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Ubiquitylation and Activation of a Rab Gtpase Is Promoted by a B2ar–HACE1 Complex

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Ubiquitylation and Activation of a Rab Gtpase Is Promoted by a B2ar–HACE1 Complex ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 111–123 doi:10.1242/jcs.132944 RESEARCH ARTICLE Ubiquitylation and activation of a Rab GTPase is promoted by a b2AR–HACE1 complex Ve´ronik Lachance1,2, Jade Degrandmaison1,2,Se´bastien Marois1,2,Me´lanie Robitaille3, Samuel Ge´nier1,2, Ste´phanie Nadeau1,2, Ste´phane Angers3 and Jean-Luc Parent1,2,* ABSTRACT neurotransmitters, lipids, nucleotides, peptides, ions and photons. All GPCRs share a common molecular topology with We and others have shown that trafficking of G-protein-coupled a hydrophobic core of seven transmembrane a-helices, three receptors is regulated by Rab GTPases. Cargo-mediated regulation intracellular loops, three extracellular loops, an extracellular N- of vesicular transport has received great attention lately. Rab terminus and an intracellular C-terminus. GPCRs are typically GTPases, which form the largest branch of the Ras GTPase delivered to the plasma membrane in a ligand-responsive and superfamily, regulate almost every step of vesicle-mediated signaling-competent form. Following agonist stimulation, the trafficking. Rab GTPases are well-recognized targets of human majority of GPCRs internalize into endosomes and can then diseases but their regulation and the mechanisms connecting them undergo recycling to the cell surface or lysosomal degradation to cargo proteins are still poorly understood. Here, we show by (Costanzi et al., 2009; Pierce et al., 2002; Ritter and Hall, 2009). overexpression and depletion studies that HACE1, a HECT- The fact that more than 30% of prescribed drugs target GPCRs domain-containing ubiquitin ligase, promotes the recycling of the highlights their importance in the treatment of disease (Hopkins b2-adrenergic receptor (b2AR), a prototypical G-protein-coupled and Groom, 2002). Therefore, a better comprehension of the receptor, through a Rab11a-dependent mechanism. Interestingly, cellular events controlling their intracellular trafficking is the b2AR in conjunction with HACE1 triggered ubiquitylation of essential to improve the actual drug efficacy and specificity, Rab11a, as observed by western blot analysis. LC-MS/MS but also to identify new pharmaceutical targets. experiments determined that Rab11a is ubiquitylated on Lys145. Our laboratory and others have previously characterized Rab GTPases as key regulators of GPCR trafficking (Hamelin et al., A Rab11a-K145R mutant failed to undergo b2AR–HACE1-induced ubiquitylation and inhibited the HACE1-mediated recycling of the 2005; Lachance et al., 2011; Parent et al., 2009; Seachrist et al., 2002; Wikstro¨m et al., 2008; Zhang et al., 2009). More than 60 b AR. Rab11a, but not Rab11a-K145R, was activated by b AR– 2 2 Rab GTPases, forming the largest branch of Ras-related small HACE1, indicating that ubiquitylation of Lys145 is involved in GTPases, are involved in almost every step of vesicle-mediated activation of Rab11a. Co-expression of b2AR–HACE1 also transport (Zerial and McBride, 2001). Each Rab GTPase has a potentiated ubiquitylation of Rab6a and Rab8a, but not of other distinct subcellular localization that correlates with the Rab GTPases that were tested. We report a novel regulatory compartments between which they coordinate transport mechanism of Rab GTPases through their ubiquitylation, with (Hutagalung and Novick, 2011). These proteins are well- associated functional effects demonstrated on Rab11a. This recognized targets in human disease, but to date are suggests a new pathway whereby a cargo protein, such as a G- underexplored therapeutically. Elucidation of how dysregulated protein-coupled receptor, can regulate its own trafficking by inducing Rab proteins and accessory proteins contribute to disease remains the ubiquitylation and activation of a Rab GTPase. an area of intensive study and an essential foundation for effective drug targeting. Cancer, neurodegeneration, diabetes and KEY WORDS: GPCR, G-protein-coupled receptor, Rab, Trafficking bone diseases represent examples of pathologies resulting from aberrant function of Rab GTPases (Kelly et al., 2012; Li, 2011; INTRODUCTION Richards and Rutherford, 1990). G-protein-coupled receptors (GPCRs) represent ,4% of the Like other GTPases, Rab proteins shuttle between the inactive human genome and form one of the largest and most studied (GDP-bound) and active (GTP-bound) conformations. To do so, family of proteins (Fredriksson and Schio¨th, 2005; Harrow et al., distinct regulators promote the exchange of GDP to GTP (guanine 2012; Venter et al., 2001). They mediate physiological responses nucleotide exchange factors, GEFs) and GTP hydrolysis (guanine to a vast array of cellular mediators such as hormones, nucleotide activating proteins, GAPs) (Schwartz et al., 2007). Despite the large number of Rabs, very few GEFs and GAPs have 1Service de Rhumatologie, De´partement de Me´decine, Faculte´ de Me´decine et been identified for these small GTPases to date. To cite a few, des Sciences de la Sante´, Universite´ de Sherbrooke, the Centre de Recherche some DENN (differentially expressed in normal and neoplastic Clinique E´ tienne-Le Bel, Sherbrooke, QC J1H 5N4, Canada. 2Institut de cells) domain proteins such as Rab6IP1 (a Rab39 GEF), and Vsp9 Pharmacologie de Sherbrooke, Sherbrooke, QC J1H 5N4, Canada. 3Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, and the domain proteins such as Rabex5 (a Rab5 GEF), have been Department of Biochemistry, Faculty of Medicine, University of Toronto, Toronto, described as Rab GEFs. However, Rab GAPs are known to share ON M5S 3M2, Canada. a common TBC1 (Tre-2/Bub2/Cdc16) domain (Barr and *Author for correspondence ([email protected]) Lambright, 2010; Marat et al., 2011; Xiong et al., 2012). It has been shown that the GPCR angiotensin II type 1A receptor Received 12 April 2013; Accepted 12 October 2013 (AT1AR) increases the GTP-binding of Rab5a. This study not Journal of Cell Science 111 RESEARCH ARTICLE Journal of Cell Science (2014) 127, 111–123 doi:10.1242/jcs.132944 only reported the first evidence that GPCRs might control activity verifying the endogenous colocalization of HACE1 with b2AR. of Rabs, but it also underlined that a direct interaction between To do so, colocalization studies were carried out using cells the receptor and the GTPase seems necessary for this effect stably expressing HA-b2AR. Comparative analyses revealed no (Seachrist et al., 2002). However, it remains unclear whether the significant differences between the trafficking properties of receptor itself acts as a GEF or recruits proteins possessing a GEF transiently expressed b2AR compared with stably expressed activity. b2AR (supplementary material Fig. S1). Both systems were thus It was recently described that mono-ubiquitylation enhances used interchangeably in the present report. As shown in Fig. 1Fa– activation of K-Ras and facilitates its binding to specific effectors d, HACE1 and b2AR colocalize into intracellular compartments (Sasaki et al., 2011). Considering the small number of found throughout the cytoplasm, in the proximity of the cell characterized Rab GEFs, and the size of the Rab family, one membrane and in the perinuclear region, under basal conditions could speculate that activation of Rab GTPases might also be (Fig. 1Fd, extracted colocalizing pixels). Agonist stimulation of controlled by a general mechanism involving post-translational the receptor resulted in internalization of the b2AR from the modifications such as ubiquitylation. However, Rab plasma membrane into intracellular compartments and in a ubiquitylation has not been described so far. Interestingly, distribution of HACE1 that concentrated towards the perinuclear HACE1 (HECT domain and ankyrin repeat containing E3 region where it predominantly colocalized with the receptor ubiquitin protein ligase 1) was identified as a Rab interactor (Fig. 1Ff–i). The intracellular distribution of HACE1 and its (Tang et al., 2011) but no effect on Rab ubiquitylation was colocalization with the receptor was similar to that seen in basal reported. Because b2-adrenergic receptor (b2AR) trafficking is conditions as receptor recycling progressed (Fig. 1Fk–s). regulated by various Rab proteins (Awwad et al., 2007; Dong and The functional implication of endogenous HACE1 on the Wu, 2007; Dong et al., 2010; Hammad et al., 2012; Moore et al., trafficking of stably expressed b2AR was assessed in cells 2004; Parent et al., 2009; Seachrist et al., 2000), we investigated transfected with HACE1 siRNA. Depletion of HACE1 whether HACE1 was able to modulate b2AR trafficking. Here, significantly reduced the cell surface expression of the b2AR we report that co-expression of b2AR with HACE1 induces the compared with expression in control cells (Fig. 2A). There was a ubiquitylation and activation of Rab11a, which in turn regulates ,33% increase in apparent agonist-induced internalization of the b2AR recycling. Ubiquitylation of other specific Rabs was also receptor when cells were transfected with HACE1 siRNA observed in the presence of b2AR–HACE1. Together, our data compared with cells transfected with control siRNA (60% uncover a new regulatory mechanism for Rab GTPases by which compared with 45% internalization, respectively) (Fig. 2B). a cargo protein can direct its own trafficking. b2AR recycling after agonist-induced internalization was inhibited by ,25% in cells depleted of HACE1 compared with RESULTS the control (Fig. 2C). These data validated the results obtained HACE1 interacts with and
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