Abstract
The clathrin-mediated sequestration pathway is used by non-G protein-coupled receptors (e.g., transferrin receptors) and a large number of G protein-coupled receptors, including beta-2 adrenoceptors and various muscarinic acetylcholine receptor (mAChR) subtypes. Recently, the ubiquitously expressed small GTPase RhoA has been implicated as a negative regulator of transferrin receptor internalization. Because mAChRs and other G protein-coupled receptors are able to activate RhoA, we investigated in HEK-293 cells whether RhoA regulates the sequestration of m1 and m2 mAChRs, which internalize via clathrin-coated and nonclathrin-coated vesicles in HEK-293 cells, respectively. Overexpression of wild-type RhoA inhibited agonist-induced sequestration of both m1 and m2 mAChRs by as much as 70%. Inhibition could be reversed by coexpression ofClostridium botulinum C3 transferase, which inactivates RhoA by ADP-ribosylation. Overexpression of C3 transferase alone had no effect on m1 and m2 mAChR sequestration. In addition, overexpression of RhoA inhibited m1 and m2 mAChR transport to the plasma membrane by 60 and 31%, respectively, which was blocked by coexpression of C3 transferase. We conclude that RhoA is not an endogenous regulator of mAChR sequestration, but when overexpressed, strongly inhibits mAChR trafficking (i.e., sequestration and transport to the plasma membrane) in HEK-293 cells.
Footnotes
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Send reprint requests to: Chris J. van Koppen, Ph.D., Institut für Pharmakologie, Universitätsklinikum Essen, D-45122 Essen, Germany. E-mail:van_koppen{at}uni-essen.de
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↵1 This work was supported by the Deutsche Forschungsgemeinschaft, and the IFORES program of the Universitätsklinikum Essen.
- Abbreviations:
- DME
- Dulbecco’s modified Eagle’s
- mAChR
- muscarinic acetylcholine receptor
- G protein
- guanine nucleotide-binding protein
- GPCR
- G protein-coupled receptor
- NMS
- N-methylscopolamine
- PBS
- phosphate-buffered saline
- QNB
- quinuclidinyl-benzilate
- SDS
- sodium dodecyl sulfate
- Received May 12, 1998.
- Accepted July 30, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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