Research ArticleDEVELOPMENTAL DISORDERS

Dominant-negative Gα subunits are a mechanism of dysregulated heterotrimeric G protein signaling in human disease

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Science Signaling  12 Apr 2016:
Vol. 9, Issue 423, pp. ra37
DOI: 10.1126/scisignal.aad2429
  • Fig. 1 ACS mutations affect residues that cluster around the nucleotide binding pocket of Gαi3.

    Top: Schematic diagram depicting structural elements of Gαi3 and the location of all five mutations found to date in patients with ACS. Switch regions I, II, and III involved in activation-induced conformational changes are depicted in gray, and the five conserved G boxes involved in nucleotide binding are shown in blue. Bottom: Three-dimensional representation of the Gαi nucleotide binding pocket [Protein Data Bank (PDB): 1GIA]. Amino acids mutated in ACS (beige) cluster around the nucleotide binding pocket (GTP in purple).

  • Fig. 2 ACS mutations increase the frequency of developmental defects induced by ectopic expression of Gα i3 in X. laevis embryos.

    (A) Validation of an assay to monitor Gαi3-induced developmental defects in X. laevis. Left: Schematic diagram illustrating the relationship between tissue remodeling defects and inversion of the gravitational orientation of Gαi-injected embryos. Top right: Representative embryos injected with mRNA encoding wild-type Gαi3 (Gαi3 WT; 60 pg) compared with uninjected controls at the late neurula stage. Arrowhead marks inverted gravitational orientation [neural tube (NT) facing downward]. Bottom right: Sagittal sections of a representative control and a Gαi3-injected embryo with impaired archenteron (arc) inflation and incomplete blastocoel (bc) removal. (B and C) Assessment of the percentage of embryos displaying a gravitational defect after injection with 30 or 60 pg of mRNA encoding Gαi3 WT or ACS mutant. Representative images (B) are presented and marked as in (A). Quantification is shown in (C). Data are means ± SE of the phenotype proportion of the indicated total number (n) of embryos from each test group. *P < 0.01 and **P < 0.001 for Gαi3 mutants compared to WT using Fisher’s exact test. Immunoblots (IB; C) confirm protein abundance in embryos injected with the indicated mRNAs.

  • Fig. 3 ACS-associated Gαi3 mutants fail to bind GTP in vitro.

    (A) Coomassie blue–stained gel of purified proteins treated as indicated in a trypsin protection assay to assess whether Gαi3 S47R is activated by nucleotides. Arrow marks full-length His-Gαi3; * marks trypsin-resistant fragment of active His-Gαi3. (B) Effect of S47R mutation on the steady-state GTPase activity of Gαi3. Purified His-Gαi3 proteins were incubated in the presence of radiolabeled GTP, and activity was determined by measuring the release of radioactive phosphate. (C and D) GTPγS binding by Gαi3 mutant (S47R) and WT was determined by intrinsic fluorescence measurements (F0, basal fluorescence) (C) or radioligand binding (D). (E) Assessment of GDP occupation on Gαi3 WT and S47R. Nucleotide content of equimolar amounts of His-Gαi3 WT (black) or His-Gαi3 S47R (gray) (top) was compared to GDP (black) or GTP (gray) standards (bottom) by HPLC (A280, nucleotide absorbance at 280 nm). (F and G) GTPγS binding as assessed by trypsin protection assays by Gαi3 ACS mutants G40R, G45V, S47R, T48N, and N269Y. Representative immunoblots of Gαi3 (arrow, full-length Gαi3; *, trypsin-resistant fragment of active Gαi3) in HEK293T cell lysates treated as indicated in (F) and quantified in (G). One experiment representative of three is shown in (A) to (D) and (F). Data in (G) are means ± SEM (n = 4 experiments). *P < 0.05 and **P < 0.01 compared to WT using Student’s t test. Endo, endogenous.

  • Fig. 4 ACS-associated Gαi3 mutants are not activated by the GPCR A1R.

    (A) Schematic diagram depicting the BRET assay used to monitor the dissociation of Gαi3:Gβγ trimers upon GPCR stimulation. Under resting conditions, Venus-tagged Gβγ (V-Gβγ) associates with Gαi3 and BRET signals are low. Upon stimulation of A1R with adenosine, Gi trimers dissociate, and free V-Gβγ binds to mas-GRK3ct-Nluc (GRK), leading to an increase of BRET signal. (B) Assessment of A1R-induced dissociation of Gi trimers containing Gαi3 WT or ACS mutant subunits. HEK293T cells were transfected with plasmids encoding for Venus(155–239)-Gβ1 (VC-Gβ1), Venus(1–155)-Gγ2 (VN-Gγ2), mas-GRK3ct-Nluc, and A1R along with Gαi3 WT or ACS mutants (G40R, G45V, S47R, T48N, or N269Y), and BRET was measured every second (as described in Materials and Methods). Equal amounts of V-Gβγ and the different Gαi3 proteins were verified by immunoblotting (fig. S2). After 30 s under resting conditions, cells were stimulated with adenosine (1 μM). The average of BRET signal during the first 30 s (basal BRET) was subtracted from each data point to present the data as increase of BRET (ΔBRET). One representative experiment of four is shown.

  • Fig. 5 i3 ACS mutations increase binding to GEFs.

    (A) Schematic of Ric-8A binding to different conformations of Gαi3 during its activation cycle and description of constructs that mimic each one of these conformations. (B) Ric-8A binding by Gαi3 WT or ACS mutant using yeast two-hybrid assays (schematic, top; AD, Gal4 activation domain; BD, Gal4 DNA binding domain). Data are means ± SEM (n = 3 experiments: *P < 0.05 and **P < 0.01, Student’s t test). (C) Immunoblot of the strains used in (B). (D) Relative affinity of ACS-associated Gαi3 mutants for the ETAR GPCR as determined by immunoprecipitation (IP) with myc or immunoglobulin G (IgG) antibody in lysates of HEK293T cells transfected with myc-ETAR and the indicated Gαi3 constructs. Blots are representative of three experiments.

  • Fig. 6 ACS-associated Gαi3 mutants prevent ETAR-mediated activation of Gαq.

    (A and B) BRET assay in HEK293T cells transfected with Gαq (left) or Gαi3 (right) along with V-Gβγ, mas-GRK3-Nuc, and ETAR, stimulated (arrow) with the indicated concentrations of ET-1, analyzed as in Fig. 4B. One representative experiment is shown in (A). Data in (B) are means ± SEM (n = 3 experiments). (C) Putative mechanism of dominant-negative action of ACS-associated Gαi3 mutants on Gαq activation by ETAR as determined by BRET assays. In cells expressing Gαi3 WT (top), ETAR couples predominantly to Gq. In cells expressing Gαi3 mutants (bottom), high-affinity binding of mutants to ETAR precludes Gq activation. (D) HEK293T cells transfected with Gαq (left) or Gαi3 WT (right) (1 μg) and substoichiometric amounts of Gαi3 WT or an ACS mutant (0.12 μg) along with V-Gβγ, mas-GRK3-Nuc, and ETAR were stimulated (arrow) with ET-1 (0.3 μM Gαq/3 μM Gαi3). One representative of three experiments, with respective immunoblots (below), is shown. HA, hemagglutinin.

  • Fig. 7 Proposed model.

    Left: In healthy individuals, ETAR couples predominantly to Gαq/11, which promotes PLCβ4 activation and the expression of DLX5/6 transcription factors required for neural crest cell differentiation and normal craniofacial development. Right: In type I ACS patients, ACS-associated Gαi3 mutants bind with high affinity to ETAR and form an unproductive complex. This reduces the availability of ETAR for Gαq activation, which blocks the signaling pathway required for normal craniofacial development.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/9/423/ra37/DC2

    Fig. S1. ACS mutations in Gαi3 affect residues conserved across all Gα proteins in humans.

    Fig. S2. ACS-associated Gαi3 mutants bind to Gβγ.

    Fig. S3. The subcellular localization of ACS-associated Gαi3 mutants is similar to that of wild-type Gαi3.

    Fig. S4. Gαq and Gαi3 associate similarly with Gβγ subunits.

  • Supplementary Materials for:

    Dominant-negative Gα subunits are a mechanism of dysregulated heterotrimeric G protein signaling in human disease

    Arthur Marivin, Anthony Leyme, Kshitij Parag-Sharma, Vincent DiGiacomo, Anthony Y. Cheung, Lien T. Nguyen, Isabel Dominguez, Mikel Garcia-Marcos*

    *Corresponding author. E-mail: mgm1{at}bu.edu

    This PDF file includes:

    • Fig. S1. ACS mutations in Gαi3 affect residues conserved across all Gα proteins in humans.
    • Fig. S2. ACS-associated Gαi3 mutants bind to Gβγ
    • Fig. S3. The subcellular localization of ACS-associated Gαi3 mutants is similar to that of wild-type Gαi3.
    • Fig. S4. Gαq and Gαi3 associate similarly with Gβγ subunits.

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    Citation: A. Marivin, A. Leyme, K. Parag-Sharma, V. DiGiacomo, A. Y. Cheung, L. T. Nguyen, I. Dominguez, M. Garcia-Marcos, Dominant-negative Gα subunits are a mechanism of dysregulated heterotrimeric G protein signaling in human disease. Sci. Signal. 9, ra37 (2016).

    © 2016 American Association for the Advancement of Science

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