Geneviève Laroche

Peroxiredoxin-4 interacts with and regulates the thromboxane A2 receptor

We identified peroxiredoxin‐4 (Prx‐4) as a protein interacting with the β isoform of the thromboxane A2 receptor (TPβ) by yeast two‐hybrid analysis. Prx‐4 co‐immunoprecipitated constitutively with TPβ in HEK293 cells. The second and third intracellular loops as well as the C‐terminus of TPβ interacted directly with Prx‐4. Co‐expression of Prx‐4 caused a 60% decrease in cell surface expression of TPβ. Prx‐4 and TPβ predominantly co‐localized in the endoplasmic reticulum. Co‐expression of Prx‐4 in cells treated with H2O2 targeted TPβ for degradation. We show for the first time an interaction between a receptor involved in oxidative stress and Prx‐4, an anti‐oxidative enzyme.

WDR36 acts as a scaffold protein tethering a G-protein-coupled receptor, Gαq and phospholipase Cβ in a signalling complex.

We identified the WD-repeat-containing protein, WDR36, as an interacting partner of the β isoform of thromboxane A2 receptor (TPβ) by yeast two-hybrid screening. We demonstrated that WDR36 directly interacts with the C-terminus and the first intracellular loop of TPβ by in vitro GST-pulldown assays. The interaction in a cellular context was observed by co-immunoprecipitation, which was positively affected by TPβ stimulation. TPβ–WDR36 colocalization was detected by confocal microscopy at the plasma membrane in non-stimulated HEK293 cells but the complex translocated to intracellular vesicles following receptor stimulation. Coexpression of WDR36 and its siRNA-mediated knockdown, respectively, increased and inhibited TPβ-induced Gαq signalling. Interestingly, WDR36 co-immunoprecipitated with Gαq, and promoted TPβ–Gαq interaction. WDR36 also associated with phospholipase Cβ (PLCβ) and increased the interaction between Gαq and PLCβ, but prevented sequestration of activated Gαq by GRK2. In addition, the presence of TPβ in PLCβ immunoprecipitates was augmented by expression of WDR36. Finally, disease-associated variants of WDR36 affected its ability to modulate Gαq-mediated signalling by TPβ. We report that WDR36 acts as a new scaffold protein tethering a G-protein-coupled receptor, Gαq and PLCβ in a signalling complex.

G-protein signaling modulator-3, a gene linked to autoimmune diseases, regulates monocyte function and its deficiency protects from inflammatory arthritis

Polymorphism at the GPSM3 gene locus is inversely associated with four systemic autoimmune diseases, including rheumatoid arthritis and ankylosing spondylitis. G-protein signaling modulator-3 (GPSM3) expression is most pronounced in myeloid cells, in which it targets heterotrimeric G-protein Gαi subunits of chemokine receptors, critical to immune function. To begin to explore the regulatory role of GPSM3 in monocytes, human THP-1 and primary mouse myeloid cells were cultured under stimulus conditions; GPSM3 was found by immunoblotting to be expressed at highest levels in the mature monocyte. To evaluate the effects of GPSM3 deficiency on a myeloid-dependent autoimmune disease, collagen antibody-induced arthritis (CAIA) was induced in Gpsm3-/- and control mice, which were then analyzed for clinical score, paw swelling, intra-articular proinflammatory markers, and histopathology. Mice lacking GPSM3 were protected from CAIA, and expression of monocyte-representative pro-inflammatory chemokine receptors and cytokines in paws of Gpsm3-/- mice were decreased. Flow cytometry, apoptosis, and transwell chemotaxis experiments were conducted to further characterize the effect of GPSM3 deficiency on survival and chemokine responsiveness of monocytes. GPSM3-deficient myeloid cells had reduced migration ex vivo to CCL2, CX3CL1, and chemerin and enhanced apoptosis in vitro. Our results suggest that GPSM3 is an important regulator of monocyte function involving mechanisms of differentiation, survival, and chemotaxis, and deficiency in GPSM3 expression is protective in acute inflammatory arthritis.

G-protein signaling modulator-3 regulates heterotrimeric G-protein dynamics through dual association with Gβ and Gαi protein subunits.

Background: GPSM3 is known to bind inactive Gαi and act as a GDP dissociation inhibitor, preventing Gβγ association. Results: GPSM3 also interacts with Gβ, independent of Gαi binding and excluding a Gγ interaction. Conclusion: GPSM3 stabilizes Gβ until formation of the Gβγ dimer. Significance: GPSM3 may establish a novel checkpoint in the regulation of heterotrimeric protein subunit dynamics. Regulation of the assembly and function of G-protein heterotrimers (Gα·GDP/Gβγ) is a complex process involving the participation of many accessory proteins. One of these regulators, GPSM3, is a member of a family of proteins containing one or more copies of a small regulatory motif known as the GoLoco (or GPR) motif. Although GPSM3 is known to bind Gαi·GDP subunits via its GoLoco motifs, here we report that GPSM3 also interacts with the Gβ subunits Gβ1 to Gβ4, independent of Gγ or Gα·GDP subunit interactions. Bimolecular fluorescence complementation studies suggest that the Gβ-GPSM3 complex is formed at, and transits through, the Golgi apparatus and also exists as a soluble complex in the cytoplasm. GPSM3 and Gβ co-localize endogenously in THP-1 cells at the plasma membrane and in a juxtanuclear compartment. We provide evidence that GPSM3 increases Gβ stability until formation of the Gβγ dimer, including association of the Gβ-GPSM3 complex with phosducin-like protein PhLP and T-complex protein 1 subunit eta (CCT7), two known chaperones of neosynthesized Gβ subunits. The Gβ interaction site within GPSM3 was mapped to a leucine-rich region proximal to the N-terminal side of its first GoLoco motif. Both Gβ and Gαi·GDP binding events are required for GPSM3 activity in inhibiting phospholipase-Cβ activation. GPSM3 is also shown in THP-1 cells to be important for Akt activation, a known Gβγ-dependent pathway. Discovery of a Gβ/GPSM3 interaction, independent of Gα·GDP and Gγ involvement, adds to the combinatorial complexity of the role of GPSM3 in heterotrimeric G-protein regulation.

G Protein signaling modulator-3 inhibits the inflammasome activity of NLRP3.

Background: NLRP3 is a key regulator of innate inflammation and is linked to inflammatory diseases. Results: GPSM3 associates with NLRP3 and inhibits its function. Conclusion: GPSM3 specifically inhibits NLRP3-dependent inflammasome activity by interacting with its leucine-rich repeat domain. Significance: This association uncovers a putative new mechanism of NLRP3 control, linking a G protein modulator to NLRP3-dependent inflammatory diseases. Inflammasomes are multi-protein complexes that regulate maturation of the interleukin 1β-related cytokines IL-1β and IL-18 through activation of the cysteine proteinase caspase-1. NOD-like receptor family, pyrin domain containing 3 (NLRP3) protein is a key component of inflammasomes that assemble in response to a wide variety of endogenous and pathogen-derived danger signals. Activation of the NLRP3-inflammasome and subsequent secretion of IL-1β is highly regulated by at least three processes: transcriptional activation of both NLRP3 and pro-IL-1β genes, non-transcriptional priming of NLRP3, and final activation of NLRP3. NLRP3 is predominantly expressed in cells of the hematopoietic lineage. Using a yeast two-hybrid screen, we identified the hematopoietic-restricted protein, G protein signaling modulator-3 (GPSM3), as a NLRP3-interacting protein and a negative regulator of IL-1β production triggered by NLRP3-dependent inflammasome activators. In monocytes, GPSM3 associates with the C-terminal leucine-rich repeat domain of NLRP3. Bone marrow-derived macrophages lacking GPSM3 expression exhibit an increase in NLRP3-dependent IL-1β, but not TNF-α, secretion. Furthermore, GPSM3-null mice have enhanced serum and peritoneal IL-1β production following Alum-induced peritonitis. Our findings suggest that GPSM3 acts as a direct negative regulator of NLRP3 function.

The N-terminal coiled-coil domain of the cytohesin/ARNO family of guanine nucleotide exchange factors interacts with Gαq

Cytohesins are guanine-nucleotide exchange factors (GEF) for the Arf family of GTPases. One member of the Arf family, ARF6, plays an active role in the intracellular trafficking of G protein-coupled receptors. We have previously reported that Gαq signaling leads to the activation of ARF6, possibly through a direct interaction with cytohesin-2/ARNO. Here, we report that Gαq can directly interact with cytohesin-1, another Arf-GEF of the ARNO/cytohesin family. Cytohesin-1 preferentially associated with a constitutively active mutant of Gαq (Gαq-Q209L) compared to wild-type Gαq in HEK293 cells. Stimulation of TPβ, a Gαq-coupled receptor, to activate Gαq resulted in the promotion of a protein complex between Gαq and cytohesin-1. Confocal immunofluorescence microscopy revealed that wild-type Gαq and cytohesin-1 co-localized in intracellular compartments and at or near the plasma membrane. In contrast, expression of Gαq-Q209L induced a drastic increase in the localization of cytohesin-1 at the plasma membrane. Expression of a dominant-negative mutant of cytohesin-1 reduced by 40% the agonist-induced internalization of TPβ, a process that we previously demonstrated to be dependent on Gαq-mediated signaling and Arf6 activation. Using deletion mutants, we show that cytohesin-1 interacts with Gαq through its N-terminal coiled-coil domain. Cytohesin-1 and cytohesin-2/ARNO mutants lacking the coiled-coil domain were unable to relay Gαq-mediated activation of Arf6. This is the first report of an interaction between the coiled-coil domain of the cytohesin/ARNO family of Arf-GEFs and a member of the heterotrimeric G proteins.

Regulation of the Subcellular Localization of the G-protein Subunit Regulator GPSM3 through Direct Association with 14-3-3 Protein

Background: GPSM3, a guanine nucleotide dissociation inhibitor acting on Gαi subunit family members, is both cytoplasm- and nucleus-localized. Results: GPSM3 interacts directly with 14-3-3 through a phosphorylation-dependent mechanism. Conclusion: Interaction with 14-3-3 stabilizes a cytoplasmic pool of GPSM3. Significance: Our findings represent the first demonstration of post-translational modulation of GPSM3, a hematopoietic-restricted G-protein subunit regulator. G-protein signaling modulator-3 (GPSM3), also known as G18 or AGS4, is a member of the Gαi/o-Loco (GoLoco) motif containing proteins. GPSM3 acts through its two GoLoco motifs to exert GDP dissociation inhibitor activity over Gαi subunits; recently revealed is the existence of an additional regulatory site within GPSM3 directed toward monomeric Gβ subunits during their biosynthesis. Here, using in silico and proteomic approaches, we have found that GPSM3 also interacts directly with numerous members of the 14-3-3 protein family. This interaction is dependent on GPSM3 phosphorylation, creating a mode II consensus 14-3-3 binding site. 14-3-3 binding to the N-terminal disordered region of GPSM3 confers stabilization from protein degradation. The complex of GPSM3 and 14-3-3 is exclusively cytoplasmic, and both moieties mutually control their exclusion from the nucleus. Phosphorylation of GPSM3 by a proline-directed serine/threonine kinase and the resultant association of 14-3-3 is the first description of post-translational regulation of GPSM3 subcellular localization, a process that likely regulates important spatio-temporal aspects of G-protein-coupled receptor signaling modulation by GPSM3.