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Nucleotide exchange-dependent and nucleotide exchange-independent functions of plant heterotrimeric GTP-binding proteins.

ABSTRACT: Heterotrimeric guanine nucleotide-binding proteins (G proteins), which are composed of ?, ?, and ? subunits, are versatile, guanine nucleotide-dependent, molecular on-off switches. In animals and fungi, the exchange of GDP for GTP on G? controls G protein activation and is crucial for normal cellular responses to diverse extracellular signals. The model plant Arabidopsis thaliana has a single canonical G? subunit, AtGPA1. We found that, in planta, the constitutively active, GTP-bound AtGPA1(Q222L) mutant and the nucleotide-free AtGPA1(S52C) mutant interacted with G??1 and G??2 dimers with similar affinities, suggesting that G protein heterotrimer formation occurred independently of nucleotide exchange. In contrast, AtGPA1(Q222L) had a greater affinity than that of AtGPA1(S52C) for G??3, suggesting that the GTP-bound conformation of AtGPA1(Q222L) is distinct and tightly associated with G??3. Functional analysis of transgenic lines expressing either AtGPA1(S52C) or AtGPA1(Q222L) in the gpa1-null mutant background revealed various mutant phenotypes that were complemented by either AtGPA1(S52C) or AtGPA1(Q222L). We conclude that, in addition to the canonical GDP-GTP exchange-dependent mechanism, plant G proteins can function independently of nucleotide exchange.

SUBMITTER: Maruta N

PROVIDER: S-EPMC6988777 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Nucleotide exchange-dependent and nucleotide exchange-independent functions of plant heterotrimeric GTP-binding proteins.

Maruta Natsumi N Trusov Yuri Y Chakravorty David D Urano Daisuke D Assmann Sarah M SM Botella Jose R JR

Science signaling 20191105 606

Heterotrimeric guanine nucleotide-binding proteins (G proteins), which are composed of α, β, and γ subunits, are versatile, guanine nucleotide-dependent, molecular on-off switches. In animals and fungi, the exchange of GDP for GTP on Gα controls G protein activation and is crucial for normal cellular responses to diverse extracellular signals. The model plant <i>Arabidopsis thaliana</i> has a single canonical Gα subunit, AtGPA1. We found that, in planta, the constitutively active, GTP-bound AtGP ...[more]

PMID: 31690635

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Project description:BackgroundC3G is a guanine nucleotide exchange factor (GEF) that activates Rap1 to promote cell adhesion. Resting C3G is autoinhibited and the GEF activity is released by stimuli that signal through tyrosine kinases. C3G is activated by tyrosine phosphorylation and interaction with Crk adaptor proteins, whose expression is elevated in multiple human cancers. However, the molecular details of C3G activation and the interplay between phosphorylation and Crk interaction are poorly understood.MethodsWe combined biochemical, biophysical, and cell biology approaches to elucidate the mechanisms of C3G activation. Binding of Crk adaptor proteins to four proline-rich motifs (P1 to P4) in C3G was characterized in vitro using isothermal titration calorimetry and sedimentation velocity, and in Jurkat and HEK293T cells by affinity pull-down assays. The nucleotide exchange activity of C3G over Rap1 was measured using nucleotide-dissociation kinetic assays. Jurkat cells were also used to analyze C3G translocation to the plasma membrane and the C3G-dependent activation of Rap1 upon ligation of T cell receptors.ResultsCrkL interacts through its SH3N domain with sites P1 and P2 of inactive C3G in vitro and in Jurkat and HEK293T cells, and these sites are necessary to recruit C3G to the plasma membrane. However, direct stimulation of the GEF activity requires binding of Crk proteins to the P3 and P4 sites. P3 is occluded in resting C3G and is essential for activation, while P4 contributes secondarily towards complete stimulation. Tyrosine phosphorylation of C3G alone causes marginal activation. Instead, phosphorylation primes C3G lowering the concentration of Crk proteins required for activation and increasing the maximum activity. Unexpectedly, optimal activation also requires the interaction of CrkL-SH2 domain with phosphorylated C3G.ConclusionOur study revealed that phosphorylation of C3G by Src and Crk-binding form a two-factor mechanism that ensures tight control of C3G activation. Additionally, the simultaneous SH2 and SH3N interaction of CrkL with C3G, required for the activation, reveals a novel adaptor-independent function of Crk proteins relevant to understanding their role in physiological signaling and their deregulation in diseases. Video abstract.

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Nucleotide exchange-dependent and nucleotide exchange-independent functions of plant heterotrimeric GTP-binding proteins.

Publications

Nucleotide exchange-dependent and nucleotide exchange-independent functions of plant heterotrimeric GTP-binding proteins.

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