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

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. Colocalization 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 siRNAmediated 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 (TP␣), and ␤2-adrenergic receptor), whereas it did not affect the expression of green fluorescent protein, GRK2 (G proteincoupled receptor kinase 2), and VSVG (vesicular stomatitis virus glycoprotein), showing specificity toward G proteincoupled receptors. Altogether, these results suggest that ANKRD13C acts as a molecular chaperone for G proteincoupled receptors, regulating their biogenesis and exit from the ER.As the largest family of membrane receptors, G proteincoupled receptors (GPCRs) 4 mediate physiological responses to a vast array of cellular mediators such as hormones, neurotransmitters, lipids, nucleotides, peptides, ions, and photons. To be fully functional, GPCRs need to be delivered to the plasma membrane in a ligand-responsive and signaling-competent form. The synthesis and maturation of these receptors require a complex combination of processes that include protein folding, posttranslational modifications, and transport through distinct cellular compartments of the secretory pathway (1). In recent years, GPCRs were found to interact with many proteins besides G proteins, and such interactions were shown to play important roles in receptor biogenesis and trafficking. For example, the Drosophila cyclophilin protein ninaA (neither inactivation nor afterpotential A) and its mammalian homolog, RanBP2 (Ran-binding protein 2), associate with rhodopsin and opsins, respectively. They have been characterized as chaperones that can facilitate receptor folding and are essential for efficient localization of opsins at the cell surface (2-4). The receptor-activity-modifying proteins represent another example of GPCR-interacting proteins implicated in the trafficking and functionality of receptors....

Pharmacological chaperoning: A primer on mechanism and pharmacology

“…In this regard, PC stabilization of A1 adenosine receptor folding intermediates promotes the dissociation of the receptor from HSP 40 protein D1 receptor interacting protein 78 (DRiP78) [91]. Intriguingly, PCs promote the interaction of the prostanoid DP1 receptor and ANKRD13C [92], a cytoplasmically localized molecular chaperone for GPCRs that aids in forward trafficking [93]. …”

Ubiquitination and activation of a Rab GTPase promoted by a β2-Adrenergic Receptor/HACE1 complex

“…HACE1 contains six putative ankyrin repeat domains usually known for their scaffolding properties in protein complex assembly (Mosavi et al, 2004). We recently showed that ankyrin-repeat-containing proteins can promote export of GPCRs (Parent et al, 2010). It is thus possible that HACE1 increases cell surface targeting of the b 2 AR through protein interactions with its ankyrin repeats.…”

“…For quantification of expression of cell-surface receptors, 6.5610 5 HEK293 cells were plated in 24-well plates pre-coated with 0.1 mg/ml poly-L-lysine (Sigma), transfected with the indicated constructs and then maintained for an additional 48 hours and then processed for ELISA as described previously (Hamelin et al, 2005;Lachance et al, 2011;Parent et al, 2009;Parent et al, 2010;Parent et al, 1999;Thériault et al, 2004).…”

“…Both buffers were supplemented with protease inhibitors (9 nM pepstatin, 9 nM antipain, 10 nM leupeptin and 10 nM chymostatin) (Sigma Aldrich). Immunoprecipitations were then carried out as we described previously (Lachance et al, 2011;Parent et al, 2010).…”

“…HEK293 cells were transfected with 10 nM oligonucleotide using the Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's instructions. Protein expression analysis by western blotting and ELISA experiments were performed at 72 hours posttransfection as usual (Lachance et al, 2011;Parent et al, 2010).…”