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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.


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The proteosomal inhibitor lactacystin does not affect agonist-promoted down-regulation internalization of the M2 mAChR in MEFwt. MEFwt cells were transfected with HA-M2 mAChR and, after 24 hr cells were incubated for 20 min with or without 10 μM lactacystin then treated with 1 mM carbachol for 30 min. Lactacystin has no effect on the internalization of the M2 mAChR receptor in response to agonist. Internalization was determined using [3H]-NMS binding in whole cells as described in methods. Data are [3H]-NMS bound per well (plated at 1 × 105 cells) and are presented as mean ± standard deviation from three independent experiments with duplicate data points. Statistical analysis was performed using a paired t-test, * indicates p ≤ 0.05; (compared to paired untreated control), ns indicates not significant.
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Figure 5: The proteosomal inhibitor lactacystin does not affect agonist-promoted down-regulation internalization of the M2 mAChR in MEFwt. MEFwt cells were transfected with HA-M2 mAChR and, after 24 hr cells were incubated for 20 min with or without 10 μM lactacystin then treated with 1 mM carbachol for 30 min. Lactacystin has no effect on the internalization of the M2 mAChR receptor in response to agonist. Internalization was determined using [3H]-NMS binding in whole cells as described in methods. Data are [3H]-NMS bound per well (plated at 1 × 105 cells) and are presented as mean ± standard deviation from three independent experiments with duplicate data points. Statistical analysis was performed using a paired t-test, * indicates p ≤ 0.05; (compared to paired untreated control), ns indicates not significant.

Mentions: To determine whether or not the effects of lactacystin were occurring at the level of receptor internalization, we examined the effect of lactacystin on the agonist-promoted internalization of mAChRs using the membrane impermeable muscarinic antagonist N-methylscopolamine (NMS). MEFwt cells were transfected with HA-M2 mAChR, and after 24 hr cells were incubated for 20 min in the absence or presence of 10 μM lactacystin prior to treatment with 1 mM carbachol for 30 min. Pretreatment with inhibitor had no effect on agonist-promoted internalization (Figure 5). These results indicate that agonist-promoted down-regulation of mAChRs involves receptor β-arrestin ubiquitination.


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)

The proteosomal inhibitor lactacystin does not affect agonist-promoted down-regulation internalization of the M2 mAChR in MEFwt. MEFwt cells were transfected with HA-M2 mAChR and, after 24 hr cells were incubated for 20 min with or without 10 μM lactacystin then treated with 1 mM carbachol for 30 min. Lactacystin has no effect on the internalization of the M2 mAChR receptor in response to agonist. Internalization was determined using [3H]-NMS binding in whole cells as described in methods. Data are [3H]-NMS bound per well (plated at 1 × 105 cells) and are presented as mean ± standard deviation from three independent experiments with duplicate data points. Statistical analysis was performed using a paired t-test, * indicates p ≤ 0.05; (compared to paired untreated control), ns indicates not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2648954&req=5

Figure 5: The proteosomal inhibitor lactacystin does not affect agonist-promoted down-regulation internalization of the M2 mAChR in MEFwt. MEFwt cells were transfected with HA-M2 mAChR and, after 24 hr cells were incubated for 20 min with or without 10 μM lactacystin then treated with 1 mM carbachol for 30 min. Lactacystin has no effect on the internalization of the M2 mAChR receptor in response to agonist. Internalization was determined using [3H]-NMS binding in whole cells as described in methods. Data are [3H]-NMS bound per well (plated at 1 × 105 cells) and are presented as mean ± standard deviation from three independent experiments with duplicate data points. Statistical analysis was performed using a paired t-test, * indicates p ≤ 0.05; (compared to paired untreated control), ns indicates not significant.
Mentions: To determine whether or not the effects of lactacystin were occurring at the level of receptor internalization, we examined the effect of lactacystin on the agonist-promoted internalization of mAChRs using the membrane impermeable muscarinic antagonist N-methylscopolamine (NMS). MEFwt cells were transfected with HA-M2 mAChR, and after 24 hr cells were incubated for 20 min in the absence or presence of 10 μM lactacystin prior to treatment with 1 mM carbachol for 30 min. Pretreatment with inhibitor had no effect on agonist-promoted internalization (Figure 5). These results indicate that agonist-promoted down-regulation of mAChRs involves receptor β-arrestin ubiquitination.

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