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

Background Sustained agonist-promoted ubiquitination of β-arrestin has been correlated with increased stability of the GPCR – β-arrestin complex. Moreover, abrogation of β-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 β-arrestin ubiquitination but no stable co-localization with β-arrestin. In contrast, sustained ubiquitination of β-arrestin by activation of M2 mAChRs does result in stable co-localization between the M2 mAChR and β-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 β-arrestin following agonist treatment, we sought to determine the effects of β-arrestin ubiquitination on M1 and M2 mAChR down-regulation. A constitutively ubiquitinated β-arrestin 2 chimera in which ubiquitin is fused to the C-terminus of β-arrestin 2 (YFP-β-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 β-arrestin sequence on receptor down-regulation. Agonist-promoted internalization of the M2 mAChR was not affected by expression of β-arrestin lysine mutants lacking putative ubiquitination sites, β-arrestin 2K18R, K107R, K108R, K207R, K296R, while down-regulation and stable co-localiztion of the receptor with this β-arrestin lysine mutant were significantly reduced. Interestingly, expression of β-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 β-arrestin lysine mutant. Conclusion These findings indicate that ubiquitination of β-arrestin has a distinct role in the differential trafficking and degradation of M1 and M2 mAChRs.


Background
There are five subtypes of muscarinic acetylcholine receptors (M 1 -M 5 mAChR) with distinct yet overlapping tissue distributions. Muscarinic receptors regulate a variety of physiological responses ranging from cardiac homeostasis to cholinergic signaling in the brain [1]. A common feature of mAChRs, and in fact all GPCRs, is that both their expression and activation are tightly regulated. Agonistpromoted trafficking of mAChRs, and most other GPCRs can be broken down into five distinct phases: agonistbinding promotes G protein dissociation (I) from the receptor which allows phosphorylation of specific serine and threonine residues (II) on internal loops of the receptor by G protein receptor kinases (GRKs). This phosphorylation allows the binding of β-arrestins (III) which promotes homologous desensitization and subsequent internalization of the receptor into clathrin coated pits. Following internalization, the receptor can either be dephosphorylated and recycled (IV) to the cell surface or targeted for degradation (V) in proteasomes or lysosomes [1].
β-arrestins have emerged as a central control point in the trafficking of nearly all GPCRs [2]. In addition to mediating desensitization of GPCRs, β-arrestin also participates in clathrin-dependent endocytosis of activated receptors by directly interacting with clathrin and the clathrin-associated adaptor AP-2 [3,4]. Once internalized, β-arrestins are involved in regulation of post-endocytotic trafficking and recently have been shown to function as scaffolding proteins interacting with cellular trafficking machinery [2,5].
Initial reports examining the role of β-arrestin, clathrin and dynamin indicated that agonist-promoted internalization of the M 2 mAChR proceeded via both arrestindependent and -independent pathways [6,7]. Later work, however, indicated the essential role of β-arrestin in M 2 mAChR internalization, and suggested that all M 2 mAChR internalization, like other mAChR subtypes, is dynamindependent [8,9].
Two classes of GPCRs have been identified with respect to kinetics of receptor recycling and interaction with βarrestins. Class A receptors, such as the β 2 adrenergic (β 2 AR), dopamine D1A and endothelin 1A receptors recycle rapidly and dissociate from β-arrestin prior to receptor internalization. Class B receptors, such as the vasopressin 2 (V2R), angiotensin 1a and neurotensin 1 receptors, recycle slowly and internalize in a stable association with βarrestin [10,11]. Recently, it has become clear that the classification of receptors as A or B directly correlates with patterns of β-arrestin ubiquitination and deubiquitination [12]. Stimulation of Class A β 2 ARs leads to transient ubiquitination of β-arrestin with deubiquitination occur-ring shortly (minutes) after internalization [12]. In contrast, stimulation of Class B V2Rs leads to a stable ubiquitination of β-arrestin [12].
Ubiquitination of proteins is a signal for degradation that leads to delivery to and degradation of proteins in the 26S proteasome [13]. There are a number of examples where ubiquitination has been shown to be involved in the regulation of GPCRs, including the opiod receptors [14], yeast pheromone receptor [15], human immunodeficiency virus co-receptor CXCR4 [16], and β 2 ARs [17].
Class A receptors, which do not internalize with β-arrestin, display a pattern of transient β-arrestin ubiquitination. Class B receptors, on the other hand, do internalize with β-arrestin, and display a sustained β-arrestin ubiquitination pattern [12]. Shenoy et al. [17] showed that agonist stimulation led to the ubiquitination of both βarrestin and the β 2 AR. Their data suggested that it was the ubiquitination of β-arrestin that was involved in the initial receptor internalization step whereas ubiquitination of the receptor itself was required for subsequent receptor degradation.
In the present study, we wanted to address several questions regarding the role of β-arrestin ubiquitination in the agonist-promoted down-regulation of mAChRs. First we wanted to determine if there was a role for β-arrestin ubiquitination in receptor down-regulation. Collectively, our data support a role for β-arrestin in differential targeting of mAChRs towards down-regulation. In addition, the data indicate that this differential targeting may be controlled by preferential interaction of mAChR subtypes with the different β-arrestin subtypes which in turn may be mediated by specific patterns of β-arrestin ubiquitination.

Results
For a number of years there has been some controversy as to the role of β-arrestin in M 2 mAChR specific internalization and down-regulation. Our recent publication [9] established the essential role of β-arrestin in the internalization of the M 2 mAChR. The current study is a follow up which not only addresses the role of β-arrestin in agonistpromoted mAChR down-regulation, but also indicates a differential role for β-arrestin in M 1 versus M 2 mAChR down-regulation. As in the previous study, we performed our down-regulation experiments in mouse embryonic fibroblasts (MEFs) derived from β-arrestin 1/2 double knockout mice (MEF KO1/2) [18]. Experiments performed using this cell line offer a significant advantage over previous studies of the role of β-arrestin in GPCR internalization and down-regulation that used dominantnegative or knockdown strategies because they avoid any possible complications that could arise from the presence of low levels of endogenous arrestin proteins. These cells have been characterized to confirm the absence of mAChR expression using both PCR and radioligand binding assays [9].
To determine whether or not MEF cells could be used in the current study, we performed agonist-promoted downregulation time courses for both the M 1 and M 2 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-M 1 mAChR or HA-M 2 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 M 2 mAChR occurred after 6 hr of stimulation ( Figure 1). In contrast, maximal down-regulation of the M 1 subtype did not occur until after 12 hr of carbachol stimulation. In addition, M 2 mAChRs were maximally decreased by only 22% while the M 1 subtype was decreased by 55%. These results demonstrate that exogenously expressed M 1 and M 2 mAChRs undergo agonistpromoted 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 M 2 mAChRs were maximally down-regulated by 6 hr of stimulation, subsequent single time point experiments used the 12 hr time point so that the M 1 and M 2 subtype experiments would be compared at a standardized time point.
Several recent studies have reported that mAChR downregulation occurs independently of agonist-promoted internalization [19][20][21]. Recently, we demonstrated that agonist-promoted internalization of M 2 mAChRs in MEF cells was β-arrestin dependent [9]. To examine whether or not agonist-promoted down-regulation of M 1 and M 2 mAChRs is β-arrestin dependent, we performed downregulation studies in MEF β-arrestin double knockout cells transiently expressing either M 1 or M 2 mAChRs. MEF KO1/2 cells were transfected with eGFP-M 1 mAChR or HA-M 2 mAChR and either FLAG-β-arrestin 1 or 2. After 24 hr, cells were treated with 1 mM carbachol for 12 hr. In the absence of exogenous β-arrestin, there was no down-regulation in response to agonist stimulation in the double knockout cell line ( Recent studies have demonstrated that agonist stimulation of GPCRs results in ubiquitination of β-arrestin 2 [12]. This group demonstrated receptor specific agonistpromoted ubiquitination of β-arrestin 2 that was either transient (peaked at 1 min of agonist stimulation) or stable (still present after 15 min of agonist stimulation) [12]. We performed experiments in order to determine if agonist stimulation of M 1 or M 2 mAChRs promoted the ubiquitination of β-arrestin and if so, examine whether the observed ubiquitination was transient or stable. MEF KO1/2 cells were transfected with FLAG-β-arrestin 2 and eGFP-M 1 or HA-M 2 mAChR and treated with carbachol for 0, 1, 3, 15 and 30 min. Stimulation of M 1 and M 2 mAChRs significantly increased ubiquitination of β-arrestin 2 (Figure 3). This increase is clearly visible at both the 15 and 30 min time point for both mAChR subtypes. These results demonstrate that stimulation of M 1 or M 2 mAChRs promotes a stable ubiquitination of β-arrestin 2.
Having established that β-arrestin is required for mAChR down-regulation and that muscarinic stimulation leads to the ubiquitination of β-arrestin, we wanted to examine if there was a direct role for β-arrestin ubiquitination in receptor down-regulation. Since ubiquitination is known to serve as a signal for protein degradation [13] we were interested in determining whether disruption of the ubiquitin/proteasome pathway would affect the agonist-pro-Time-course of agonist-promoted down-regulation of mAChRs in MEF Figure 1 Time-course of agonist-promoted down-regulation of mAChRs in MEF. MEF wt cells were transfected with eGFP-M 1 (r) or HA-M 2 (n) mAChR. 24 hr following transfection, cells were treated with 1 mM carbachol for the indicated time. Down-regulation was determined using [ 3 H]-QNB binding (fmol/mg protein) in crude membranes as described in methods. The M 1 mAChR displays nearly threefold the down-regulation of the M 2 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 M 1 and M 2 mAChRs. 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-M 2 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 βarrestin ubiquitination.
Rescue of mAChR down-regulation in MEF KO1/2 with β-arrestin 1 or 2 Figure 2 Rescue of mAChR down-regulation in MEF KO1/2 with β-arrestin 1 or 2. MEF KO1/2 cells were transfected with eGFP-M 1 mAChR (A-B) or HA-M 2 mAChR (C-D) and either FLAG-β-arrestin 1 (top) or 2 (bottom). 24 hr following transfection, cells were treated with 1 mM carbachol for 12 hr. Down-regulation was determined using [ 3 H]-QNB binding (fmol/mg protein) in crude membranes as described in methods. No down-regulation occurs in the absence of β-arrestin and either isoform (β-arrestin 1 or 2) rescues both constitutive and agonist-promoted down-regulation. Data are expressed as percent of [ 3 H]-QNB bound (fmol/mg total protein) compared to untreated, no β-arrestin control and are presented as mean ± standard error of the mean from three independent experiments with duplicate data points. Statistical analysis was performed using a paired t-test. *: p ≤ 0.05 versus the untreated, no β-arrestin control. Total M 1 mAChR expressed (fmol/mg) at t = 0 was 400 -900 (M 1 ) and 1000 -2000 (M 2 ) for both the β-arrestin 1 and β-arrestin 2 rescue experiments.

Percent [ 3 H]-QNB Bound
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-M 1 or HA-M 2 mAChR and either FLAGβ-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 M 1 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 M 2 subtype (Figure7). β-arrestin 2-Ub also increased the constitutive receptor down-regulation, and again the effect was much larger for the M 2 (90%) vs M 1 (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 agonistpromoted down-regulation of both the M 1 and M 2 mAChRs.
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 agonistpromoted down-regulation, we examined the ability of specific β-arrestin 2 lysine mutants to mediate agonistpromoted down-regulation of the M 1 and M 2 mAChR. MEF KO1/2 cells were transfected with eGFP-M 1 or HA-M 2 mAChR and either empty vector (control), FLAG-β-arrestin 2 (wild-type), FLAG-β-arrestin 2 K18R, K107R, K108R, K207R, K296R or FLAG-β-arrestin 2 K11R, 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 downregulation of the M 1 subtype. We also noted a large constitutive effect on down-regulation with both mutant βarrestins. In contrast, for the M 2 subtype only wild-type βarrestin 2 (24%) and β-arrestin 2 K11R, K12R (27%) were able to rescue agonist-promoted down-regulation ( Figure 7). Since it was possible that the effect of β-arrestin 2 K18R, K107R, K108R, K207R, K296R on M 2 mAChR down-regulation seen in the previous experiment was actually occurring at the receptor internalization step, we performed receptor internalization experiments with [ 3 H]-NMS. Transfections were identical to the previous experiment. After 24 hr, cells were treated with 1 mM carbachol for 1 hr. All βarrestin 2 constructs (wild-type or lysine mutants) were able to rescue agonist-promoted internalization of M 2 mAChRs in MEF KO1/2 ( Figure 8).
Since β-arrestin 2 K18R, K107R, K108R, K207R, K296R not only appears to interfere with the agonist promoted down-regulation of the M 2 mAChR but also increased the constitutive down-regulation of the M 1 mAChR we examined the ability of this mutant to co-localize with the M 1 /M 2 mAChR compared to wt and β-arrestin 2 K11R, K12R following agonist stimulation. MEF KO1/2 were plated on cover slips in 6-well plates. 24 hrs after transfection with eGFP-M 1 mAChR or HA-M 2 mAChR and FLAG tagged β-arrestin (wt or lysine mutants) cells were treated for 30 min or 12 hrs with 1 mM carbachol. Cells were fixed and processed for indirect immunofluorescence as described in methods. After 30 minutes of agonist exposure there is clear overlap of the M 2 mAChR receptor and β-arrestin signal signified by the yellow puncta ( Figure 9) for the wt and βarrestin 2 K11, K12R . This overlap is absent with β-arrestin 2 K18R, K107R, K108R, K207R, K296R which indicates a disrupted or impaired interaction between this β-arrestin mutant and the M 2 mAChR. Surprisingly, despite our previous observation of the effects of β-arrestin 2 K18R, K107R, K108R, K207R, K296R on constitutive M 1 down-regulation, no co-localization was observed between β-arrestin and the M 1 mAChR ( Figure 10).
Finally we used immunocytochemistry with the lysosomal marker LAMP-1 to examine the effects of the β- After 24 hr, cells were treated with 1 mM carbachol for 6 hr. There was significant co-localization of the receptor with LAMP-1 in the presence of wild-type β-arrestin 2 or β-arrestin 2 K11R, K12R (Figure 11). It is clear, however, that β-arrestin 2 K18R, K107R, K108R, K207R, K296R shows reduced colocalization of the M 2 mAChR with LAMP ( Figure 11).
Collectively, these data indicate that the agonist-promoted down-regulation of the M 1 and M 2 mAChRs involves differential sorting of the receptor by ubiquitinated β-arrestin.

Discussion
Our recent work established the essential role of β-arrestin in the internalization of the M 2 mAChR [9]. The present study extends the observations of previous work and demonstrates that the agonist-promoted down-regulation of M 1 and M 2 mAChRs is β-arrestin dependent, and that the ubiquitination pattern of β-arrestin has a critical role in the differential down-regulation for M 1 vs M 2 mAChRs.
It has been previously established in a variety of cell lines that a prolonged activation of M 1 or M 2 mAChRs induces receptor down-regulation. In agreement with these findings, long-term stimulation of M 1 or M 2 mAChRs induced receptor down-regulation with the M 1 mAChR showing significantly more down-regulation than M 2 subtype. This observation suggests that the two subtypes are differentially regulated by endogenous β-arrestins. No down-regulation of either mAChR subtype occurred in the absence of β-arrestin and either β-arrestin subtype was able to rescue receptor down-regulation.
The observations of Shenoy and co-workers suggest that the ubiquitination of β-arrestin is a critical control point in the trafficking of different classes of GPCRs [12,17,22]. The time course of ubiquitination of β-arrestin 2 in response to muscarinic stimulation in MEF KO1/2 cells transiently expressing the M 1 or M 2 mAChRs was slow in onset and stable over time. This observation suggests that both M 1 and M 2 mAChRs display a Class B β-arrestin ubiquitination pattern despite the fact that the M 1 mAChR is known to recycle rapidly compared to M 2 [23], and does not show a stable endocytotic co-localization with β-arrestin 2 [9] -both characteristics of Class A GPCRs. This observation suggests that there is some flexibility in the Class A vs B distinction. This same flexibility has been observed in the categorization of somatostatin receptor subtypes [24].
To confirm that agonist-promoted down-regulation of mAChRs was ubiquitin-dependent we performed internalization/down-regulation experiments in the presence of the proteosomal inhibitor lactacystin. Lactacystin is a specific inhibitor of the 26S proteasome that functions by covalently modifying the active site and inhibiting the enzymatic activity of the proteasome [13]. This inhibition of the enzyme would inhibit recycling and thus deplete the available ubiquitin. Lactacystin was able to completely block agonist-promoted down-regulation with no effect on agonist-promoted internalization. These results suggested that the availability of free ubiquitin has a role in agonist-promoted down-regulation of the mAChRs. This effect is most likely due to effects on ubiquitin-dependent lysosomal sorting as evidenced by a later experiment that demonstrated the co-localization of the agonist-internalized M 2 mAChR with the lysosomal membrane protein LAMP-1.  Having established that β-arrestin is ubiquitinated via mAChR activation, we then examined whether the ubiquitination of β-arrestin enhances its ability to mediate agonist-promoted down-regulation. M 1 mAChR levels were reduced and M 2 levels were nearly completely ablated in the presence of β-arrestin 2-Ub compared to wild-type β-arrestin. The effect was seen on both constitutive and agonist promoted down-regulation.
In order to examine the role of β-arrestin ubiquitination in targeting mAChRs toward degradation, we used β-arrestin lysine mutants that have been previously shown to have a role in the endocytotic trafficking of other GPCRs. The constructs, generated by Shenoy and co-workers, contain mutations of potential ubiquitination sites near the amino terminus (β-arrestin 2 K11R, K12R ) and at five other sites in the protein (β-arrestin 2 K18R, K107R, K108R, K207R, K296R ) [22]. We were particularly interested in the possible differential effects of β-arrestin ubiquitination patterns on M 1 vs M 2 down-regulation given the fact that M 1 and M 2 mAChRs did not fit neatly into the classical Class A vs B distinction.
Both of the β-arrestin lysine mutants were able to mediate agonist-promoted down-regulation of the M 1 mAChR to the same extent as wild-type β-arrestin. While our data clearly indicates an essential role for β-arrestin ubiquitination in M 1 mAChR down-regulation, other lysine residues than the ones examined must be the targets for the necessary ubiquitination required for targeting the M 1 mAChRs . 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 2 K18R, K107R, K108R, K207R, K296R mutant when co-expressed with the M 2 mAChR. Data are expressed as percent of [ 3 H]-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 M 1 (300-500 fmol/mg) and M 2 (1500-2500 fmol/mg) mAChR expressed in the absence of β-arrestin constructs was similar. toward down-regulation. We did observe increased constitutive down-regulation of the M 1 mAChR with both mutant β-arrestin constructs. It is possible that the pattern of ubiquitination of these mutant β-arrestins altered the constitutive targeting of the M 1 mAChR toward lysosomal down-regulation. This observation is one of several in this study that suggests that the pattern of ubiquitination on βarrestin is directly implicated in the downstream targeting of mAChRs. Previously it has been suggested that the only role for β-arrestin ubiquitination is to promote the internalization of GPCRs.
We obtained surprisingly different results with the effects of the β-arrestin lysine mutants on the agonist-promoted down-regulation of the M 2 mAChR subtype. Mutation of the two lysine residues in β-arrestin 2 K11R, K12R had no effect on either agonist-promoted internalization or down-regulation of M 2 mAChRs. This result is similar to those seen with other Class B receptors such as the V2R and NK1R [22]. The results are different however from those seen with another Class B receptor, the AT1a recep-tor. When this receptor was co-expressed with β-arrestin 2 K11R, K12R , agonist stimulation resulted in transient association of the receptor with β-arrestin 2, thus converting the receptor to a Class A type.
The fact that β-arrestin 2 K18R, K107R, K108R, K207R, K296R was able to internalize but not down-regulate the M 2 mAChR suggests that one or more of these lysines has a role in down-regulation but not internalization for this subtype. These data suggest a direct role for the ubiquitination state of β-arrestin in the lysosomal targeting of the M 2 mAChR.
Unlike effects observed with the M 2 mAChR, β-arrestin 2 K18R, K107R, K108R, K207R, K296R was able to mediate down-regulation of M 1 mAChRs. This observation clearly demonstrates that the role of these lysines in down-regulation is subtype specific and that a specific pattern of ubiquitination on β-arrestin can differentially target the two mAChR subtypes for down-regulation.
Agonist-promoted internalization of M 2 mAChR is unaffected by β-arrestin 2 lysine mutants in MEF KO1/2  which may indicate a disrupted or impaired interaction between this β-arrestin mutant and the M 2 mAChR. βarrestin 2 K18R, K107R, K108R, K207R, K296R was also shown to disrupt the co-localization of the M 2 mAChR and the lysosomal marker LAMP-1 confirming our observation that βarrestin 2 K18R, K107R, K108R, K207R, K296R does not mediate the agonist-promoted degradation of the M 2 mAChR. Surprisingly, no co-localization of M 1 mAChR was observed with any of the three β-arrestin constructs. Time points from 1 min to 12 hrs of carbachol treatment were performed to attempt to image co-localization between the M 1 mAChR and the β-arrestins (data not shown). This observation seems to indicate that the role of β-arrestin in M 1 mAChR down-regulation may involve a transient interaction between β-arrestin and the receptor which fates the receptor toward degradation.
Our observations of M 2 mAChR trafficking are different from the results of work with the Class A β 2 AR [17] which suggests that ubiquitination of β-arrestin is required for receptor internalization and that ubiquitination of the receptor itself is required for receptor degradation. Our data indicate that, for the M 1 mAChR, the ubiquitination state of β-arrestin appears to have a role in the level of constitutive (agonist-independent) down-regulation and for the M 2 mAChR the ubiquitination state of β-arrestin has a direct role in the agonist-promoted down-regulation of the receptor. Of the seven lysine residues we examined, at least one (or more) of the group 18, 107, 108, 207 and 296 are required for this M 2 mAChR down-regulation. We also observed that M 2 mAChR internalization was independent of β-arrestin ubiquitination at these same lysine residues that have been shown to interfere with the association of β-arrestin with other Class B receptors [22]. The possibility that other lysine residues than those we examined are ubiquitinated in order to internalize the receptor would preserve the role for β-arrestin in receptor internalization. The absence of down-regulation of M 2 mAChRs with β-arrestin 2 K18R, K107R, K108R, K207R, K296R however, clearly indicates a direct role for the ubiquitination of βarrestin in receptor down-regulation.

Conclusion
We conclude that agonist-promoted down-regulation of both M 1 and M 2 mAChRs is β-arrestin-dependent. We further conclude that this down-regulation is specifically modulated by the ubiquitination state of β-arrestin and that this β-arrestin ubiquitination differentially targets the receptor subtypes for degradation in lysosomes. Significantly, where it has been suggested that Class A GPCR down-regulation proceeds primarily via receptor ubiquitination, these data indicate that the ubiquitination state of β-arrestin 2 has an essential role in the differential targeting the M 1 and M 2 mAChR toward down-regulation. Future studies will examine the role of receptor ubiquitination in M 1 and M 2 mAChR down-regulation. Specifically, we will examine whether expression of wild type vs the β-arrestin 2 K18R, K107R, K108R, K207R, K296R mutant differentially affects M 1 and M 2 mAChR ubiquitination and subsequent down-regulation.