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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

β-arrestin 2K18R, K107R, K108R, K207R, K296Rdifferentially affects down-regulation of M1 and M2 mAChRs in MEF KO1/2. MEF KO1/2 cells were transfected with eGFP-M1 (A) mAChR or HA-M2 (B) mAChR and either empty vector (control), FLAG-β-arrestin 2 (WT), FLAG-β-arrestin 2K18R, K107R, K108R, K207R, K296R or FLAG-β-arrestin 2K11R, K12R. 24 hr after transfection, cells were treated with 1 mM carbachol for 12 hr. Down-regulation was determined in crude membranes (fmol/mg protein) as described in methods. All β-arrestin 2 constructs were able to mediate agonist-promoted down-regulation of mAChR with the exception of the β-arrestin 2K18R, K107R, K108R, K207R, K296R mutant when co-expressed with the M2 mAChR. Data are expressed as percent of [3H]-QNB bound (compared to untreated, no β-arrestin control) and presented as mean ± standard deviation from three independent experiments with duplicate or quadruplicate data points. Statistical analysis was performed using a repeated measures ANOVA with Bonferroni post test; * indicates p ≤ 0.05 ** indicates p ≤ 0.001 (compared to untreated, no β-arrestin control), ns indicates not significant. Total M1 (300–500 fmol/mg) and M2 (1500–2500 fmol/mg) mAChR expressed in the absence of β-arrestin constructs was similar.
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Figure 7: β-arrestin 2K18R, K107R, K108R, K207R, K296Rdifferentially affects down-regulation of M1 and M2 mAChRs in MEF KO1/2. MEF KO1/2 cells were transfected with eGFP-M1 (A) mAChR or HA-M2 (B) mAChR and either empty vector (control), FLAG-β-arrestin 2 (WT), FLAG-β-arrestin 2K18R, K107R, K108R, K207R, K296R or FLAG-β-arrestin 2K11R, K12R. 24 hr after transfection, cells were treated with 1 mM carbachol for 12 hr. Down-regulation was determined in crude membranes (fmol/mg protein) as described in methods. All β-arrestin 2 constructs were able to mediate agonist-promoted down-regulation of mAChR with the exception of the β-arrestin 2K18R, K107R, K108R, K207R, K296R mutant when co-expressed with the M2 mAChR. Data are expressed as percent of [3H]-QNB bound (compared to untreated, no β-arrestin control) and presented as mean ± standard deviation from three independent experiments with duplicate or quadruplicate data points. Statistical analysis was performed using a repeated measures ANOVA with Bonferroni post test; * indicates p ≤ 0.05 ** indicates p ≤ 0.001 (compared to untreated, no β-arrestin control), ns indicates not significant. Total M1 (300–500 fmol/mg) and M2 (1500–2500 fmol/mg) mAChR expressed in the absence of β-arrestin constructs was similar.

Mentions: Having demonstrated a role for ubiquitin in agonist-promoted mAChR degradation, we were interested in the effects of disrupting β-arrestin 2 ubiquitination on receptor down-regulation. Several lysine residues on β-arrestin are known to be sites of ubiquitination [22]. To further confirm the essential role of ubiquitination in agonist-promoted down-regulation, we examined the ability of specific β-arrestin 2 lysine mutants to mediate agonist-promoted down-regulation of the M1 and M2 mAChR. MEF KO1/2 cells were transfected with eGFP-M1 or HA-M2 mAChR and either empty vector (control), FLAG-β-arrestin 2 (wild-type), FLAG-β-arrestin 2K18R, K107R, K108R, K207R, K296R or FLAG-β-arrestin 2K11R, K12R. After 24 hr, cells were treated with 1 mM carbachol for 12 hr. As shown previously, there was no down-regulation in the control cells in the absence of β-arrestin 2 (Figure 7). All three β-arrestin 2 constructs were able to rescue agonist-promoted down-regulation of the M1 subtype. We also noted a large constitutive effect on down-regulation with both mutant β-arrestins. In contrast, for the M2 subtype only wild-type β-arrestin 2 (24%) and β-arrestin 2K11R, K12R (27%) were able to rescue agonist-promoted down-regulation (Figure 7). β-arrestin 2K18R, K107R, K108R, K207R, K296R, had lost the ability to rescue agonist-promoted down-regulation of the M2 mAChR (Figure 7).


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)

β-arrestin 2K18R, K107R, K108R, K207R, K296Rdifferentially affects down-regulation of M1 and M2 mAChRs in MEF KO1/2. MEF KO1/2 cells were transfected with eGFP-M1 (A) mAChR or HA-M2 (B) mAChR and either empty vector (control), FLAG-β-arrestin 2 (WT), FLAG-β-arrestin 2K18R, K107R, K108R, K207R, K296R or FLAG-β-arrestin 2K11R, K12R. 24 hr after transfection, cells were treated with 1 mM carbachol for 12 hr. Down-regulation was determined in crude membranes (fmol/mg protein) as described in methods. All β-arrestin 2 constructs were able to mediate agonist-promoted down-regulation of mAChR with the exception of the β-arrestin 2K18R, K107R, K108R, K207R, K296R mutant when co-expressed with the M2 mAChR. Data are expressed as percent of [3H]-QNB bound (compared to untreated, no β-arrestin control) and presented as mean ± standard deviation from three independent experiments with duplicate or quadruplicate data points. Statistical analysis was performed using a repeated measures ANOVA with Bonferroni post test; * indicates p ≤ 0.05 ** indicates p ≤ 0.001 (compared to untreated, no β-arrestin control), ns indicates not significant. Total M1 (300–500 fmol/mg) and M2 (1500–2500 fmol/mg) mAChR expressed in the absence of β-arrestin constructs was similar.
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Figure 7: β-arrestin 2K18R, K107R, K108R, K207R, K296Rdifferentially affects down-regulation of M1 and M2 mAChRs in MEF KO1/2. MEF KO1/2 cells were transfected with eGFP-M1 (A) mAChR or HA-M2 (B) mAChR and either empty vector (control), FLAG-β-arrestin 2 (WT), FLAG-β-arrestin 2K18R, K107R, K108R, K207R, K296R or FLAG-β-arrestin 2K11R, K12R. 24 hr after transfection, cells were treated with 1 mM carbachol for 12 hr. Down-regulation was determined in crude membranes (fmol/mg protein) as described in methods. All β-arrestin 2 constructs were able to mediate agonist-promoted down-regulation of mAChR with the exception of the β-arrestin 2K18R, K107R, K108R, K207R, K296R mutant when co-expressed with the M2 mAChR. Data are expressed as percent of [3H]-QNB bound (compared to untreated, no β-arrestin control) and presented as mean ± standard deviation from three independent experiments with duplicate or quadruplicate data points. Statistical analysis was performed using a repeated measures ANOVA with Bonferroni post test; * indicates p ≤ 0.05 ** indicates p ≤ 0.001 (compared to untreated, no β-arrestin control), ns indicates not significant. Total M1 (300–500 fmol/mg) and M2 (1500–2500 fmol/mg) mAChR expressed in the absence of β-arrestin constructs was similar.
Mentions: Having demonstrated a role for ubiquitin in agonist-promoted mAChR degradation, we were interested in the effects of disrupting β-arrestin 2 ubiquitination on receptor down-regulation. Several lysine residues on β-arrestin are known to be sites of ubiquitination [22]. To further confirm the essential role of ubiquitination in agonist-promoted down-regulation, we examined the ability of specific β-arrestin 2 lysine mutants to mediate agonist-promoted down-regulation of the M1 and M2 mAChR. MEF KO1/2 cells were transfected with eGFP-M1 or HA-M2 mAChR and either empty vector (control), FLAG-β-arrestin 2 (wild-type), FLAG-β-arrestin 2K18R, K107R, K108R, K207R, K296R or FLAG-β-arrestin 2K11R, K12R. After 24 hr, cells were treated with 1 mM carbachol for 12 hr. As shown previously, there was no down-regulation in the control cells in the absence of β-arrestin 2 (Figure 7). All three β-arrestin 2 constructs were able to rescue agonist-promoted down-regulation of the M1 subtype. We also noted a large constitutive effect on down-regulation with both mutant β-arrestins. In contrast, for the M2 subtype only wild-type β-arrestin 2 (24%) and β-arrestin 2K11R, K12R (27%) were able to rescue agonist-promoted down-regulation (Figure 7). β-arrestin 2K18R, K107R, K108R, K207R, K296R, had lost the ability to rescue agonist-promoted down-regulation of the M2 mAChR (Figure 7).

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