A Rab10-ACAP1-Arf6 GTPases cascade modulates M4 muscarinic acetylcholine receptor trafficking and signaling.

Membrane trafficking processes regulate the G protein-coupled receptor activity. The muscarinic acetylcholine receptors (mAChRs) are highly pursued drug targets for neurological diseases, but the cellular machineries that control the trafficking of these receptors remain largely elusive. Here, we revealed the role of the small GTPase Rab10 as a negative regulator for the post-activation trafficking of M4 mAChR and the underlying mechanism. We show that constitutively active Rab10 arrests the receptor within Rab5-positive early endosomes and significantly hinders the resensitization of M4-mediated Ca signaling. Mechanistically, M4 binds to Rab10-GTP, which requires the motif RKKRQMAA (R-A) within the third intracellular loop. Moreover, Rab10-GTP inactivates Arf6 by recruiting the Arf6 GTPase-activating protein, ACAP1. Strikingly, deletion of the motif R-A causes M4 to bypass the control by Rab10 and switch to the Rab4-facilitated fast recycling pathway, thus reusing the receptor. Therefore, Rab10 couples the cargo sorting and membrane trafficking regulation through cycle between GTP-bound and GDP-bound state. Our findings suggest a model that Rab10 binds to the M4 like a molecular brake and controls the receptor's transport through endosomes, thus modulating the signaling, and this regulation is specific among the mAChR subtypes.