Agonist-dependent delivery of M(2) muscarinic acetylcholine receptors to the cell surface after pertussis toxin treatment.

The internalization of the M(2) muscarinic cholinergic receptor (mAChR) proceeds through an atypical pathway that is independent of arrestin and clathrin function and shows a unique sensitivity to dynamin when the receptor is expressed in human embryonic kidney 293 cells. In this report we demonstrate that the internalization of the M(2) mAChR was modulated by activation of heterotrimeric G proteins, because treatment with pertussis toxin, which ADP-ribosylates G proteins of the G(i/o) family, caused a significant delay in the onset of internalization of the M(2) mAChR. The effects of pertussis toxin could not be explained by alteration of the agonist-dependent phosphorylation of the M(2) mAChR. The modulation of internalization by pertussis toxin was revealed to be due to recruitment of intracellular receptors to the cell surface upon agonist treatment. Pretreatment with pertussis toxin also greatly increased both the rate and extent of recovery of M(2) mAChRs to the cell surface after agonist-mediated internalization. These results demonstrate a novel aspect involved in the regulation of GPCRs. As with the tightly controlled internalization of GPCRs, the delivery of GPCRs to the cell surface is also highly regulated.

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

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.

Trafficking of M(2) muscarinic acetylcholine receptors.

Internalization is an important mechanism regulating the agonist-dependent responses of G-protein-coupled receptors. The internalization of the M(2) muscarinic cholinergic receptors (mAChR) in HEK293 cells has been demonstrated to occur by an unknown mechanism that is independent of arrestins and dynamin. In this study we examined various aspects of the trafficking of the M(2) mAChR in HEK293 cells to characterize this unknown pathway of internalization. Internalization of the M(2) mAChR was rapid and extensive, but prolonged incubation with agonist did not lead to appreciable down-regulation (a decrease in total receptor number) of the receptors. Recovery of M(2) mAChRs to the cell surface following agonist-mediated internalization was a very slow process that contained protein synthesis-dependent and -independent components. The protein synthesis-dependent component of the recovery of receptors to the cell surface did not appear to reflect a requirement for synthesis of new receptors, as no changes in total receptor number were observed either in the presence or absence of cycloheximide. Phosphorylation of the M(2) mAChR did not appear to influence the rate or extent of the recovery of receptors to the cell surface, as the recovery of a phosphorylation-deficient mutant M(2) mAChR, the N,C(Ala-8) mutant, was similar to the recovery of the wild type M(2) mAChR. Finally, the constitutive, nonagonist-dependent internalization and recycling of the M(2) mAChR was very slow and also contained protein synthesis-dependent and -independent components, suggesting that a similar pathway controls the recovery from agonist-dependent and -independent internalization. Overall, these data demonstrated a variety of previously unappreciated facets involved in the regulation of M(2) mAChRs.

Desensitization of G-protein-coupled receptors in the cardiovascular system.

Multiple mechanisms exist to control the signaling and density of G-protein-coupled receptors (GPRs). Upon agonist binding and receptor activation, a series of reactions participate in the turn off or desensitization of GPRs. Many GPRs are phosphorylated by protein kinases and consequently uncoupled from G proteins. In addition, many GPRs are sequestered from the cell surface and become inaccessible to their activating ligands. Both receptor:G protein uncoupling and receptor sequestration may involve the participation of arrestins or other proteins. A model for receptor regulation has been developed from studies of the beta-adrenergic receptor. However, recent studies suggest that other GPRs important in the cardiovascular system, such as the muscarinic cholinergic receptors that regulate heart rate, might be regulated by mechanisms other than those that regulate the beta-adrenergic receptors. This review summarizes our current understanding of the processes involved in the desensitization of GPRs.

Molecular events associated with the regulation of signaling by M2 muscarinic receptors.

Multiple events are associated with the regulation of signaling by the M2 muscarinic cholinergic receptors (mAChRs). Desensitization of the attenuation of adenylyl cyclase by the M2 mAChRs appears to involve agonist-dependent phosphorylation of M2 mAChRs by G-protein coupled receptor kinases (GRKs) that phosphorylate the receptors in a serine/threonine rich motif in the 3rd intracellular domain of the receptors. Mutation of residues 307-311 from TVSTS to AVAAA in this domain of the human M2 mAChR results in a loss of receptor/G-protein uncoupling and a loss of arrestin binding. Agonist-induced sequestration of receptors away from their normal membrane environment is also regulated by agonist-induced phosphorylation of the M2 mAChRs on the 3rd intracellular domain, but in HEK cells, the predominant pathway of internalization is not regulated by GRKs or arrestins. This pathway of internalization is not inhibited by a dominant negative dynamin, and does not appear to involve either clathrin coated pits or caveolae. The signaling of the M2 mAChR to G-protein regulated inwardly rectifying K channels (GIRKs) can be modified by RGS proteins. In HEK cells, expression of RGS proteins leads to a constitutive activation of the channels through a mechanism that depends on Gbetagamma. RGS proteins appear to increase the concentration of free Gbetagamma in addition to acting as GAPs. Thus multiple mechanisms acting at either the level of the M2 mAChRs or the G-proteins can contribute to the regulation of signaling via the M2 mAChRs.