Abstract
BackgroundArabidopsis, 7-transmembrane Regulator of G signaling protein 1 (AtRGS1) modulates canonical G protein signaling by promoting the inactive state of heterotrimeric G protein complex on the plasma membrane. It is known that plant leucine-rich repeat receptor–like kinases (LRR RLKs) phosphorylate AtRGS1 in vitro but little is known about the in vivo interaction, molecular dynamics, or the cellular consequences of this interaction.MethodsTherefore, a subset of the known RLKs that phosphorylate AtRGS1 were selected for elucidation, namely, BAK1, BIR1, FLS2. Several microscopies for both static and dynamic protein-protein interactions were used to follow in vivo interactions between the RLKs and AtRGS1 after the presentation of the Pathogen-associated Molecular Pattern, Flagellin 22 (Flg22). These microscopies included Förster Resonance Energy Transfer, Bimolecular Fluoresence Complementation, and Cross Number and Brightness Fluorescence Correlation Spectroscopy. In addition, reactive oxygen species and calcium changes in living cells were quantitated using luminometry and R-GECO1 microscopy.ResultsThe LRR RLKs BAK1 and BIR1, interact with AtRGS1 at the plasma membrane. The RLK ligand flg22 sets BAK1 in motion toward AtRGS1 and BIR1 away, both returning to the baseline orientations by 10 minutes. The C-terminal tail of AtRGS1 is important for the interaction with BAK1 and for the tempo of the AtRGS1/BIR1 dynamics. This window of time corresponds to the flg22-induced transient production of reactive oxygen species and calcium release which are both attenuated in the rgs1 and the bak1 null mutants.ConclusionsA temporal model of these interactions is proposed. flg22 binding induces nearly instantaneous dimerization between FLS2 and BAK1. Phosphorylated BAK1 interacts with and enables AtRGS1 to move away from BIR1 and AtRGS1 becomes phosphorylated leading to its endocytosis thus leading to de-repression by permitting AtGPA1 to exchange GDP for GTP. Finally, the G protein complex becomes dissociated thus AGB1 interacts with its effector proteins leading to changes in reactive oxygen species and calcium.
Highlights
Heterotrimeric G proteins couple extracellular signals to cytoplasmic changes in multiple abiotic [1,2,3,4] and biotic stress responses [5,6,7,8], and developmental cues
This window of time corresponds to the flg22-induced transient production of reactive oxygen species and calcium release which are both attenuated in the rgs1 and the bak1 null mutants
Phosphorylated BRI1-associated Kinase 1 (BAK1) interacts with and enables AtRGS1 to move away from BAK1-interacting receptor-like kinase1 (BIR1) and AtRGS1 becomes phosphorylated leading to its endocytosis leading to de-repression by permitting Arabidopsis thaliana Gα subunit (AtGPA1) to exchange GDP for GTP
Summary
Heterotrimeric G proteins couple extracellular signals to cytoplasmic changes in multiple abiotic [1,2,3,4] and biotic stress responses [5,6,7,8], and developmental cues. A growing body of evidence indicates that signal specificity is achieved by Leucine-Rich Repeat Receptor-Like Kinases (LRR-RLK) and G protein complexes in related pathways [9,10,11,12,13,14,15,16]. A set of AtRGS1 LRR-RLK partners and their dynamics in canonical G signal transduction is used to model ligand-induced dynamics of heterotrimeric G protein-coupled receptor-like kinase complexes. XLGs consist of an N-terminal domain of unknown function and a C-terminal Gα- like domain [25,26] Both structural and experimental evidence indicate that XLG proteins bind Gβγ dimers but have lost the guanine nucleotide dependency [27]. The non-canonical heterotrimeric G protein complex (XLG2/3, AGB1, and AGG1/2) interacts with the FLS2-BIK1-RbohD complex and flg leads to phosphorylation of the amino terminal domain of XLG2 by BIK1. It is known that plant leucine-rich repeat receptor–like kinases (LRR RLKs) phosphorylate AtRGS1 in vitro but little is known about the in vivo interaction, molecular dynamics, or the cellular consequences of this interaction.
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