gγ subunits among the growing list of intrinsically disordered proteins that exploit combinatorial post-translational modification to govern signaling pathway output.more » « less
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Abstract Heterotrimeric G-proteins regulate multiple aspects of plant growth, development, and response to biotic and abiotic stresses. While the core components of heterotrimeric G-proteins and their basic biochemistry are similar in plants and metazoans, key differences exist in their regulatory mechanisms. In particular, the activation mechanisms of plant G-proteins appear diverse and may include both canonical and novel modes. Classical G-protein-coupled receptor-like proteins exist in plants and interact with Gα proteins, but their ability to activate Gα by facilitating GDP to GTP exchange has not been demonstrated. Conversely, there is genetic and functional evidence that plant G-proteins interact with the highly prevalent receptor-like kinases (RLKs) and are phosphorylated by them. This suggests the exciting scenario that in plants the G-proteins integrate RLK-dependent signal perception at the plasma membrane with downstream effectors. Because RLKs are active kinases, it is also likely that the activity of plant G-proteins is regulated via phosphorylation/dephosphorylation rather than GTP–GDP exchange as in metazoans. This review discusses our current knowledge of the possible RLK-dependent regulatory mechanisms of plant G-protein signaling in the context of several biological systems and outlines the diversity that might exist in such regulation.more » « less
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Intrinsically disordered regions (IDRs) in proteins are often targets of combinatorial posttranslational modifications, which serve to regulate protein structure and function. Emerging evidence suggests that the N-terminal tails of G protein γ subunits, which are essential components of heterotrimeric G proteins, are intrinsically disordered, phosphorylation-dependent determinants of G protein signaling. Here, we found that the yeast Gγ subunit Ste18 underwent combinatorial, multisite phosphorylation events within its N-terminal IDR. G protein–coupled receptor (GPCR) activation and osmotic stress induced phosphorylation at Ser7, whereas glucose and acid stress induced phosphorylation at Ser3, which was a quantitative indicator of intracellular pH. Each site was phosphorylated by a distinct set of kinases, and phosphorylation of one site affected phosphorylation of the other, as determined through exposure to serial stimuli and through phosphosite mutagenesis. Last, we showed that phosphorylation resulted in changes in IDR structure and that different combinations of phosphorylation events modulated the activation rate and amplitude of the downstream mitogen-activated protein kinase Fus3. These data place Gγ subunits among intrinsically disordered proteins that undergo combinatorial posttranslational modifications that govern signaling pathway output.
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