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Drosophila Ric-8 interacts with the Gα12/13 subunit, Concertina, during activation of the Folded gastrulation pathway.

Peters KA, Rogers SL - Mol. Biol. Cell (2013)

Bottom Line: A component of this pathway, the Drosophila Gα12/13 subunit, Concertina (Cta), is necessary to trigger actomyosin contractility during gastrulation events.Ric-8 mutants exhibit similar gastrulation defects to Cta mutants.We show that Ric-8 regulates this pathway through physical interaction with Cta and preferentially interacts with inactive Cta and directs its localization within the cell.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 Lineberger Comprehensive Cancer Center, Chapel Hill, NC 27514.

ABSTRACT
Heterotrimeric G proteins, composed of α, β, and γ subunits, are activated by exchange of GDP for GTP on the Gα subunit. Canonically, Gα is stimulated by the guanine-nucleotide exchange factor (GEF) activity of ligand-bound G protein-coupled receptors. However, Gα subunits may also be activated in a noncanonical manner by members of the Ric-8 family, cytoplasmic proteins that also act as GEFs for Gα subunits. We used a signaling pathway active during Drosophila gastrulation as a model system to study Ric-8/Gα interactions. A component of this pathway, the Drosophila Gα12/13 subunit, Concertina (Cta), is necessary to trigger actomyosin contractility during gastrulation events. Ric-8 mutants exhibit similar gastrulation defects to Cta mutants. Here we use a novel tissue culture system to study a signaling pathway that controls cytoskeletal rearrangements necessary for cellular morphogenesis. We show that Ric-8 regulates this pathway through physical interaction with Cta and preferentially interacts with inactive Cta and directs its localization within the cell. We also use this system to conduct a structure-function analysis of Ric-8 and identify key residues required for both Cta interaction and cellular contractility.

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Individual residues derived from Ric-8 cluster mutants comprise key interaction sites for Cta binding and function. (A) Individual Ric-8 point mutants from cluster mutants (1, 9, and 13) negatively regulate binding to Myc-CtaGA. Cells were transfected with GFP, Ric-8–GFP, or individual Ric-8–GFP point mutants and CtaGA. IPs were performed with GFP-binding protein and probed with anti-GFP and anti-Myc. (B) Quantification of the IP experiments in A (black bars). The pull-down:input ratios were determined using quantitative densitometry and normalized to Ric-8–GFP (±SEM; error bars, p < 0.05). S2R+ cells were depleted of endogenous Ric-8, transfected with Ric-8–GFP or individual Ric-8–GFP point mutants, and scored for the percentage of transfected cells constricting within the population (±SEM; hatched bars). Dashed lines indicate where two separate gels have been combined. (C) Proposed model for Ric-8 function within the Fog signaling pathway. Ric-8 initially acts to chaperone the folding of Cta and is released before Cta association with β13F and γ1. The heterotrimer is targeted to the plasma membrane, where it interacts with a GPCR for Fog. Fog binding activates Cta through exchange of GDP for GTP. Cta-GTP activates RhoGEF2, and RhoGEF2’s GAP activity catalyzes hydrolysis of GTP to GDP. Ric-8 may then either bind Cta-GDP or stabilize a nucleotide-free version of Cta. Ric-8 then localizes the inactive Cta for reactivation and reinsertion into the Fog signaling pathway, either by returning it to the heterotrimer to be reactivated by the GPCR (A) or through disassociation of GDP, which facilitates GTP binding, and subsequent pathway reinsertion directly upstream of RhoGEF2 (B).
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Figure 6: Individual residues derived from Ric-8 cluster mutants comprise key interaction sites for Cta binding and function. (A) Individual Ric-8 point mutants from cluster mutants (1, 9, and 13) negatively regulate binding to Myc-CtaGA. Cells were transfected with GFP, Ric-8–GFP, or individual Ric-8–GFP point mutants and CtaGA. IPs were performed with GFP-binding protein and probed with anti-GFP and anti-Myc. (B) Quantification of the IP experiments in A (black bars). The pull-down:input ratios were determined using quantitative densitometry and normalized to Ric-8–GFP (±SEM; error bars, p < 0.05). S2R+ cells were depleted of endogenous Ric-8, transfected with Ric-8–GFP or individual Ric-8–GFP point mutants, and scored for the percentage of transfected cells constricting within the population (±SEM; hatched bars). Dashed lines indicate where two separate gels have been combined. (C) Proposed model for Ric-8 function within the Fog signaling pathway. Ric-8 initially acts to chaperone the folding of Cta and is released before Cta association with β13F and γ1. The heterotrimer is targeted to the plasma membrane, where it interacts with a GPCR for Fog. Fog binding activates Cta through exchange of GDP for GTP. Cta-GTP activates RhoGEF2, and RhoGEF2’s GAP activity catalyzes hydrolysis of GTP to GDP. Ric-8 may then either bind Cta-GDP or stabilize a nucleotide-free version of Cta. Ric-8 then localizes the inactive Cta for reactivation and reinsertion into the Fog signaling pathway, either by returning it to the heterotrimer to be reactivated by the GPCR (A) or through disassociation of GDP, which facilitates GTP binding, and subsequent pathway reinsertion directly upstream of RhoGEF2 (B).

Mentions: We identified four Ric-8–GFP mutants with clustered point mutations (1, 9, 10, and 13) that had significantly reduced binding to Myc-CtaGA (Figure 5, B and C, and Table 1). To further parse out the individual residues responsible for this interaction, we made single point mutants for each cluster of more than one mutated amino acid (Supplemental Figure S6 and Table 1). Mutant 10 is a singular mutation, so it was not tested again. We identified specific residues within mutants 1, 9, and 13 that attenuated the ability of Cta to bind Ric-8 (Figure 6, A and B, and Table 1). Although not statistically significant, both Myc-Cta and Myc-CtaQL variants exhibited decreased binding to mutant 1 and moderate to high binding to mutants 9, 10, and 13 (Supplemental Figure S7, A–D). The fact that mutants 9, 10, and 13 inhibited binding to Myc-CtaGA but did not strongly affect Myc-Cta or Myc-CtaQL binding indicates that the C-terminal region of Ric-8 may be important for high-affinity binding seen specifically in the Ric-8/GTP–free Cta interaction, whereas the N-terminal residues found in mutant 1 could be important for global Ric-8 association and function.


Drosophila Ric-8 interacts with the Gα12/13 subunit, Concertina, during activation of the Folded gastrulation pathway.

Peters KA, Rogers SL - Mol. Biol. Cell (2013)

Individual residues derived from Ric-8 cluster mutants comprise key interaction sites for Cta binding and function. (A) Individual Ric-8 point mutants from cluster mutants (1, 9, and 13) negatively regulate binding to Myc-CtaGA. Cells were transfected with GFP, Ric-8–GFP, or individual Ric-8–GFP point mutants and CtaGA. IPs were performed with GFP-binding protein and probed with anti-GFP and anti-Myc. (B) Quantification of the IP experiments in A (black bars). The pull-down:input ratios were determined using quantitative densitometry and normalized to Ric-8–GFP (±SEM; error bars, p < 0.05). S2R+ cells were depleted of endogenous Ric-8, transfected with Ric-8–GFP or individual Ric-8–GFP point mutants, and scored for the percentage of transfected cells constricting within the population (±SEM; hatched bars). Dashed lines indicate where two separate gels have been combined. (C) Proposed model for Ric-8 function within the Fog signaling pathway. Ric-8 initially acts to chaperone the folding of Cta and is released before Cta association with β13F and γ1. The heterotrimer is targeted to the plasma membrane, where it interacts with a GPCR for Fog. Fog binding activates Cta through exchange of GDP for GTP. Cta-GTP activates RhoGEF2, and RhoGEF2’s GAP activity catalyzes hydrolysis of GTP to GDP. Ric-8 may then either bind Cta-GDP or stabilize a nucleotide-free version of Cta. Ric-8 then localizes the inactive Cta for reactivation and reinsertion into the Fog signaling pathway, either by returning it to the heterotrimer to be reactivated by the GPCR (A) or through disassociation of GDP, which facilitates GTP binding, and subsequent pathway reinsertion directly upstream of RhoGEF2 (B).
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Related In: Results  -  Collection

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Figure 6: Individual residues derived from Ric-8 cluster mutants comprise key interaction sites for Cta binding and function. (A) Individual Ric-8 point mutants from cluster mutants (1, 9, and 13) negatively regulate binding to Myc-CtaGA. Cells were transfected with GFP, Ric-8–GFP, or individual Ric-8–GFP point mutants and CtaGA. IPs were performed with GFP-binding protein and probed with anti-GFP and anti-Myc. (B) Quantification of the IP experiments in A (black bars). The pull-down:input ratios were determined using quantitative densitometry and normalized to Ric-8–GFP (±SEM; error bars, p < 0.05). S2R+ cells were depleted of endogenous Ric-8, transfected with Ric-8–GFP or individual Ric-8–GFP point mutants, and scored for the percentage of transfected cells constricting within the population (±SEM; hatched bars). Dashed lines indicate where two separate gels have been combined. (C) Proposed model for Ric-8 function within the Fog signaling pathway. Ric-8 initially acts to chaperone the folding of Cta and is released before Cta association with β13F and γ1. The heterotrimer is targeted to the plasma membrane, where it interacts with a GPCR for Fog. Fog binding activates Cta through exchange of GDP for GTP. Cta-GTP activates RhoGEF2, and RhoGEF2’s GAP activity catalyzes hydrolysis of GTP to GDP. Ric-8 may then either bind Cta-GDP or stabilize a nucleotide-free version of Cta. Ric-8 then localizes the inactive Cta for reactivation and reinsertion into the Fog signaling pathway, either by returning it to the heterotrimer to be reactivated by the GPCR (A) or through disassociation of GDP, which facilitates GTP binding, and subsequent pathway reinsertion directly upstream of RhoGEF2 (B).
Mentions: We identified four Ric-8–GFP mutants with clustered point mutations (1, 9, 10, and 13) that had significantly reduced binding to Myc-CtaGA (Figure 5, B and C, and Table 1). To further parse out the individual residues responsible for this interaction, we made single point mutants for each cluster of more than one mutated amino acid (Supplemental Figure S6 and Table 1). Mutant 10 is a singular mutation, so it was not tested again. We identified specific residues within mutants 1, 9, and 13 that attenuated the ability of Cta to bind Ric-8 (Figure 6, A and B, and Table 1). Although not statistically significant, both Myc-Cta and Myc-CtaQL variants exhibited decreased binding to mutant 1 and moderate to high binding to mutants 9, 10, and 13 (Supplemental Figure S7, A–D). The fact that mutants 9, 10, and 13 inhibited binding to Myc-CtaGA but did not strongly affect Myc-Cta or Myc-CtaQL binding indicates that the C-terminal region of Ric-8 may be important for high-affinity binding seen specifically in the Ric-8/GTP–free Cta interaction, whereas the N-terminal residues found in mutant 1 could be important for global Ric-8 association and function.

Bottom Line: A component of this pathway, the Drosophila Gα12/13 subunit, Concertina (Cta), is necessary to trigger actomyosin contractility during gastrulation events.Ric-8 mutants exhibit similar gastrulation defects to Cta mutants.We show that Ric-8 regulates this pathway through physical interaction with Cta and preferentially interacts with inactive Cta and directs its localization within the cell.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 Lineberger Comprehensive Cancer Center, Chapel Hill, NC 27514.

ABSTRACT
Heterotrimeric G proteins, composed of α, β, and γ subunits, are activated by exchange of GDP for GTP on the Gα subunit. Canonically, Gα is stimulated by the guanine-nucleotide exchange factor (GEF) activity of ligand-bound G protein-coupled receptors. However, Gα subunits may also be activated in a noncanonical manner by members of the Ric-8 family, cytoplasmic proteins that also act as GEFs for Gα subunits. We used a signaling pathway active during Drosophila gastrulation as a model system to study Ric-8/Gα interactions. A component of this pathway, the Drosophila Gα12/13 subunit, Concertina (Cta), is necessary to trigger actomyosin contractility during gastrulation events. Ric-8 mutants exhibit similar gastrulation defects to Cta mutants. Here we use a novel tissue culture system to study a signaling pathway that controls cytoskeletal rearrangements necessary for cellular morphogenesis. We show that Ric-8 regulates this pathway through physical interaction with Cta and preferentially interacts with inactive Cta and directs its localization within the cell. We also use this system to conduct a structure-function analysis of Ric-8 and identify key residues required for both Cta interaction and cellular contractility.

Show MeSH
Related in: MedlinePlus