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Internalization of the m2 muscarinic acetylcholine receptor. Arrestin-independent and -dependent pathways
R Pals-Rylaarsdam1, V V Gurevich, K B Lee
1Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Abstract:
Recent studies have identified agonist-dependent phosphorylation as a critical event in the rapid uncoupling of the m2 muscarinic cholinergic receptors (mAChR) from G-proteins and sequestration of the receptors away from the cell surface. However, mutant m2 mAChRs were identified that were phosphorylated but unable to desensitize in adenylyl cyclase assays, while they internalized like wild type (WT) mAChRs. We have tested whether these properties might stem from differences in the abilities of the WT and mutant mAChR to bind arrestins, proteins implicated in both receptor/G-protein uncoupling and internalization. We have determined that arrestin binding requires phosphorylation at a cluster of Ser/Thr residues in amino acids 307-311 in the m2 mAChR. A strong correlation was found between the ability of WT and mutant receptors to bind arrestins in vitro or in vivo and to desensitize in adenylyl cyclase assays. However, the phosphorylation-dependent internalization of the m2 mAChR in HEK-tsA201 cells did not require arrestins and did not proceed via clathrin-mediated endocytosis. While the m2 mAChR was able to enter a clathrin- and arrestin-dependent pathway when arrestin 2 or arrestin 3 was significantly overexpressed, the preferred pathway of internalization of WT and certain mutant m2 mAChR in HEK-tsA201 cells did not involve participation of arrestins. The results suggest that the phosphorylation-mediated regulation of the m2 mAChR may involve arrestin-dependent and -independent events.
Insights
Agonist-dependent phosphorylation of muscarinic acetylcholine receptors (mAChR) is key for their uncoupling and internalization. Arrestin binding, crucial for desensitization, requires specific phosphorylation sites on m2 mAChR, but internalization can occur independently of arrestins.
Area of Science:
- Pharmacology
- Cell Biology
- Molecular Biology
Background:
- Agonist-dependent phosphorylation is critical for m2 muscarinic acetylcholine receptor (mAChR) uncoupling and cell surface sequestration.
- Mutant m2 mAChRs can be phosphorylated but resist desensitization, yet internalize similarly to wild-type (WT) receptors.
Purpose of the Study:
- To investigate if differential arrestin binding explains the distinct desensitization and internalization properties of WT and mutant m2 mAChRs.
- To elucidate the role of arrestins in m2 mAChR phosphorylation-mediated regulation.
Main Methods:
- Investigated m2 mAChR-arrestin interactions in vitro and in vivo.
- Assessed receptor desensitization using adenylyl cyclase assays.
- Examined receptor internalization pathways, including clathrin-mediated endocytosis, in HEK-tsA201 cells.
Main Results:
- Arrestin binding to m2 mAChR necessitates phosphorylation at Ser/Thr residues 307-311.
- A strong correlation exists between arrestin binding and receptor desensitization.
- M2 mAChR internalization is largely arrestin-independent and does not require clathrin-mediated endocytosis, although these pathways can be engaged under specific overexpression conditions.
Conclusions:
- Phosphorylation-mediated regulation of m2 mAChR involves both arrestin-dependent (desensitization) and arrestin-independent (internalization) mechanisms.
- Receptor internalization can proceed via pathways distinct from canonical arrestin and clathrin involvement.