Regulation of GPCR expression through an interaction with CCT7, a subunit of the CCT/TRiC complex.

Mechanisms that prevent aggregation and promote folding of nascent G protein-coupled receptors (GPCRs) remain poorly understood. We identified chaperonin containing TCP-1 subunit eta (CCT7) as an interacting partner of the ß-isoform of thromboxane A2 receptor (TPß) by yeast two-hybrid screening. CCT7 coimmunoprecipitated with overexpressed TPß and ß2-adrenergic receptor (ß2AR) in HEK 293 cells, but also with endogenous ß2AR. CCT7 depletion by small interfering RNA reduced total and cell-surface expression of both receptors and caused redistribution of the receptors to juxtanuclear aggresomes, significantly more so for TPß than ß2AR. Interestingly, Hsp90 coimmunoprecipitated with ß2AR but virtually not with TPß, indicating that nascent GPCRs can adopt alternative folding pathways. In vitro pull-down assays showed that both receptors can interact directly with CCT7 through their third intracellular loops and C-termini. We demonstrate that Trp334 in the TPß C-terminus is critical for the CCT7 interaction and plays an important role in TPß maturation and cell-surface expression. Of note, introducing a tryptophan in the corresponding position of the TPα isoform confers the CCT7-binding and maturation properties of TPß. We show that an interaction with a subunit of the CCT/TCP-1 ring complex (TRiC) chaperonin complex is involved in regulating aggregation of nascent GPCRs and in promoting their proper maturation and expression.

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 Gα(s)-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 Gα(i/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.

Regulation of ß2-adrenergic receptor maturation and anterograde trafficking by an interaction with Rab geranylgeranyltransferase: modulation of Rab geranylgeranylation by the receptor.

Previous reports by us and others demonstrated that G protein-coupled receptors interact functionally with Rab GTPases. Here, we show that the ß(2)-adrenergic receptor (ß(2)AR) interacts with the Rab geranylgeranyltransferase α-subunit (RGGTA). Confocal microscopy showed that ß(2)AR co-localizes with RGGTA in intracellular compartments and at the plasma membrane. Site-directed mutagenesis revealed that RGGTA binds to the L(339)L(340) motif in the ß(2)AR 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 ß(2)AR, which requires the interaction of RGGTA with the ß(2)AR L(339)L(340) motif. Furthermore, the ß(2)AR modulates the geranylgeranylation of Rab6a, Rab8a, and Rab11a, but not of other Rab proteins tested in this study. Regulation of Rab geranylgeranylation by the ß(2)AR was dependent on the RGGTA-interacting L(339)L(340) motif. Interestingly, a RGGTA-Y107F mutant was unable to regulate Rab geranylgeranylation but still promoted ß(2)AR 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.

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

We identified the WD-repeat-containing protein, WDR36, as an interacting partner of the ß isoform of thromboxane A(2) receptor (TPß) by yeast two-hybrid screening. We demonstrated that WDR36 directly interacts with the C-terminus and the first intracellular loop of TPß by in vitro GST-pulldown assays. The interaction in a cellular context was observed by co-immunoprecipitation, which was positively affected by TPß stimulation. TPß-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 TPß-induced Gαq signalling. Interestingly, WDR36 co-immunoprecipitated with Gαq, and promoted TPß-Gαq interaction. WDR36 also associated with phospholipase Cß (PLCß) and increased the interaction between Gαq and PLCß, but prevented sequestration of activated Gαq by GRK2. In addition, the presence of TPß in PLCß immunoprecipitates was augmented by expression of WDR36. Finally, disease-associated variants of WDR36 affected its ability to modulate Gαq-mediated signalling by TPß. We report that WDR36 acts as a new scaffold protein tethering a G-protein-coupled receptor, Gαq and PLCß in a signalling complex.

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 D(2). 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 A(2) (TPα), and ß2-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.

Peroxiredoxin-4 interacts with and regulates the thromboxane A(2) receptor.

We identified peroxiredoxin-4 (Prx-4) as a protein interacting with the beta isoform of the thromboxane A(2) receptor (TPbeta) by yeast two-hybrid analysis. Prx-4 co-immunoprecipitated constitutively with TPbeta in HEK293 cells. The second and third intracellular loops as well as the C-terminus of TPbeta interacted directly with Prx-4. Co-expression of Prx-4 caused a 60% decrease in cell surface expression of TPbeta. Prx-4 and TPbeta predominantly co-localized in the endoplasmic reticulum. Co-expression of Prx-4 in cells treated with H(2)O(2) targeted TPbeta for degradation. We show for the first time an interaction between a receptor involved in oxidative stress and Prx-4, an anti-oxidative enzyme.

Small interfering RNA-mediated reduction in heterogeneous nuclear ribonucleoparticule A1/A2 proteins induces apoptosis in human cancer cells but not in normal mortal cell lines.

To prevent their recognition as DNA breaks, the ends of linear chromosomes are organized into telomeres, which are made of proteins bound to telomere-specific, double-stranded repeats and to single-stranded DNA extensions, the G-tails. The mammalian heterogeneous nuclear ribonucleoparticule A1 and A2 proteins can bind with high affinity to such G-tails. Moreover, previous work established that in certain mouse cells a severe reduction in the level of A1 is associated with shortened telomeric repeat tracts, and restoring A1 expression increases telomere length. Here, we document that the expression of A1/A2 proteins is elevated in a variety of human cancers, whereas A1/A2 expression is lower or absent in normal tissues. To determine whether the status of A1/A2 proteins could be improved from cancer markers to cancer targets, we used small interfering RNA-mediated RNA interference to elicit a reduction in A1/A2 proteins in a variety of human cell lines. We show that this treatment provoked specific and rapid cell death by apoptosis in cell lines derived from cervical, colon, breast, ovarian, and brain cancers. Cancer cell lines that lack p53 or express a defective p53 protein were equally sensitive to a small interfering RNA-mediated decrease in A1/A2 expression. The reduction in A1/A2 levels in HeLa cells was associated with a change in the distribution of the lengths of G-tails, an event not observed when apoptosis was induced with staurosporine. Remarkably, comparable decreases in the expression of A1/A2 in several mortal human fibroblastic and epithelial cell lines did not promote cell death. Thus, manipulating the level and activity of A1/A2 proteins may constitute a potent and specific approach in the treatment of human cancers of various origins.