Pascale Labrecque

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.

Regulation of β2-Adrenergic Receptor Maturation and Anterograde Trafficking by an Interaction with Rab Geranylgeranyltransferase MODULATION OF Rab GERANYLGERANYLATION BY THE RECEPTOR

Background: Interacting partners and regulation of Rab geranylgeranyltransferase are poorly characterized. Mechanisms of GPCR maturation and anterograde trafficking are not fully understood. Results: RGGTA interacts with a dileucine motif in the β2AR to regulate β2AR maturation/anterograde trafficking and β2AR-mediated Rab geranylgeranylation. Conclusion: RGGTA and the β2AR interact functionally. Significance: This is the first demonstration of a functional interaction between RGGTA and a transmembrane receptor. Previous reports by us and others demonstrated that G protein-coupled receptors interact functionally with Rab GTPases. Here, we show that the β2-adrenergic receptor (β2AR) interacts with the Rab geranylgeranyltransferase α-subunit (RGGTA). Confocal microscopy showed that β2AR co-localizes with RGGTA in intracellular compartments and at the plasma membrane. Site-directed mutagenesis revealed that RGGTA binds to the L339L340 motif in the β2AR C terminus known to be involved in the transport of the receptor from the endoplasmic reticulum to the cell surface. Modulation of the cellular levels of RGGTA protein by overexpression or siRNA-mediated knockdown of the endogenous protein demonstrated that RGGTA has a positive role in the maturation and anterograde trafficking of the β2AR, which requires the interaction of RGGTA with the β2AR L339L340 motif. Furthermore, the β2AR modulates the geranylgeranylation of Rab6a, Rab8a, and Rab11a, but not of other Rab proteins tested in this study. Regulation of Rab geranylgeranylation by the β2AR was dependent on the RGGTA-interacting L339L340 motif. Interestingly, a RGGTA-Y107F mutant was unable to regulate Rab geranylgeranylation but still promoted β2AR maturation, suggesting that RGGTA may have functions independent of Rab geranylgeranylation. We demonstrate for the first time an interaction between a transmembrane receptor and RGGTA which regulates the maturation and anterograde transport of the receptor, as well as geranylgeranylation of Rab GTPases.

Inverse agonist and pharmacochaperone properties of MK-0524 on the prostanoid DP1 receptor.

Prostaglandin D2 (PGD2) acts through two G protein-coupled receptors (GPCRs), the prostanoid DP receptor and CRTH2 also known as DP1 and DP2, respectively. Several previously characterized GPCR antagonists are now classified as inverse agonists and a number of GPCR ligands are known to display pharmacochaperone activity towards a given receptor. Here, we demonstrate that a DP1 specific antagonist, MK-0524 (also known as laropiprant), decreased basal levels of intracellular cAMP produced by DP1, a Gαs-coupled receptor, in HEK293 cells. This reduction in cAMP levels was not altered by pertussis toxin treatment, indicating that MK-0524 did not induce coupling of DP1 to Gαi/o proteins and that this ligand is a DP1 inverse agonist. Basal ERK1/2 activation by DP1 was not modulated by MK-0524. Interestingly, treatment of HEK293 cells expressing Flag-tagged DP1 with MK-0524 promoted DP1 cell surface expression time-dependently to reach a maximum increase of 50% compared to control after 24 h. In contrast, PGD2 induced the internalization of 75% of cell surface DP1 after the same time of stimulation. The increase in DP1 cell surface targeting by MK-0524 was inhibited by Brefeldin A, an inhibitor of transport from the endoplasmic reticulum-Golgi to the plasma membrane. Confocal microscopy confirmed that a large population of DP1 remained trapped intracellularly and co-localized with calnexin, an endoplasmic reticulum marker. Redistribution of DP1 from intracellular compartments to the plasma membrane was observed following treatment with MK-0524 for 24 h. Furthermore, MK-0524 promoted the interaction between DP1 and the ANKRD13C protein, which we showed previously to display chaperone-like effects towards the receptor. We thus report that MK-0524 is an inverse agonist and a pharmacochaperone of DP1. Our findings may have important implications during therapeutic treatments with MK-0524 and for the development of new molecules targeting DP1.

ANKRD13C Acts as a Molecular Chaperone for G Protein-coupled Receptors

Although the mechanisms that regulate folding and maturation of newly synthesized G protein-coupled receptors are crucial for their function, they remain poorly characterized. By yeast two-hybrid screening, we have isolated ANKRD13C, a protein of unknown function, as an interacting partner for the DP receptor for prostaglandin D2. In the present study we report the characterization of this novel protein as a regulator of DP biogenesis and trafficking in the biosynthetic pathway. Co-localization by confocal microscopy with an endoplasmic reticulum (ER) marker, subcellular fractionation experiments, and demonstration of the interaction between ANKRD13C and the cytoplasmic C terminus of DP suggest that ANKRD13C is a protein associated with the cytosolic side of ER membranes. Co-expression of ANKRD13C with DP initially increased receptor protein levels, whereas siRNA-mediated knockdown of endogenous ANKRD13C decreased them. Pulse-chase experiments indicated that ANKRD13C can promote the biogenesis of DP by inhibiting the degradation of newly synthesized receptors. However, a prolonged interaction between ANKRD13C and DP resulted in ER retention of misfolded/unassembled forms of the receptor and to their proteasome-mediated degradation. ANKRD13C also regulated the expression of other GPCRs tested (CRTH2, thromboxane A2 (TPα), and β2-adrenergic receptor), whereas it did not affect the expression of green fluorescent protein, GRK2 (G protein-coupled receptor kinase 2), and VSVG (vesicular stomatitis virus glycoprotein), showing specificity toward G protein-coupled receptors. Altogether, these results suggest that ANKRD13C acts as a molecular chaperone for G protein-coupled receptors, regulating their biogenesis and exit from the ER.

Adenovirus protease expressed in insect cells cleaves adenovirus proteins, ovalbumin and baculovirus protease in the absence of activating peptide

The adenovirus type 2 protease (EP) was expressed by infecting insect cells with a recombinant baculovirus. Immunoblot and activity analysis showed EP to be present in both the nucleus and cytoplasm. While the insect cell expressed EP was more soluble than the Escherichia coli expressed EP, its activity was one quarter of the latter, suggesting that eukaryotic postsynthetic modifications are not essential for enzyme activity. EP inactivated a cytoplasmic cathepsin-like baculovirus-encoded cysteine protease which carries a single EP cleavage site and which was capable of digesting most adenovirus structural proteins in vitro. In addition to cleavage of the baculovirus protease, the adenovirus EP was also able to cleave ovalbumin and canine adenovirus protein pre-VII, in the absence of activating peptide. EP activation therefore may occur by means of factors other than the specific activating peptide.

β-Trace Protein Assays: A Comparison Between Nephelometric and ELISA Methodologies

To the Editor: b-Trace protein (BTP) is a low-molecular-weight glycoprotein with multiple isoforms of varying molecular weight (23-29 kDa) and an emerging marker of glomerular filtration rate (GFR). Equations for estimated GFR (eGFR) that incorporate BTP have been proposed. As of 2013, assays for BTP in biological fluids were commercially available from Cayman Chemicals (an immunometric ELISA using monoclonal murine antibodies) and Siemens (a particle-enhanced nephelometric immunoassay using polyclonal rabbit antibodies against human urinary BTP). The assays use different analytical techniques and different antibodies, which may recognize different glycoprotein epitopes and therefore may not bind to all BTP isoforms.Unlike creatinine and cystatinC, there are no higher order referencematerials available for BTP. The aim of this studywas to examine differences in these BTP assays and their impact on eGFR. We also sought to examine differences in the Siemens BTP assay in different laboratories and over time. Detailed methods are in Item S1. Frozen serum was measured in 2015 and came from the following adult sources: kidney transplant recipients (sampled 2007-2012; n 5 77), patients with cirrhosis (sampled 2014; n 5 103), and patients with chronic kidney disease (CKD; sampled 2014; n 5 101). Serum was split 3 ways, frozen, and shipped to the participating laboratories. BTP was measured using the Siemens N Latex BTP assay at the Children’s Hospital of Eastern Ontario (CHEO), Canada, and at the University of Minnesota, United States. The University of Sherbrooke, Canada, measured BTP using the Cayman assay. Absolute differences between paired BTP values were calculated. Paired t tests were used to compare mean differences. This analysis was repeated after stratifying patients by BTP less than and greater than the median of the average of the 2 assays being compared and by population. Percentages of samples with paired values within 10% (P10) and 30% (P30) of each other were calculated. A similar analysis was performed using eGFR calculated using the CKD-EPI BTP equation, assuming a 65-year-old woman. Relationships between paired BTP and paired eGFR results were also examined using Deming regression and Bland-Altman analysis, respectively. Siemens CHEO concentrations were 0.77 6 1.01 mg/L lower than Cayman concentrations (P , 0.001), with greater absolute difference in the higher BTP subgroup (Table 1). Similar