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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

Expression of a chimeric ubiquitinated form of β-arrestin 2 enhances the agonist-promoted down-regulation of M1 and M2 mAChRs in MEF KO1/2. MEF KO1/2 cells were transfected with eGFP-M1 (A) or HA-M2 (B) mAChRs and either no β-arrestin, FLAG-β-arrestin 2 or YFP-β-arrestin 2-Ub. 24 hr following transfection, cells were treated with 1 mM carbachol for 12 hr. Down-regulation was determined using [3H]-QNB binding (fmol/mg protein) in crude membranes as described in methods. Expression of the constitutively ubiquitinated form of β-arrestin (β-arrestin 2-Ub) had a greater effect on down-regulation of the M2 mAChR compared to the M1 subtype. Data are expressed as percent of [3H]-QNB bound compared to the untreated control with no β-arrestin and are presented as mean ± standard deviation from two independent experiments with duplicate data points. Statistical analysis was performed using a repeated measures ANOVA with Bonferroni post test; * indicates p ≤ 0.05 and ** indicates p ≤ 0.001 (compared to untreated, no β-arrestin control), ns indicates not significant. Total mAChR expressed (fmol/mg) in the absence of β-arrestin was 1000 – 2000 for both receptor subtypes.
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Figure 6: Expression of a chimeric ubiquitinated form of β-arrestin 2 enhances the agonist-promoted down-regulation of M1 and M2 mAChRs in MEF KO1/2. MEF KO1/2 cells were transfected with eGFP-M1 (A) or HA-M2 (B) mAChRs and either no β-arrestin, FLAG-β-arrestin 2 or YFP-β-arrestin 2-Ub. 24 hr following transfection, cells were treated with 1 mM carbachol for 12 hr. Down-regulation was determined using [3H]-QNB binding (fmol/mg protein) in crude membranes as described in methods. Expression of the constitutively ubiquitinated form of β-arrestin (β-arrestin 2-Ub) had a greater effect on down-regulation of the M2 mAChR compared to the M1 subtype. Data are expressed as percent of [3H]-QNB bound compared to the untreated control with no β-arrestin and are presented as mean ± standard deviation from two independent experiments with duplicate data points. Statistical analysis was performed using a repeated measures ANOVA with Bonferroni post test; * indicates p ≤ 0.05 and ** indicates p ≤ 0.001 (compared to untreated, no β-arrestin control), ns indicates not significant. Total mAChR expressed (fmol/mg) in the absence of β-arrestin was 1000 – 2000 for both receptor subtypes.

Mentions: To examine the consequences of β-arrestin ubiquitination on receptor down-regulation, we expressed a yellow fluorescent protein-tagged β-arrestin 2-ubiquitin chimera that cannot be deubiquitinated by cellular deubiquitinases (YFP-β-arrestin 2-Ub) [12]. MEF KO1/2 cells were transfected with eGFP-M1 or HA-M2 mAChR and either FLAG-β-arrestin 2 or YFP-β-arrestin 2-Ub. After 24 hr, cells were treated with 1 mM carbachol for 12 hr. There was a 40% agonist-promoted down-regulation of the M1 mAChR by β-arrestin 2 alone which increased to 70% in the presence of β-arrestin 2-Ub (Figure 6). These values were 62 and 95%, respectively, for the M2 subtype (Figure7). β-arrestin 2-Ub also increased the constitutive receptor down-regulation, and again the effect was much larger for the M2 (90%) vs M1 (50%) subtype (Figure 6A and 6B). It is clear from these data that ubiquitination enhanced the ability of β-arrestin 2 to mediate both constitutive and agonist-promoted down-regulation of both the M1 and M2 mAChRs.


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)

Expression of a chimeric ubiquitinated form of β-arrestin 2 enhances the agonist-promoted down-regulation of M1 and M2 mAChRs in MEF KO1/2. MEF KO1/2 cells were transfected with eGFP-M1 (A) or HA-M2 (B) mAChRs and either no β-arrestin, FLAG-β-arrestin 2 or YFP-β-arrestin 2-Ub. 24 hr following transfection, cells were treated with 1 mM carbachol for 12 hr. Down-regulation was determined using [3H]-QNB binding (fmol/mg protein) in crude membranes as described in methods. Expression of the constitutively ubiquitinated form of β-arrestin (β-arrestin 2-Ub) had a greater effect on down-regulation of the M2 mAChR compared to the M1 subtype. Data are expressed as percent of [3H]-QNB bound compared to the untreated control with no β-arrestin and are presented as mean ± standard deviation from two independent experiments with duplicate data points. Statistical analysis was performed using a repeated measures ANOVA with Bonferroni post test; * indicates p ≤ 0.05 and ** indicates p ≤ 0.001 (compared to untreated, no β-arrestin control), ns indicates not significant. Total mAChR expressed (fmol/mg) in the absence of β-arrestin was 1000 – 2000 for both receptor subtypes.
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Figure 6: Expression of a chimeric ubiquitinated form of β-arrestin 2 enhances the agonist-promoted down-regulation of M1 and M2 mAChRs in MEF KO1/2. MEF KO1/2 cells were transfected with eGFP-M1 (A) or HA-M2 (B) mAChRs and either no β-arrestin, FLAG-β-arrestin 2 or YFP-β-arrestin 2-Ub. 24 hr following transfection, cells were treated with 1 mM carbachol for 12 hr. Down-regulation was determined using [3H]-QNB binding (fmol/mg protein) in crude membranes as described in methods. Expression of the constitutively ubiquitinated form of β-arrestin (β-arrestin 2-Ub) had a greater effect on down-regulation of the M2 mAChR compared to the M1 subtype. Data are expressed as percent of [3H]-QNB bound compared to the untreated control with no β-arrestin and are presented as mean ± standard deviation from two independent experiments with duplicate data points. Statistical analysis was performed using a repeated measures ANOVA with Bonferroni post test; * indicates p ≤ 0.05 and ** indicates p ≤ 0.001 (compared to untreated, no β-arrestin control), ns indicates not significant. Total mAChR expressed (fmol/mg) in the absence of β-arrestin was 1000 – 2000 for both receptor subtypes.
Mentions: To examine the consequences of β-arrestin ubiquitination on receptor down-regulation, we expressed a yellow fluorescent protein-tagged β-arrestin 2-ubiquitin chimera that cannot be deubiquitinated by cellular deubiquitinases (YFP-β-arrestin 2-Ub) [12]. MEF KO1/2 cells were transfected with eGFP-M1 or HA-M2 mAChR and either FLAG-β-arrestin 2 or YFP-β-arrestin 2-Ub. After 24 hr, cells were treated with 1 mM carbachol for 12 hr. There was a 40% agonist-promoted down-regulation of the M1 mAChR by β-arrestin 2 alone which increased to 70% in the presence of β-arrestin 2-Ub (Figure 6). These values were 62 and 95%, respectively, for the M2 subtype (Figure7). β-arrestin 2-Ub also increased the constitutive receptor down-regulation, and again the effect was much larger for the M2 (90%) vs M1 (50%) subtype (Figure 6A and 6B). It is clear from these data that ubiquitination enhanced the ability of β-arrestin 2 to mediate both constitutive and agonist-promoted down-regulation of both the M1 and M2 mAChRs.

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