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Differential role of beta-arrestin ubiquitination in agonist-promoted down-regulation of M1 vs M2 muscarinic acetylcholine receptors.

Mosser VA, Jones KT, Hoffman KM, McCarty NA, Jackson DA - J Mol Signal (2008)

Bottom Line: Internalization of receptors was unaffected by proteasome inhibitors, but down-regulation was significantly reduced, suggesting a role for the ubiquitination machinery in promoting down-regulation of the receptors.Agonist-promoted internalization of the M2 mAChR was not affected by expression of beta-arrestin lysine mutants lacking putative ubiquitination sites, beta-arrestin 2K18R, K107R, K108R, K207R, K296R, while down-regulation and stable co-localiztion of the receptor with this beta-arrestin lysine mutant were significantly reduced.Interestingly, expression of beta-arrestin 2K18R, K107R, K108R, K207R, K296R increased the agonist-promoted down-regulation of the M1 mAChR but did not result in a stable co-localiztion of the receptor with this beta-arrestin lysine mutant.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical and Pharmaceutical Sciences, College of Health Professions and Biomedical Science, Skaggs Building Room 376, The University of Montana, Missoula, MT 59812, USA. darrell.jackson@umontana.edu.

ABSTRACT

Background: Sustained agonist-promoted ubiquitination of beta-arrestin has been correlated with increased stability of the GPCR - beta-arrestin complex. Moreover, abrogation of beta-arrestin ubiquitination has been reported to inhibit receptor internalization with minimal effects on receptor degradation.

Results: Herein we report that agonist activation of M1 mAChRs produces a sustained beta-arrestin ubiquitination but no stable co-localization with beta-arrestin. In contrast, sustained ubiquitination of beta-arrestin by activation of M2 mAChRs does result in stable co-localization between the M2 mAChR and beta-arrestin. Internalization of receptors was unaffected by proteasome inhibitors, but down-regulation was significantly reduced, suggesting a role for the ubiquitination machinery in promoting down-regulation of the receptors. Given the ubiquitination status of beta-arrestin following agonist treatment, we sought to determine the effects of beta-arrestin ubiquitination on M1 and M2 mAChR down-regulation. A constitutively ubiquitinated beta-arrestin 2 chimera in which ubiquitin is fused to the C-terminus of beta-arrestin 2 (YFP-beta-arrestin 2-Ub) significantly increased agonist-promoted down-regulation of both M1 and M2 mAChRs, with the effect substantially higher on the M2 mAChR. Based on this observation, we were interested in examining the effects of disruption of potential ubiquitination sites in the beta-arrestin sequence on receptor down-regulation. Agonist-promoted internalization of the M2 mAChR was not affected by expression of beta-arrestin lysine mutants lacking putative ubiquitination sites, beta-arrestin 2K18R, K107R, K108R, K207R, K296R, while down-regulation and stable co-localiztion of the receptor with this beta-arrestin lysine mutant were significantly reduced. Interestingly, expression of beta-arrestin 2K18R, K107R, K108R, K207R, K296R increased the agonist-promoted down-regulation of the M1 mAChR but did not result in a stable co-localiztion of the receptor with this beta-arrestin lysine mutant.

Conclusion: These findings indicate that ubiquitination of beta-arrestin has a distinct role in the differential trafficking and degradation of M1 and M2 mAChRs.

No MeSH data available.


Related in: MedlinePlus

Time-course of agonist-promoted down-regulation of mAChRs in MEF. MEF wt cells were transfected with eGFP-M1 (◆) or HA-M2 (■) mAChR. 24 hr following transfection, cells were treated with 1 mM carbachol for the indicated time. Down-regulation was determined using [3H]-QNB binding (fmol/mg protein) in crude membranes as described in methods. The M1 mAChR displays nearly threefold the down-regulation of the M2 subtype over the same time course. Data are expressed as percent down-regulation compared to t = 0 control and are presented as mean ± standard error of the mean for three independent experiments with duplicate data points. Total mAChR expressed (fmol/mg) at t = 0 was 4000 for both M1 and M2 mAChRs.
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Figure 1: Time-course of agonist-promoted down-regulation of mAChRs in MEF. MEF wt cells were transfected with eGFP-M1 (◆) or HA-M2 (■) mAChR. 24 hr following transfection, cells were treated with 1 mM carbachol for the indicated time. Down-regulation was determined using [3H]-QNB binding (fmol/mg protein) in crude membranes as described in methods. The M1 mAChR displays nearly threefold the down-regulation of the M2 subtype over the same time course. Data are expressed as percent down-regulation compared to t = 0 control and are presented as mean ± standard error of the mean for three independent experiments with duplicate data points. Total mAChR expressed (fmol/mg) at t = 0 was 4000 for both M1 and M2 mAChRs.

Mentions: To determine whether or not MEF cells could be used in the current study, we performed agonist-promoted down-regulation time courses for both the M1 and M2 mAChR subtypes. Total receptor numbers were measured using the non-selective membrane permeable muscarinic antagonist quinuclidinyl benzilate (QNB), which binds to both surface and intracellular pools of mAChR. Wild-type MEF cells that were transfected with either eGFP-M1 mAChR or HA-M2 mAChR resulted in similar expression levels (~4000 fmol/mg). After 24 hr, cells were treated with 1 mM carbachol for the indicated time. Maximal down-regulation of the M2 mAChR occurred after 6 hr of stimulation (Figure 1). In contrast, maximal down-regulation of the M1 subtype did not occur until after 12 hr of carbachol stimulation. In addition, M2 mAChRs were maximally decreased by only 22% while the M1 subtype was decreased by 55%. These results demonstrate that exogenously expressed M1 and M2 mAChRs undergo agonist-promoted down-regulation in MEFwt cells. The different time course and extent of down-regulation suggest the possibility that down-regulation of the two subtypes may involve distinct pathways. Despite the fact that M2 mAChRs were maximally down-regulated by 6 hr of stimulation, subsequent single time point experiments used the 12 hr time point so that the M1 and M2 subtype experiments would be compared at a standardized time point.


Differential role of beta-arrestin ubiquitination in agonist-promoted down-regulation of M1 vs M2 muscarinic acetylcholine receptors.

Mosser VA, Jones KT, Hoffman KM, McCarty NA, Jackson DA - J Mol Signal (2008)

Time-course of agonist-promoted down-regulation of mAChRs in MEF. MEF wt cells were transfected with eGFP-M1 (◆) or HA-M2 (■) mAChR. 24 hr following transfection, cells were treated with 1 mM carbachol for the indicated time. Down-regulation was determined using [3H]-QNB binding (fmol/mg protein) in crude membranes as described in methods. The M1 mAChR displays nearly threefold the down-regulation of the M2 subtype over the same time course. Data are expressed as percent down-regulation compared to t = 0 control and are presented as mean ± standard error of the mean for three independent experiments with duplicate data points. Total mAChR expressed (fmol/mg) at t = 0 was 4000 for both M1 and M2 mAChRs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2648954&req=5

Figure 1: Time-course of agonist-promoted down-regulation of mAChRs in MEF. MEF wt cells were transfected with eGFP-M1 (◆) or HA-M2 (■) mAChR. 24 hr following transfection, cells were treated with 1 mM carbachol for the indicated time. Down-regulation was determined using [3H]-QNB binding (fmol/mg protein) in crude membranes as described in methods. The M1 mAChR displays nearly threefold the down-regulation of the M2 subtype over the same time course. Data are expressed as percent down-regulation compared to t = 0 control and are presented as mean ± standard error of the mean for three independent experiments with duplicate data points. Total mAChR expressed (fmol/mg) at t = 0 was 4000 for both M1 and M2 mAChRs.
Mentions: To determine whether or not MEF cells could be used in the current study, we performed agonist-promoted down-regulation time courses for both the M1 and M2 mAChR subtypes. Total receptor numbers were measured using the non-selective membrane permeable muscarinic antagonist quinuclidinyl benzilate (QNB), which binds to both surface and intracellular pools of mAChR. Wild-type MEF cells that were transfected with either eGFP-M1 mAChR or HA-M2 mAChR resulted in similar expression levels (~4000 fmol/mg). After 24 hr, cells were treated with 1 mM carbachol for the indicated time. Maximal down-regulation of the M2 mAChR occurred after 6 hr of stimulation (Figure 1). In contrast, maximal down-regulation of the M1 subtype did not occur until after 12 hr of carbachol stimulation. In addition, M2 mAChRs were maximally decreased by only 22% while the M1 subtype was decreased by 55%. These results demonstrate that exogenously expressed M1 and M2 mAChRs undergo agonist-promoted down-regulation in MEFwt cells. The different time course and extent of down-regulation suggest the possibility that down-regulation of the two subtypes may involve distinct pathways. Despite the fact that M2 mAChRs were maximally down-regulated by 6 hr of stimulation, subsequent single time point experiments used the 12 hr time point so that the M1 and M2 subtype experiments would be compared at a standardized time point.

Bottom Line: Internalization of receptors was unaffected by proteasome inhibitors, but down-regulation was significantly reduced, suggesting a role for the ubiquitination machinery in promoting down-regulation of the receptors.Agonist-promoted internalization of the M2 mAChR was not affected by expression of beta-arrestin lysine mutants lacking putative ubiquitination sites, beta-arrestin 2K18R, K107R, K108R, K207R, K296R, while down-regulation and stable co-localiztion of the receptor with this beta-arrestin lysine mutant were significantly reduced.Interestingly, expression of beta-arrestin 2K18R, K107R, K108R, K207R, K296R increased the agonist-promoted down-regulation of the M1 mAChR but did not result in a stable co-localiztion of the receptor with this beta-arrestin lysine mutant.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical and Pharmaceutical Sciences, College of Health Professions and Biomedical Science, Skaggs Building Room 376, The University of Montana, Missoula, MT 59812, USA. darrell.jackson@umontana.edu.

ABSTRACT

Background: Sustained agonist-promoted ubiquitination of beta-arrestin has been correlated with increased stability of the GPCR - beta-arrestin complex. Moreover, abrogation of beta-arrestin ubiquitination has been reported to inhibit receptor internalization with minimal effects on receptor degradation.

Results: Herein we report that agonist activation of M1 mAChRs produces a sustained beta-arrestin ubiquitination but no stable co-localization with beta-arrestin. In contrast, sustained ubiquitination of beta-arrestin by activation of M2 mAChRs does result in stable co-localization between the M2 mAChR and beta-arrestin. Internalization of receptors was unaffected by proteasome inhibitors, but down-regulation was significantly reduced, suggesting a role for the ubiquitination machinery in promoting down-regulation of the receptors. Given the ubiquitination status of beta-arrestin following agonist treatment, we sought to determine the effects of beta-arrestin ubiquitination on M1 and M2 mAChR down-regulation. A constitutively ubiquitinated beta-arrestin 2 chimera in which ubiquitin is fused to the C-terminus of beta-arrestin 2 (YFP-beta-arrestin 2-Ub) significantly increased agonist-promoted down-regulation of both M1 and M2 mAChRs, with the effect substantially higher on the M2 mAChR. Based on this observation, we were interested in examining the effects of disruption of potential ubiquitination sites in the beta-arrestin sequence on receptor down-regulation. Agonist-promoted internalization of the M2 mAChR was not affected by expression of beta-arrestin lysine mutants lacking putative ubiquitination sites, beta-arrestin 2K18R, K107R, K108R, K207R, K296R, while down-regulation and stable co-localiztion of the receptor with this beta-arrestin lysine mutant were significantly reduced. Interestingly, expression of beta-arrestin 2K18R, K107R, K108R, K207R, K296R increased the agonist-promoted down-regulation of the M1 mAChR but did not result in a stable co-localiztion of the receptor with this beta-arrestin lysine mutant.

Conclusion: These findings indicate that ubiquitination of beta-arrestin has a distinct role in the differential trafficking and degradation of M1 and M2 mAChRs.

No MeSH data available.


Related in: MedlinePlus