RhoA-sensitive trafficking of muscarinic acetylcholine receptors

The clathrin-mediated sequestration pathway is used by non-G protein-couple d receptors (e.g., transferrin receptors) and a large number of G protein-c oupled receptors, including beta-2 adrenoceptors and various muscarinic ace tytcholine receptor (mAChR) subtypes. Recently, the ubiquitously expressed small GTPase RhoA has been implicated as a negative regulator of transferri n receptor internalization. Because mAChRs and other G protein-coupled rece ptors are able to activate RhoA, we investigated in HEK-293 cells whether R hoA regulates the sequestration of m1 and m2 mAChRs, which internalize via clathrin-coated and nonclathrin-coated vesicles in HEK-293 cells, respectiv ely. Overexpression of wild-type RhoA inhibited agonist-induced sequestrati on of both m1 and m2 mAChRs by as much as 70%. Inhibition could be reversed by coexpression of Clostridium botulinum C3 transferase, which inactivates RhoA by ADP-ribosylation. Overexpression of C3 transferase alone had no ef fect on mi and m2 mAChR sequestration. In addition, overexpression of RhoA inhibited mi and m2 mAChR transport to the plasma membrane by 60 and 31 %, respectively, which was blocked by coexpression of C3 transferase. We concl ude that RhoA is not an endogenous regulator of mAChR sequestration. but wh en overexpressed, strongly inhibits mAChR trafficking (i.e., sequestration and transport to the plasma membrane) in HEK-293 cells.