<p>To obtain a &#x003b2;-arrestin2 mutant that is not ubiquitinated upon 7TMR stimulation, we made conservative changes of groups of lysines to arginines, overexpressed FLAG-tagged mutants in COS-7 cells and tested the &#x003b2;-arrestin precipitates for the ubiquitination signal induced by one-minute isoproterenol stimulation ([Fig 1 A, B,.figureReference+reference+grf=toolXmlRef]). Surprisingly, elimination of such a signal required replacement of all of beta-arrestin2's 31 lysine residues (mutant beta-arrestin2^0K [Fig 1B,.figureReference+reference+grf=toolXmlRef]). When a FLAG epitope-tagged beta-arrestin2^0K is over expressed in HEK-293 cells, no ubiquitination smear is detected upon isoproterenol stimulation ([Fig 1C,.figureReference+reference+grf=toolXmlRef]). Although these experiments indicate that beta-arrestin2^0K can be expressed as a properly folded protein that is isolated and detected by the epitope tag, a concern still remains whether beta-arrestin2^0K despite its 31 lysine to arginine changes is a bona fide form of beta-arrestin.</p><p>To test whether the basic folding and binding properties of beta-arrestin2^0K are retained, we compared the binding of in vitro translated beta-arrestin2 and beta-arrestin2^0K to purified recombinant beta2AR reconstituted in vesicles. We also tested beta-arrestin2-Ub for receptor binding under the same conditions. In these in vitro assays, both WT and beta-arrestin2^0K represent non-ubiquitinated forms and only beta-arrestin2-Ub chimera constitutes the ubiquitinated form. As shown in [Fig 2A and B,.figureReference+reference+grf=toolXmlRef], beta-arrestin2^0K bound to the beta2AR to the same extent as beta-arrestin2. However the presence of a single ubiquitin moiety increased the binding by fourfold ([Fig 2A and B,.figureReference+reference+grf=toolXmlRef]). These experiments suggest that while both non-ubiquitinated forms of beta-arrestin2 (i.e. WT and 0K) are equipotent for beta2AR binding, there is more binding between the beta2AR and the ubiquitinated form (i.e. beta-arrestin2-Ub). When binding was performed in the presence of isoproterenol, a small increase was observed for all three beta-arrestin forms (data not shown). We hypothesized that reconstituted beta2AR was already in an activated conformation due to the presence of zinterol in purification buffers. If so, inclusion of an antagonist could alter the observed binding. When beta-arrestin-receptor complex formation was tested in the presence of propranolol, we found a dramatic decrease in binding for all three beta-arrestin forms ([Fig 2A and B,.figureReference+reference+grf=toolXmlRef]), suggesting that propranolol destabilizes but does not eliminate receptor-beta-arrestin binding in these experiments. Moreover, when reconstituted receptor samples were probed with a beta2AR specific phosphoserine antibody (serines 355,356), a small amount of phosphorylation was detected ([Fig 2C,.figureReference+reference+grf=toolXmlRef] upper panel). Addition of GRK2 leads to an increase in the phosphorylation signal and isoproterenol augments it further ([Fig 2C,.figureReference+reference+grf=toolXmlRef]). We observed a comparable increase in binding above basal conditions for all three beta-arrestin forms upon GRK2 phosphorylation and isoproterenol treatment of the reconstituted beta2AR ([Fig 2D,.figureReference+reference+grf=toolXmlRef]). Collectively, these in vitro binding assays confirm that although ubiquitinated beta-arrestin2 forms a tight complex with the beta2AR, nonubiquitinated beta-arrestin2 can bind reconstituted beta2AR and that the protein-protein interaction domain(s) between the receptor and beta-arrestin2^0K is mostly unperturbed.</p><p>To determine the isoproterenol-stimulated binding of beta-arrestin2^0K to the beta2AR in a cellular context we employed immunoprecipitation assays utilizing chemical crosslinking with a sufhydryl-reactive compound, DTME ([Fig 3,.figureReference+reference+grf=toolXmlRef]). We used COS-7 cells transiently transfected with FLAG-beta2AR and beta-arrestin2^0K-GFP, immunoprecipitated the receptors under nonstimulated or stimulated conditions (5 min, 1 μM isoproterenol) and detected beta-arrestin2^0K-GFP by Western blotting ([Fig 3A,.figureReference+reference+grf=toolXmlRef]). beta-arrestin2^0K-GFP binds to activated receptors with a 2-3 fold agonist-induced recruitment ([Fig 3B,.figureReference+reference+grf=toolXmlRef]). In similar assays, the WT and beta-arrestin2-Ub were recruited 10-12 and 12-15 fold, respectively (data not shown). These experiments further suggest that beta-arrestin2^0K-GFP albeit much weaker than the WT nevertheless binds the beta2AR upon agonist stimulation. Likely, the robust association of beta-arrestin2^0K and the beta2AR does not occur in cells due to a lack of beta-arrestin2 ubiquitination, which helps to stabilize the complex.</p><p>We next transfected HEK-293 cells stably expressing the beta2AR with either beta-arrestin2-GFP, GFP-beta-arrestin2-Ub (stable ubiquitination), or beta-arrestin2^0K-GFP (no ubiquitination) and examined the translocation patterns induced by isoproterenol. All three beta-arrestin variants are uniformly distributed in the cytosol prior to agonist treatment ([Fig 4 A-C,.figureReference+reference+grf=toolXmlRef]). Within one-minute of agonist-stimulation, both WT and GFP-beta-arrestin2-Ub are recruited to the cell membrane and form distinct puncta and at 30 minutes GFP-beta-arrestin2-Ub is recruited to endosomal vesicles ([Fig 4B,.figureReference+reference+grf=toolXmlRef]), while the WT remains at the membrane ([Fig 4A,.figureReference+reference+grf=toolXmlRef]). As previously shown, with a 'Class A' receptor a stably ubiquitinated beta-arrestin traffics into endosomes while the transiently ubiquitinated WT beta-arrestin dissociates and remains at the plasma membrane. On the other hand, agonist stimulation for 1 or 30 min does not lead to a major change in the intracellular distribution of beta-arrestin2^0K-GFP ([Fig 4C,.figureReference+reference+grf=toolXmlRef], center panels).</p><p>To test whether the loss of translocation correlates with a loss of ubiquitination owing to cumulative lysine mutations, we examined isoproterenol-induced recruitment of all the mutants shown in [Fig 1A, B,.figureReference+reference+grf=toolXmlRef]. When the GFP-tagged version of each mutant was coexpressed with HA-beta2AR in HEK-293 cells, we observed normal cytosolic expression under basal conditions for all the beta-arrestin2 variants ([supplementary Fig 1,.figureReference+reference+grf=toolXmlRef]). However, upon 1 min isoproterenol stimulation, a decrease in the level of recruitment was observed correlating with the ubiquitination status of beta-arrestin2 ([Fig 5,.figureReference+reference+grf=toolXmlRef] middle panels and [Fig 1B,.figureReference+reference+grf=toolXmlRef]). Some amount of recruitment remains even when 26 lysine residues are altered. The only mutant that is totally defective in translocation is beta-arrestin2^0K where no ubiquitination sites remain. These data suggest a strong correlation between beta-arrestin ubiquitination status and its ability to bind activated receptors at the plasma membrane.</p><p>The above experiments also suggest that eliminating beta-arrestin ubiquitination decreases its binding affinity for receptors in vivo and hence only unstable receptor-beta-arrestin2^0K-GFP complexes arise at the cell membrane. In the case of 'Class A' receptors, such as the beta2AR, although receptor-beta-arrestin complexes are initially formed at the plasma membrane, these complexes are not long-lived. Thus, beta-arrestin is rapidly deubiquitinated, dissociates from the receptor, and the receptor alone traffics into endosomes. This indicates that events occurring during pit/vesicle formation (e.g. beta-arrestin deubiquitination) can influence the stability of beta-arrestin-receptor complexes. Possibly, the lack of ubiquitin moieties on beta-arrestin2^0K-GFP leads to its rapid disengagement from the receptor complex.</p><p>We hypothesized that beta-arrestin2^0K binds activated receptor at the plasma membrane but that its deficiency in ubiquitination results in decreased stability of the complex as the receptor moves into pits. If this were true, then blocking the internalization of receptors should result in the retention of beta-arrestin2^0K-receptor complexes at the plasma membrane. Indeed, when we inhibited the internalization of either the beta2AR ([Fig 6A,.figureReference+reference+grf=toolXmlRef]) or the 'Class B' V2R ([Fig 6B,.figureReference+reference+grf=toolXmlRef]) by co-expressing dynamin^K44A (a classical inhibitor of endocytosis, ([19,.reference+grf=toolXmlRef],[20,.reference+grf=toolXmlRef])) we trapped activated receptors as well as beta-arrestin2^0K-GFP at the membrane. These experiments clearly indicate that the deficiency in ubiquitination does not inhibit translocation of cytosolic beta-arrestin2^0K to activated receptors at the cell membrane, but rather decreases the stability of the receptor-beta-arrestin complexes that are formed.</p><p>A characteristic feature of beta-arrestin2 is its ability to augment receptor internalization upon over-expression ([9,.reference+grf=toolXmlRef]). This effect is particularly striking in COS-7 cells, which express very low levels of endogenous beta-arrestin2 ([21,.reference+grf=toolXmlRef]). To characterize the ability of beta-arrestin2^0K to promote receptor internalization, we over-expressed it together with HA-tagged beta2AR or V2R and measured the decrease in cell surface receptors after a 30 min agonist treatment. Over expression of beta-arrestin2^0K does not lead to any increase in receptor internalization whereas WT beta-arrestin2 leads to an approximately 2.5-fold increase in both beta2AR and V2R internalization ([Fig 7 A, B,.figureReference+reference+grf=toolXmlRef]). Similarly, over-expression of beta-arrestin2^0K results in no change in receptor internalization in HEK-293 cells ([Fig 7 C, D,.figureReference+reference+grf=toolXmlRef]). Predictably, because of the unstable interaction of beta-arrestin2^0K with activated receptors than the WT, the mutant does not have any inhibitory effect on receptor internalization in both cell types. We have previously demonstrated that the stably ubiquitinated form of beta-arrestin2 (beta-arrestin2-Ub) enhances receptor internalization compared to the WT ([13,.reference+grf=toolXmlRef]). In contrast, beta-arrestin2^0K, which is not ubiquitinated, forms unstable complexes with activated receptors, does not support internalization of either the beta2AR or the V2R.</p><p>We further tested if the above lack of effect of beta-arrestin2^0K to enhance receptor internalization was due to altered binding to endocytic proteins such as clathrin and AP-2. Clathrin binds beta-arrestin directly and stoichiometrically and beta-arrestin-clathrin binding is essential for receptor internalization via clathrin-coated vesicles ([7,.reference+grf=toolXmlRef]). AP-2-beta-arrestin interaction is required for the movement of receptors to clathrin-coated pits ([8,.reference+grf=toolXmlRef]). When beta-arrestin2, beta-arrestin2^0K or beta-arrestin2-Ub were immunoprecipitated from COS-7 cells transfected with HA-beta2AR after 0, 1 and 10 minutes of isoproterenol treatment, an agonist-dependent increase in clathrin binding was observed for both WT and beta-arrestin2-Ub but not for beta-arrestin2^0K ([Fig 8A, B,.figureReference+reference+grf=toolXmlRef]). beta-arrestin2^0K displayed only a weak interaction and a decrease in binding in the presence of isoproterenol ([Fig 8A, B,.figureReference+reference+grf=toolXmlRef]). For the wild type beta-arrestin2, we found a five-fold increase in AP-2 binding at 1 min of agonist treatment and this binding decreased to basal levels at 10 min ([Fig 8A, C,.figureReference+reference+grf=toolXmlRef]). A similar time course of AP-2-beta-arrestin2 interaction has been previously reported ([8,.reference+grf=toolXmlRef]). Surprisingly, both beta-arrestin2-Ub and beta-arrestin2^0K displayed robust binding to AP-2 under both basal and stimulated conditions ([Fig 8A and 8C,.figureReference+reference+grf=toolXmlRef]). Understandably, beta-arrestin2^0K's weak interaction with clathrin upon isoproterenol stimulation could be a major factor in its inability to promote receptor endocytosis. Unlike the previously reported beta-arrestin1^319-418 which binds clathrin but lacks receptor interaction ([22,.reference+grf=toolXmlRef]), beta-arrestin2^0K did not act as an inhibitor of receptor internalization.</p><p>Previous studies have shown that beta-arrestin-Raf complexes are stable since their isolation is possible by gel filtration as well as by coimmunoprecipitation ([18,.reference+grf=toolXmlRef],[23,.reference+grf=toolXmlRef]). We tested the interaction of the above three beta-arrestin forms with the MAPKKK, cRaf ([Fig 9A,.figureReference+reference+grf=toolXmlRef]) and did not observe any differences between the WT and beta-arrestin2^0K in their ability to bind myc-c-Raf1. In these assays, beta-arrestin2-Ub, however, bound more c-Raf than the WT ([Fig 9A,.figureReference+reference+grf=toolXmlRef]). The amount of c-Raf in the immunoprecipitate normalized to total input levels was significantly higher with beta-arrestin2-Ub than with the wild type as indicated by the quantification of bands from three independent experiments (data not shown).</p><p>Previous studies have also shown that by merely coexpressing beta-arrestin2 with a MAPKKK (such as c-Raf, ASK-1) and a MAPK (such as ERK2, JNK3), robust activation of MAPK could be achieved ([18,.reference+grf=toolXmlRef],[24,.reference+grf=toolXmlRef],[25,.reference+grf=toolXmlRef]). This property of beta-arrestin2 is attributed to its capacity to simultaneously bind component enzymes of a kinase cascade thus bringing them in to proximity and allowing robust phosphorylation to occur. Accordingly, cotransfection of beta-arrestin2, cRaf and GFP-ERK2 could enhance precipitation of phosphorylated ERK2 with FLAG-beta-arrestin2 ([18,.reference+grf=toolXmlRef]). To examine whether beta-arrestin2^0K was capable of a similar function, we transfected COS-7 cells with either WT, beta-arrestin2^0K or beta-arrestin2-Ub along with RFP-ERK2 and increasing amounts of myc-cRaf-1. As shown in the Western blots ([Fig 9B,.figureReference+reference+grf=toolXmlRef]) and the bar graphs depicting quantification of pERK in beta-arrestin2 precipitates ([Fig 9C,.figureReference+reference+grf=toolXmlRef]), beta-arrestin2^0K could scaffold pERK to the same extent as the WT. Interestingly, beta-arrestin2-Ub precipitates contained 60-80% more pERK than the WT suggesting either a greater level of kinase activation and/or a stronger interaction of beta-arrestin2-Ub with pERK. All the above coimmunoprecipitation data suggest that ubiquitination is not required for beta-arrestin's interaction with c-Raf and pERK and that despite the 31 lysine mutations beta-arrestin2^0K can interact with these beta-arrestin partners.</p><p>We next examined the effects of beta-arrestin2, beta-arrestin2-Ub, and beta-arrestin2^0K on the assembly of receptor/beta-arrestin2/ERK complexes. As depicted in [Fig 10A,.figureReference+reference+grf=toolXmlRef], a significant amount of pERK was associated with beta2AR immunoprecipitates from COS-7 cells upon coexpression of beta-arrestin2-Ub. Lesser amounts of pERK were detected upon wild type beta-arrestin2 expression, although this amount was still higher than the pERK detected with endogenous beta-arrestin2 in the mock-transfected samples ([Fig 10, A and B,.figureReference+reference+grf=toolXmlRef]). Expression of beta-arrestin2^0K resulted in a significant decrease in pERK in receptor complexes than what was obtained with endogenous beta-arrestin2 as seen in the bar graph representing the quantification of signals from five independent experiments ([Fig 10B,.figureReference+reference+grf=toolXmlRef]). This decrease was not due to a decline in overall ERK activation since the level of activation in whole cell lysates was identical in all transfection conditions.</p><p>In general, beta-arrestin-mediated ERK signals are retained in the cytosol and are prevented from entering the nucleus. To determine if beta-arrestin ubiquitination plays a role in the subcellular localization of agonist-stimulated pERK, we performed confocal immunofluorescence microscopy and examined the relative distribution of agonist-activated receptors, beta-arrestins and pERK. An antibody that specifically recognizes Thr202/Tyr204-phosphorylated ERK1/2 was employed to detect activated endogenous ERK. If ubiquitination of beta-arrestin2 indeed plays a role in determining the spatial distribution of active ERK, then differences should be observed in the cellular distribution of pERK stimulated in the presence of beta-arrestin2-Ub versus beta-arrestin2^0K.</p><p>As depicted in [Fig 11A,.figureReference+reference+grf=toolXmlRef], unstimulated cells show a uniform cytosolic distribution of beta-arrestin2-GFP (green), a membrane distribution of HA-beta2AR (blue) and a negligible amount of pERK (red). When the cells were stimulated for 5 minutes with isoproterenol, beta-arrestin2 redistributed to the cell membrane to colocalize with the activated receptors. A robust increase in the level of pERK was observed in both cytosol and nucleus along with a clearly demarcated pERK signal on beta-arrestin-studded cell membranes (2^nd row, [Fig 11A,.figureReference+reference+grf=toolXmlRef]). After 30 minutes of isoproterenol, the beta2ARs were visualized in intracellular vesicles. beta-arrestins are not localized to these vesicular structures but are retained at the cell membrane. A small percentage of receptors persist at the membrane, which most likely represent recycled and/or non-internalized receptors. After 30 minutes of isoproterenol treatment, a negligible amount of pERK was detected (3^rd row, [Fig 11A,.figureReference+reference+grf=toolXmlRef]). As a comparison, representative cells overexpressing both HA-beta2AR and beta-arrestin2-GFP treated with phorbol myristate acetate (PMA) are shown in the bottom row of [Fig 11A,.figureReference+reference+grf=toolXmlRef]. PMA stimulation leads to robust activation of ERK, which is distributed in both cytoplasm and nucleus. PMA stimulation does not lead to either beta2AR internalization or beta-arrestin2 translocation.</p><p>The results of similar experiments performed with GFP-beta-arrestin2-Ub and HA-beta2ARs are shown in [Fig 11B,.figureReference+reference+grf=toolXmlRef]. Under unstimulated conditions the sub-cellular distributions are identical to what is observed with the WT beta-arrestin2. Quite strikingly, at 5-minute stimulation, a distinct and robust ERK activation is observed at the cell membrane coinciding with the distinct membrane recruitment of beta-arrestin2-Ub. Although a majority of the cells (~80%) displayed such distribution at the cell membrane, some cells did have small vesicles in the vicinity of the cell membrane, which contained beta-arrestin2-Ub, beta2AR as well as pERK as shown in the figure panels (2^nd row, [Fig 11B,.figureReference+reference+grf=toolXmlRef]). Surprisingly at the 5 minute time point, unlike the case of WT beta-arrestin2 expression, little active ERK was distributed in the nucleus with beta-arrestin2-Ub overexpression. We do not know the exact mechanism by which this occurs, but possibly, beta-arrestin2-Ub can simultaneously promote beta-arrestin-dependent cytosolic ERK and curb the G protein ERK pathway leading to lesser nuclear ERK. After 30 minutes of isoproterenol treatment, a dramatic redistribution of beta-arrestin2-Ub, beta2AR and pERK was seen in intracellular vesicles. These data clearly indicate that a stably ubiquitinated beta-arrestin can remain associated with a 'Class A' receptor (i.e. beta2AR) and target activated ERK to early endosomes resulting in a pool of pERK complexed with internalized receptors</p><p>In the absence of agonist, beta-arrestin2^0K-GFP is mainly cytoplasmic, with HA-beta2AR at the plasma membrane and very little active ERK (top row, [Fig 11C,.figureReference+reference+grf=toolXmlRef]). After 5 minutes of isoproterenol-stimulation, a robust activation of ERK occurs which is seen distributed in both cytoplasmic and nuclear compartments. However, none of this active ERK is localized with beta-arrestin2^0K. Possibly, much of this activity is G protein mediated and is excluded from receptor complexes since less pERK is complexed with the beta2AR in the presence of beta-arrestin2^0K (see [Fig 10,.figureReference+reference+grf=toolXmlRef]). At 30 minutes, levels of pERK decreased but were not abolished (bottom row, [Fig 11C,.figureReference+reference+grf=toolXmlRef]). This situation contrasts with what is observed with the stably ubiquitinated beta-arrestin2-Ub ([Fig 11B,.figureReference+reference+grf=toolXmlRef]), where pERK signals are stabilized and localized on endosomal vesicles at 30 min of isoproterenol stimulation. As seen in the 30 min panels of [Fig 11C,.figureReference+reference+grf=toolXmlRef], beta2AR internalized into endosomes which is consistent with our internalization data ([Fig 7,.figureReference+reference+grf=toolXmlRef] AD), which indicate the inability of beta-arrestin2^0K to inhibit receptor internalization.</p><p>We also determined the kinetics of ERK phosphorylation in HEK-293 cells expressing the beta2AR (1 pmol per mg cellular protein) upon transfection of vector, beta-arrestin2 WT, beta-arrestin2^0K or beta-arrestin2-Ub. As shown in [Fig 12,.figureReference+reference+grf=toolXmlRef], expression of beta-arrestin2-Ub significantly increased ERK activity at 20 min of isoproterenol treatment, beta-arrestin2 led to a modest augmentation and beta-arrestin2^0K had no effect over mock conditions ([Fig 12B,.figureReference+reference+grf=toolXmlRef]). Previous studies have demonstrated that, later ERK activity induced by 7TMRs is actually beta-arrestin-mediated (reviewed in ([26,.reference+grf=toolXmlRef])). These results further support the idea that beta-arrestin ubiquitination status underlies some aspects of beta-arrestin-dependent signaling.</p><p>The beta-arrestin isoforms are mainly cytosolic proteins and are translocated to the plasma membrane upon 7TMR activation. Thus far no lipid modifications in beta-arrestins favoring macromolecular membrane interactions have been identified. One well accepted mechanism that keeps them in a membrane environment is their binding to phosphorylated domains of receptors ([5,.reference+grf=toolXmlRef]). Our current and previous results indicate that ubiquitination could be an important factor that determines the longevity of beta-arrestin's interactions with receptorsleading to colocalization on endosomal vesicles. Interestingly, when we analyzed the distribution of the ubiquitinated form of beta-arrestin by sub-cellular fractionation, we found that the ubiquitination status of beta-arrestin favors its partitioning to membrane fractions. When COS-7 cells expressing either beta-arrestin2 or beta-arrestin2-Ub were lysed in a detergent free low salt buffer (40 mM NaCl) and the soluble and insoluble fractions were further separated by differential centrifugation, nonubiquitinated beta-arrestins were mainly cytosolic. Most of the exogenously expressed beta-arrestin2 as well as YFP-beta-arrestin2 were detectable in the soluble fraction ([Fig 13,.figureReference+reference+grf=toolXmlRef]). The YFP-beta-arrestin2 band in the membrane fraction with a slightly slower mobility is unreactive to ubiquitin antibodies such as FK2, P4D1 and FK1 and its identity remains to be elucidated. On the other hand, ubiquitinated beta-arrestin2 was distributed mostly in the insoluble membrane fractions ([Fig 13 A and B,.figureReference+reference+grf=toolXmlRef]). As seen in [Fig 4B,.figureReference+reference+grf=toolXmlRef], beta-arrestin2-Ub appears to be uniformly distributed in the cytosol in an undisturbed cell. Accordingly, the membrane fractionation of beta-arrestin2-Ub is not due to its presence in inclusion bodies, but rather due to its affinity for membrane components. These results suggest that ubiquitination increases beta-arrestin's propensity for membrane association thus favoring beta-arrestin's prolonged localization in membrane microdomains. Although ubiquitination is dispensable for beta-arrestin's interactions with cytososlic partners, it may be necessary to facilitate the formation of functional 7TMR-beta-arrestin endocytic and signaling complexes in a membrane environment.</p>