G Protein-Coupled Receptor Kinases 3 and 6 Use Different Pathways to Desensitize the Endogenous M3 Muscarinic Acetylcholine Receptor in Human SH-SY5Y Cells

2001 ◽  
Vol 60 (2) ◽  
pp. 321-330 ◽  
Author(s):  
Jonathon M. Willets ◽  
R. A. John Challiss ◽  
Eamonn Kelly ◽  
Stefan R. Nahorski
Keyword(s):  
G Protein ◽  
Acetylcholine Receptor ◽  
Muscarinic Acetylcholine Receptor ◽  
G Protein Coupled Receptor ◽  
Muscarinic Acetylcholine ◽  
M3 Muscarinic Acetylcholine Receptor ◽  
Receptor Kinases ◽  
G Protein Coupled

Internalization of the M2 muscarinic acetylcholine receptor proceeds through an atypical pathway in HEK293 cells that is independent of clathrin and caveolae

2001 ◽  
Vol 114 (4) ◽  
pp. 739-746 ◽  
Author(s):  
A.G. Roseberry ◽  
M.M. Hosey
Keyword(s):  
G Protein ◽  
Acetylcholine Receptor ◽  
Muscarinic Acetylcholine Receptor ◽  
Hek293 Cells ◽  
G Protein Coupled Receptor ◽  
Coated Pits ◽  
Muscarinic Acetylcholine ◽  
G Protein Coupled

The M2 muscarinic acetylcholine receptor is a G-protein coupled receptor that undergoes agonist-induced internalization through an unidentified pathway that exhibits an atypical dependence on dynamin function in HEK293 cells. In this report we utilized several independent approaches to reveal that the internalization of the M2 muscarinic acetylcholine receptor did not utilize clathrin-coated pits or caveolae. However, we did observe that treatment with hypertonic sucrose, which is widely reported to specifically inhibit endocytosis through clathrin-coated pits, completely inhibited internalization of the M2 muscarinic acetylcholine receptor. Thus, the pathway that mediates the internalization of the M2 muscarinic acetylcholine receptor appears to be atypical in that it exhibits an unusual sensitivity to dynamin and is inhibited by hypertonic sucrose but lacks the involvement of clathrin and caveolae.

scholarly journals Disentangling bias between Gq, GRK2, and arrestin3 recruitment to the M3 muscarinic acetylcholine receptor

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Anja Floeser ◽  
Katharina Becker ◽  
Evi Kostenis ◽  
Gabriele König ◽  
Cornelius Krasel ◽  
...  
Keyword(s):  
G Protein ◽  
Acetylcholine Receptor ◽  
Rank Order ◽  
Muscarinic Acetylcholine Receptor ◽  
G Protein Coupled Receptors ◽  
Effector Proteins ◽  
Muscarinic Acetylcholine ◽  
Extracellular Signals ◽  
G Protein Coupled ◽  
Different Levels

G protein-coupled receptors (GPCRs) transmit extracellular signals to the inside by activation of intracellular effector proteins. Different agonists can promote differential receptor-induced signaling responses – termed bias – potentially by eliciting different levels of recruitment of effector proteins. As activation and recruitment of effector proteins might influence each other, thorough analysis of bias is difficult. Here, we compared the efficacy of seven agonists to induce G protein, G protein-coupled receptor kinase 2 (GRK2), as well as arrestin3 binding to the muscarinic acetylcholine receptor M3 by utilizing FRET-based assays. In order to avoid interference between these interactions, we studied GRK2 binding in the presence of inhibitors of Gi and Gq proteins and analyzed arrestin3 binding to prestimulated M3 receptors to avoid differences in receptor phosphorylation influencing arrestin recruitment. We measured substantial differences in the agonist efficacies to induce M3R-arrestin3 versus M3R-GRK2 interaction. However, the rank order of the agonists for G protein- and GRK2-M3R interaction was the same, suggesting that G protein and GRK2 binding to M3R requires similar receptor conformations, whereas requirements for arrestin3 binding to M3R are distinct.

scholarly journals Rab5-dependent Trafficking of the m4 Muscarinic Acetylcholine Receptor to the Plasma Membrane, Early Endosomes, and Multivesicular Bodies

2001 ◽  
Vol 276 (50) ◽  
pp. 47590-47598 ◽  
Author(s):  
Laura A. Volpicelli ◽  
James J. Lah ◽  
Allan I. Levey
Keyword(s):  
G Protein ◽  
Acetylcholine Receptor ◽  
Intracellular Trafficking ◽  
Intracellular Localization ◽  
Muscarinic Acetylcholine Receptor ◽  
Dominant Negative ◽  
Multivesicular Bodies ◽  
Muscarinic Acetylcholine ◽  
Early Endosomes ◽  
G Protein Coupled

The m4 subtype of muscarinic acetylcholine receptor regulates many physiological processes and is a novel therapeutic target for neurologic and psychiatric disorders. However, little is known about m4 regulation because of the lack of pharmacologically selective ligands. A crucial component of G protein-coupled receptor regulation is intracellular trafficking. We thus used subtype-specific antibodies and quantitative immunocytochemistry to characterize the intracellular trafficking of m4. We show that following carbachol stimulation, m4 co-localizes with transferrin, and the selective marker of early endosomes, EEA1. In addition, m4 intracellular localization depends on Rab5 activity. The dominant negative Rab5S34N inhibits m4 endocytosis initially following carbachol stimulation, and reduces the size of m4 containing vesicles. The constitutively active Rab5Q79L enhances m4 intracellular distribution, even in unstimulated cells. Rab5Q79L also produces strikingly enlarged vacuoles, which by electron microscopy contain internal vesicles, suggesting that they are multivesicular bodies. m4 localizes both to the perimeter and interior of these vacuoles. In contrast, transferrin localizes only to the vacuole perimeter, demonstrating divergence of m4 trafficking from the pathway followed by constitutively endocytosed transferrin. We thus suggest a novel model by which multivesicular bodies sort G protein-coupled receptors from a transferrin-positive recycling pathway to a nonrecycling, possibly degradative pathway.

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