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ABSTRACT: Previous reports by us and others demonstrated that G protein-coupled receptors interact functionally with Rab GTPases. Here, we show that the β(2)-adrenergic receptor (β(2)AR) interacts with the Rab geranylgeranyltransferase α-subunit (RGGTA). Confocal microscopy showed that β(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 β(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 β(2)AR, which requires the interaction of RGGTA with the β(2)AR L(339)L(340) motif. Furthermore, the β(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 β(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 β(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.
Journal of Biological Chemistry 11/2011; 286(47):40802-13. · 4.77 Impact Factor
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ABSTRACT: We identified the WD-repeat-containing protein, WDR36, as an interacting partner of the β isoform of thromboxane A(2) 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.
Journal of Cell Science 10/2011; 124(Pt 19):3292-304. · 6.11 Impact Factor
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ABSTRACT: 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 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.
Journal of Biological Chemistry 10/2010; 285(52):40838-51. · 4.77 Impact Factor
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ABSTRACT: 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.
FEBS Letters 09/2007; 581(20):3863-8. · 3.54 Impact Factor
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ABSTRACT: 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.
Cancer Research 12/2003; 63(22):7679-88. · 7.86 Impact Factor
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ABSTRACT: The thromboxane A2 receptor (TP) is a G protein-coupled receptor that is expressed as two alternatively spliced isoforms, α (343 residues) and
β (407 residues) that share the first 328 residues. We have previously shown that TPβ, but not TPα, undergoes agonist-induced
internalization in a dynamin-, GRK-, and arrestin-dependent manner. In the present report, we demonstrate that TPβ, but not
TPα, also undergoes tonic internalization. Tonic internalization of TPβ was temperature- and dynamin-dependent and was inhibited
by sucrose and NH4Cl treatment but unaffected by wild-type or dominant-negative GRKs or arrestins. Truncation and site-directed mutagenesis
revealed that a YX
3φ motif (whereX is any residue and φ is a bulky hydrophobic residue) found in the proximal portion of the carboxyl-terminal tail of TPβ was
critical for tonic internalization but had no role in agonist-induced internalization. Interestingly, introduction of either
a YX
2φ or YX
3φ motif in the carboxyl-terminal tail of TPα induced tonic internalization of this receptor. Additional analysis revealed
that tonically internalized TPβ undergoes recycling back to the cell surface suggesting that tonic internalization may play
a role in maintaining an intracellular pool of TPβ. Our data demonstrate the presence of distinct signals for tonic and agonist-induced
internalization of TPβ and represent the first report of a YX
3φ motif involved in tonic internalization of a cell surface receptor.
Journal of Biological Chemistry 03/2001; 276(10):7079-7085. · 4.77 Impact Factor
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ABSTRACT: Thromboxane A2(TXA2) potently stimulates platelet aggregation and smooth muscle constriction and is thought to play a role in myocardial infarction,
atherosclerosis, and bronchial asthma. The TXA2receptor (TXA2R) is a member of the G protein-coupled receptor family and is found as two alternatively spliced isoforms, α (343 residues)
and β (407 residues), which share the first 328 residues. In the present report, we demonstrate by enzyme-linked immunosorbent
assay and immunofluorescence microscopy that the TXA2Rβ, but not the TXA2Rα, undergoes agonist-induced internalization when expressed in HEK293 cells as well as several other cell types. Various
dominant negative mutants were used to demonstrate that the internalization of the TXA2Rβ is dynamin-, GRK-, and arrestin-dependent in HEK293 cells, suggesting the involvement of receptor phosphorylation and clathrin-coated
pits in this process. Interestingly, the agonist-stimulated internalization of both the α and β isoforms, but not of a mutant
truncated after residue 328, can be promoted by overexpression of arrestin-3, identifying the C-tails of both receptors as
necessary in arrestin-3 interaction. Simultaneous mutation of two dileucine motifs in the C-tail of TXA2Rβ did not affect agonist-promoted internalization. Analysis of various C-tail deletion mutants revealed that a region between
residues 355 and 366 of the TXA2Rβ is essential for agonist-promoted internalization. These data demonstrate that alternative splicing of the TXA2R plays a critical role in regulating arrestin binding and subsequent receptor internalization.
Journal of Biological Chemistry 03/1999; 274(13):8941-8948. · 4.77 Impact Factor
