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

We and others have shown that trafficking of G protein-coupled receptors is regulated by Rab GTPases. Cargo-mediated regulation of vesicular transport has received great attention lately. Rab GTPases, forming the largest branch of the Ras GTPase superfamily, regulate almost every step of vesicle-mediated trafficking. Rab GTPases are well-recognized targets of human diseases but their regulation and the mechanisms connecting them to cargo proteins are still poorly understood. Herein, we show by overexpression/depletion studies that HACE1, a HECT domain-containing ubiquitin ligase, promotes the recycling of the β2-adrenergic receptor (β2AR), a prototypical G protein-coupled receptor, through a Rab11a-dependent mechanism. Interestingly, the β2AR in conjunction with HACE1 triggered ubiquitination of Rab11a, as observed by Western blot analysis. LC-MS/MS experiments determined that Rab11a is ubiquitnatied on Lys145. A Rab11a-K145R mutant failed to undergo β2AR/HACE1-induced ubiquitination and inhibited the HACE1-mediated recycling of the β2AR. Rab11a, but not Rab11a-K145R, was activated by β2AR/HACE1 indicating that ubiquitination of Lys145 is involved in Rab11a activation. β2AR/HACE1 co-expression also potentiated ubiquitination of Rab6a and Rab8a, but not of other Rab GTPases that were tested. We report a novel regulatory mechanism of Rab GTPases by their ubiquitination with associated functional effects demonstrated on Rab11a. This partakes into a new pathway whereby a cargo protein, like a G protein-coupled receptor, can regulate its own trafficking by inducing the ubiquitination and activation of a Rab GTPase.