Agonists induce specific CB1R endocytic dwell times

The CB1R is one of the most abundant receptors in the central nervous system and a key modulator of synaptic function26. CB1R agonists favour distinct receptor conformations that can lead to selective association with intracellular signalling pathways, a mechanism proposed for functional selectivity27,28,29. Following agonist-induced activation, β-arrestins are recruited to the plasma membrane to initiate CME30,31 and signalling. Given that CB1Rs and β-arrestins interact only transiently at the plasma membrane31,32, we hypothesized that specific agonist-induced receptor conformations control the duration of clustering of receptors and β-arrestins in endocytic pits as a mechanism to influence β-arrestin signalling.

To test this hypothesis, we utilized TIRF microscopy of live cells to investigate the endocytosis of CB1Rs at the single pit level33. CB1Rs tagged at the extracellular amino terminus with the pH-sensitive super-ecliptic phluorin34,35 (SEP-CB1R) stably expressed in HEK293 cells were exposed to saturating concentrations of either the synthetic agonist WIN 55,212-2 or the endogenous eicosanoid 2-arachidonoylglycerol (2-AG), both considered to be high-efficacy agonists of G protein activation36,37. We found that 10μM 2-AG produced significantly longer endocytic dwell times, defined as the time receptors are clustered into endocytic pits before endocytosis, when compared with 5μM WIN 55,212-2 (Fig. 1a–e). Quenching experiments showed that all SEP-CB1R fluorescence was rapidly reduced by addition of MES, pH 5.5 to the imaging media. This indicated that the endocytic clusters analysed in our experiments are indeed at the cell surface and not dwelling in vesicles below the plasma membrane (Supplementary Fig. 1 and Supplementary Movie 1).

Figure 1: Agonists induce specific endocytic dwell times of the CB1R. (a) HEK293 cells expressing SEP-CB1Rs before and after 5μM WIN 55,212-2 imaged using TIRF. Kymographs from the cell surface show individual endocytic events (yellow rectangle). (b) Normalized fluorescence intensity from the event in a. s.d. from multiple normalized traces for 5μM WIN 55,212-2 is depicted in grey. (c) Representative kymograph showing individual SEP-CB1Rs endocytic events in the presence of 10μM 2-AG. (d) Normalized fluorescence intensity from an endocytic event elicited by 2-AG with s.d. from multiple events in grey. (e) Box and whiskers plot (median values with min/max range) showing dwell times of a single endocytic event elicited by 5μM WIN 55,212-2 and 10μM 2-AG (n=336 events per 12 cells and 244 events per 9 cells). (f) Endocytosis elicited by 5μM WIN 55,212-2 and 10-μM 2-AG was analysed by the decrease in total surface fluorescence intensity in HEK293 cells by time lapse TIRF microscopy (n=5 cells per treatment). (g) Dwell times were analysed for the indicated concentrations of agonist. No statistically significant difference was observed between concentrations (n=6–12 cells per condition). (The mean time±s.d. for 0.1 and 10 μM of WIN 55,212-2 was 106±63 and 117±88 s, respectively. The mean times for 0.1 and 0.5 μM of 2-AG was 177±87 and 159±80 s). (h) Kymograph from a hippocampal neuron expressing SEP-CB1Rs in the presence of 5 μM WIN 55,212-2. (i) Kymograph from a hippocampal neuron incubated with 10 μM 2-AG. (j) Representative traces of normalized fluorescence from individual endocytic events. (k) Box and whiskers plot (median values with min/max range) and cumulative probability graphs from SEP-CB1Rs dwell times elicited by the indicated agonists in hippocampal neuronal cultures (n=84 events per 7 cells and 109 events per 10 cells). Full size image

Distinct populations of CCPs have been reported at the plasma membrane: short-lived populations (τ<20–30 s) associated with abortive events and long-lived populations (τ>30–180 s) associated with productive endocytic events38,39. For GPCRs, their structure and ubiquitination levels have been shown to control the maturation process of endocytic pits40,41,42,43. However, it is not currently known whether agonists can control the kinetics of endocytic dwell times or whether the same receptor can adopt multiple dwell times.

Thus, next we sought to test whether the variability observed in dwell times reflects differences in the endocytic efficacy of the agonists or their binding properties to the receptors. First, we analysed the endocytosis of CB1Rs induced by 5μM WIN 55,212-2 and 10μM 2-AG, the same concentrations in the same cells where specific dwell times were observed. TIRF time-lapse microscopy was performed and surface fluorescence was analysed to determine the total rate of receptor removal from the cell surface (Fig. 1f). Results indicate that both agonists induced endocytosis of SEP-CB1R to the same extent with similar kinetics (τ =14–18 min; P=0.07 unpaired two-tailed t-tests throughout the manuscript unless stated). Second, we analysed whether dwell times were dependent on the concentration of the ligand. We analysed SEP-CB1R dwell times at concentrations below and close to the ligand Ki and also at near-saturating concentrations. The Ki values for WIN 55,212-2 and 2-AG were reported previously as 10–200 and 100–500 nM, respectively, with both agonists having similar potency and efficacy for GIRK current activation44,45,46,47,48. Analysis of multiple endocytic events across several experiments indicated that dwell times were not significantly different for the concentrations tested for each ligand (Fig. 1g). These results indicate that the time receptors are clustered into endocytic pits do not correlate with the endocytic efficacy or agonist concentration, suggesting a new role for dwell times during the endocytic process.

Ligand-specific dwell times are a conserved mechanism

Previous studies have shown that CB1R function is dramatically sensitive to its cellular environment and results obtained in heterologous systems do not always replicate in more complex cells such as primary neuronal cultures49,50. To test whether ligand-specific dwell times are conserved in a more complex cellular environment, we transfected dissociated hippocampal neurons with SEP-CB1Rs at DIV 4–5 and performed TIRF microscopy at DIV >15. This lag between transfection and imaging significantly reduced the expression of SEP-CB1R towards physiological levels (Supplementary Fig. 2A,B) and resulted in cellular localization comparable to endogenous receptors (Supplementary Fig. 2C,D). As observed in HEK293 cells, 5μM WIN 55,212-2 and 10 μM 2-AG elicited dramatically different CB1R dwell times in hippocampal neuronal cultures (Fig. 1h–k). Dwell times in neurons were not significantly different from those observed in HEK293 cells for the same ligand (P=0.35 for 5 μM WIN and P=0.07 for 10 μM 2-AG in HEK versus neurons). These results indicate that agonists can elicit specific endocytic dwell times that are intrinsic to the ligand–receptor interaction and not to their cellular environment.

CME has been described as the major mechanism for ligand-induced endocytosis of the CB1R31,51,52. However, other studies suggested that CB1Rs expressed in heterologous cells can also colocalize with caveolin-1, and ligand-induced endocytosis can occur in parallel via CCPs and caveolar endocytosis53,54. As different dwell times could be associated with different endocytic pathways, we investigated which mechanism mediates ligand-specific dwell times of CB1Rs. We transiently transfected fluorescently tagged Ds-Red clathrin or mCherry-caveolin-1 into HEK293 cells stably expressing SEP-CB1Rs and analysed individual endocytic events elicited by 5μM WIN 55,212-2. Dual colour simultaneous TIRF imaging showed that SEP-CB1R endocytic pits colocalized only with clathrin, and not with caveolin-1 (Fig. 2a). Kymographs from endocytic movies and intensity measurements from individual events clearly show that SEP-CB1Rs associate exclusively with Ds-Red clathrin in the presence of WIN 55,212-2 (Fig. 2b,c). Next, we elicited endocytosis with 10μM 2-AG. Single frames from endocytic movies and their kymographs show that SEP-CB1R endocytosis is again associated with the recruitment of clathrin and not caveolin-1 (Fig. 2d,e). Fluorescence intensities from individual SEP-CB1R endocytic events failed to associate with mCherry-caveolin-1 (Fig. 2f). Colocalization analysis from individual endocytic events across multiple experiments indicated that CB1Rs were highly colocalized with clathrin (Pearson’s=0.5–0.55) when compared with caveolin (Pearson’s=0.07−0.05) for both agonists (Fig. 2g,h), strongly suggesting that CME is the endocytic mechanism underlying ligand-specific dwell times.

Figure 2: Agonists control the maturation process of CB1R clathrin-coated pits. (a) HEK293 cells co-expressing SEP-CB1Rs and DsRed-clathrin (top) or mCherry-caveolin-1 (bottom) incubated with 5μM WIN 55,212-2 and imaged under TIRF microscopy. Single frames show overlay after 5 min of incubation. (b) Kymographs showing individual SEP-CB1R endocytic event with clathrin (top, yellow traces) and caveolin-1 (bottom). (c) Representative normalized fluorescence intensity scan from the overlay of SEP-CB1Rs and DsRed-clathrin. (d) HEK293 cells expressing SEP-CB1Rs and either DsRed-clathrin (top) or mCherry-caveolin-1 (bottom) were incubated with 10μM 2-AG. Single frames after 5 min show the overlay. (e) Kymographs showing individual SEP-CB1R endocytic events with clathrin (top, yellow traces) and mCherry-caveolin-1 (bottom). (f) Representative fluorescence intensity trace from an individual SEP-CB1R endocytic event showing no colocalization with mCherry-caveolin-1. (g) Colocalization was analysed in multiple cells 5 min after treatments between CB1Rs and clathrin or CB1Rs and caveolin-1 (n=5–7 cells, error bars represent s.e.m. from multiple analyses). (h) Colocalization analysis after a 5-min treatment with 10μM 2-AG between SEP-CB1Rs and DsRed-clathrin or mCherry-caveolin-1 (n=6–9 cells, error represents s.e.m. from multiple analyses). (i) Clathrin-coated pit dwell times were analysed in HEK cells expressing only Ds-Red-clathrin under basal conditions or in the presence of the indicated ligands. (j) The mean intensity from the initial 20 s from events elicited with 5μM WIN 55,212-2, black shows the mean and grey s.d. (n=50 events). (k) The mean intensity from endocytic events elicited with 10μM 2-AG (n=70 events). (l) Box and whiskers analysis comparing normalized intensities from j,k at 20 s (left) and 40 s (right) after event onset (P=0.46 and P=0.29, respectively, plot indicates the median values and min/max range). Full size image

As modulation of dwell times could be mediated by the presence of CB1Rs or by a non-CB1R-mediated effect of the agonist, we analysed the dwell times of CCPs in HEK293 cells transfected with fluorescently tagged Ds-Red clathrin in the absence of SEP-CB1Rs. Dwell times were investigated in untreated cells or in the presence of 5μM WIN55,212-2 and 10μM 2-AG. Our results showed no significant difference in dwell times between untreated control and treated cells in the absence of SEP-CB1R (Fig. 2i). Importantly, dwell times obtained from CCPs closely resemble dwell times previously reported in the literature55,56,57.

CCPs traverse distinct steps during their lifetime before removal from the cell surface. Major events are assembly or pit initiation, maturation and final separation from the cell surface55. Growing evidence suggests that these steps can be regulated by lipid kinases and phosphatases that control the formation and maturation processes of CCPs58,59,60. To investigate which of these steps agonists modulate, we compared the changes in fluorescence intensity during the assembly and maturation processes of individual SEP-CB1R endocytic pits with similar kinetics. Fluorescence intensity traces from individual events were normalized to their maximum and compared for WIN 55,212-2 and 2-AG. Analysis of multiple events indicated that maximum intensities (maximum number of receptors) were reached with similar kinetics for both agonists, with no significant difference observed during the assembly phase (Fig. 2j–l, τ ~24 s). This suggests that the recruitment of receptors into individual endocytic pits occurs with similar kinetics; however, once the coated pits reach their maximum number of receptors (at maximum intensity), pits linger for variable periods of time depending on the ligand that activated the receptor. As we cannot identify changes at the single receptor level, we cannot rule out the possibility that individual receptors move in and out from single pits during dwell times as described for other cargoes57.

β-arrestins bind to activated GPCRs to initiate the endocytic process11,12,13,14. As agonists can determine the time receptors are clustered into endocytic pits with β-arrestins, we hypothesized that this regulation could mediate β-arrestin signalling. To test this hypothesis, first we investigated the recruitment kinetics of β-arrestin-1 into CB1Rs CCPs. We have previously shown that β-arrestin-1 mediates CB1R activation of ERK1/2 (ref. 61). Cells stably expressing SEP-CB1Rs were transfected with red fluorescent protein (RFP)-tagged β-arrestin-1 and incubated with either 5μM WIN 55,212-2 or 10μM 2-AG and imaged under TIRF microscopy. Upon ligand incubation, β-arrestin-1 was rapidly recruited to the plasma membrane and into individual endocytic pits as depicted in kymographs (Fig. 3a and Supplementary Movie 2). Normalized individual fluorescence traces indicated that β-arrestin-1 was recruited into SEP-CB1R endocytic pits and remained present throughout the endocytic process (Fig. 3b). As observed with SEP-CB1Rs, 2-AG elicited longer β-arrestin-1 dwell times when compared with WIN 55,212-2 (Fig. 3c,d). Analysis of multiple experiments from cells co-expressing RFP-β-arrestin-1 and SEP-CB1Rs showed a significant increase in the dwell time of β-arrestins in the presence of 2-AG (Fig. 3e,f). This increase in dwell time was comparable to those observed with SEP-CB1R alone, suggesting that the overexpression of RFP-β-arrestin did not have a deleterious effect on the endocytic process (Figs 1e and 3e). Correlation analysis between dwell times of CB1R and β-arrestin-1 from the same endocytic events indicated that both proteins are recruited and retained at CCPs with identical ligand-dependent kinetics (Fig. 3g,h).

Figure 3: β-arrestin-1 is recruited and retained into CB1R CCPs with ligand-specific kinetics. (a) HEK293 cells co-expressing SEP-CB1R and mRFP-β-arrestin-1 were incubated with 5μM WIN 55,212-2 and analysed under dual-colour TIRF microscopy. Representative kymograph showing individual SEP-CB1R events colocalizing with mRFP-β-arrestin-1. (b) Individual normalized fluorescence intensity from SEP-CB1Rs and β-arrestin-1 during the endocytic event from a (yellow rectangle). (c) Representative kymograph from HEK293 cells expressing SEP-CB1R and mRFP-β-arrestin-1 in the presence of 10μM 2-AG. Individual SEP-CB1R endocytic events colocalize with β-arrestin-1. (d) Normalized fluorescence intensity from SEP-CB1Rs and mRFP-β-arrestin-1 from c (yellow rectangle). (e) Box and whiskers plot showing β-arrestin 1 dwell times into CCPs elicited by 5μM WIN 55,212-2 and 10μM 2-AG, n=342 events per 19 cells and 378 events per 21 cells (plot indicates the median values with min/max range). (f) Cumulative probability from β-arrestin 1 dwell times for 5μM WIN 55,212-2 and 10μM 2-AG. (g) Correlation between β-arrestin 1 dwell times and SEP-CB1R dwell times from the same endocytic event when elicited by 5μM WIN 55,212-2 (P<0.0001; n=305 endocytic events). (h) Correlation between β-arrestin 1 dwell times and SEP-CB1R dwell times from the same endocytic event when elicited by 5μM 2-AG (P<0.0001; n=197 endocytic events). Full size image

β-arrestin-2 has previously been shown to be involved in CB1R internalization28. To investigate the recruitment kinetics of β-arrestin-2 to CB1R CCPs, HEK293 cells stably expressing SEP-CB1Rs were transfected with RFP-tagged β-arrestin-2. Recruitment was initiated by either 5μM WIN 55,212-2 or 10μM 2-AG. As previously observed with β-arrestin-1, β-arrestin-2 was efficiently recruited to the plasma membrane and into individual endocytic pits (Fig. 4a,b). Analysis of β-arrestin-2 dwell times at CCPs showed identical ligand-specific kinetics to those observed before (Figs 3e,f and 4c,d). These data show that both β-arrestins can be recruited and retained into CB1R-CCPs with ligand-dependent specificity.

Figure 4: β-arrestin-2 is recruited and retained into CB1R CCPs with ligand-specific kinetics. (a) HEK293 cells co-expressing SEP-CB1R and mRFP-β-arrestin-2 were incubated with 5μM WIN 55,212-2 and analysed under dual-colour TIRF microscopy. Representative kymograph depicts an individual endocytic event and the colocalization between SEP-CB1R and mRFP-β-arrestin-2. (b) Individual normalized fluorescence intensity from SEP-CB1R and β-arrestin-2. (c) Box and whiskers plot (the median values and min/max range) showing β-arrestin-2 dwell times (n=304 events per 21 cells and 321 events per 15 cells). (d) Cumulative probability for β-arrestin 2 dwell times in the presence of 5μM WIN 55,212-2 and 10μM 2-AG. Full size image

2-AG signals through G proteins and β-arrestin-1

To test the hypothesis that prolonged dwell times influence β-arrestin signalling, we investigated ERK1/2 phosphorylation in response to WIN 55,212-2 and 2-AG along with the agonists CP55940 and its ago-allosteric modulator ORG27569 for comparison. We have previously demonstrated that ERK1/2 can be activated via CB1Rs in both a G-protein-dependent and G-protein-independent manner28,61,62. All four compounds induced ERK1/2 phosphorylation that peaked at 5 min (Fig. 5a,b). Most strikingly, 2-AG treatment resulted in sustained phosphorylation that was still substantial at 15 min (Fig. 5a bottom and Fig. 5b), while the phosphorylation elicited by CP55940, ORG27569 and WIN 55,212-2 drastically decreases after 5 min.

Figure 5: CB1R/β-arrestin-1 mediates the activation of ERK1/2 by 2-AG. (a) HEK293 cells expressing SEP-CB1Rs were exposed to 10μM CP55940, ORG27569, WIN 55212-2 and 2-AG for 5, 10 and 15 min. Cell lysates were analysed using western blots with phospho-ERK1/2 (p-ERK1/2) or total ERK1/2. (b) Quantified time course in a showing ERK1/2 phosphorylation levels induced by 10 μM of each compound. (c) HEK293 cells expressing SEP-CB1Rs were exposed to 10μM CP55940, ORG27569, WIN 55212-2 and 2-AG for 5, 10 and 15 min with PTX pre-treatment, respectively. (d) Quantified time course in c showing ERK1/2 phosphorylation levels from cells pretreated with PTX. (e,g,i,k) HEK293 cells co-expressing SEP-CB1R and either control (e), β-arrestin-1 (g), β-arrestin-2 (i) siRNAs or β-arrestin-2 siRNA with PTX pretreatment (k) were exposed to 10 μM of CP55940, ORG27569, WIN 55212-2 and 2-AG, respectively, as indicated. (f,h,j,l). Graphs in f,h,j,l provide the quantified ERK1/2 phosphorylation levels induced by 10 μM of each compound as shown in e,g,i,k. Data represent the mean±s.e.m. of at least three independent experiments. Full size image

To establish specificity, HEK293 cells lacking CB1Rs did not show changes in ERK1/2 phosphorylation upon treatment of each of the four compounds, indicating that the phosphorylation pattern shown in Fig. 5a is CB1-mediated (Supplementary Fig. 3D). We also analysed whether ORG27569 can induce endocytic events. Interestingly, 10μM ORG27569 elicited a very small number of endocytic events reflecting its ago-allosteric function. Remarkably, the dwell times elicited by ORG27569 were statistically indistinguishable from those observed with 2-AG (Supplementary Fig. 3C).

To evaluate the involvement of Gi/o proteins in ERK1/2 phosphorylation, we treated cells expressing SEP-CB1Rs with pertussis toxin (PTX). Consistent with previous findings56, PTX attenuated CP55940-induced ERK1/2 phosphorylation, while it showed no effect on ORG27569-induced phosphorylation (Fig. 5c top). PTX treatment also abolished WIN 55,212-2-induced ERK1/2 phosphorylation. Strikingly, PTX treatment inhibited 2-AG-induced ERK1/2 phosphorylation at the early time point (5 min), but not the later time points (10 and 15 min; Fig. 5c bottom and 5d). This suggests that 2-AG induces ERK1/2 activation via multiple pathways and only activation at the early time point is mediated by Gi proteins.

To test whether β-arrestins mediate 2-AG-induced ERK1/2 phosphorylation at later time points, we used silencing technology to reduce the expression of endogenous β-arrestin-1 and β-arrestin-2. Co-transfection with SEP-CB1Rs and negative control short interfering RNA (siRNA) exhibited a similar pattern of ERK1/2 phosphorylation (Fig. 5e,f) to controls (Fig. 5b). Consistent with previous data28, Fig. 5g shows that while the reduced expression of β-arrestin-1 exhibited little effect on CP55940-induced ERK1/2 phosphorylation, it substantially abrogated ORG27569-induced ERK1/2 phosphorylation, indicating that this phosphorylation is mediated by β-arrestin-1. WIN 55,212-2-induced ERK1/2 phosphorylation remained unaffected with the reduced expression of β-arrestin-1. Most strikingly, silencing β-arrestin-1 resulted in a significant decrease in the level of phosphorylation induced by 2-AG at the later time points (10 and 15 min) without affecting its phosphorylation at the 5-min time point (Fig. 5g,h). The effectiveness of isoform-specific silencing of β-arrestin 1 and 2 was confirmed (Supplementary Fig. 3E). Taken together, these results show that 2-AG induces a maximal Gi/o-mediated ERK1/2 phosphorylation at 5 min followed by a sustained ERK1/2 activation at the later time points (10 and 15 min) mediated by β-arrestin-1 providing the first reported case of canonical and non-canonical signalling pathways induced by the endogenous agonist 2-AG via CB1R.

Reduction of β-arrestin-2 using siRNA did not have a substantial effect on CP55940-, ORG27569- and WIN 55,212-2-induced ERK1/2 phosphorylation when compared with controls (Fig. 5i), although these agonists resulted in a small but sustained level of ERK1/2 phosphorylation at the later time points (10 and 15 min) compared with those shown by control siRNA transfection (Fig. 5j). This may reflect a need for β-arrestin-2 for internalization31,32 and reduced internalization may allow for extended Gi/o signalling. To test this possibility, we treated cells expressing SEP-CB1Rs with PTX. PTX treatment inhibited CP55940- and WIN 55,212-2-induced extended ERK phosphorylation suggesting that the extended ERK1/2 phosphorylation is Gi-mediated (Fig. 5k,l). In contrast, the substantially sustained levels of ERK1/2 phosphorylation at the later time points induced by 2-AG treatment (Fig. 5e bottom) was diminished to a level similar to that of CP55940, ORG27569 or WIN 55,212-2 treatment. It is possible that the reduced expression of β-arrestin-2 results in inhibition of CCP formation, thus abrogating the β-arrestin signalling that occurs in endocytic pits but prolonging ongoing Gi/o signalling. Interestingly, PTX treatment further inhibited 2-AG-induced ERK phosphorylation (Fig. 5k) suggesting that the diminished level of phosphorylation resulting from β-arrestin-2 knockdown is Gi-mediated.

Extension of dwell times enhances β-arrestin signalling

To directly test the hypothesis that dwell times dictate β-arrestin-mediated ERK1/2 signalling, we pre-treated cells with the dynamin inhibitor dyngo-4a. Inhibition of endocytosis dramatically prolonged SEP-CB1R dwell times throughout the imaging sessions (Fig. 6a) and, according to our model, it should enhance β-arrestin-mediated signalling. Since 2-AG exhibited a significantly longer endocytic dwell time than WIN 55,212-2 (Fig. 1e) and sustained β-arrestin-mediated ERK1/2 signalling (Fig. 5), the effect of prolonging the endocytic dwell time on 2-AG-induced ERK1/2 phosphorylation was evaluated up to 60 min. Figure 6b shows that 2-AG treatment (no dyngo-4a) exhibited a sustained phosphorylation pattern of ERK1/2 up to 15 min, while the phosphorylation by WIN 55,212-2 drastically decreases after the 5-min peaks. Strikingly, pre-treatment with dyngo-4a resulted not only in an extension but also in an increase in 2-AG-induced ERK1/2 phosphorylation (Fig. 6b,c). WIN 55,212-2 treatment showed essentially no increase in ERK1/2 phosphorylation in the presence of dyngo-4a, except for a small increase at later time points (45 and 60 min). One possible explanation is that prolonging dwell times results in increased interactions with either G protein or β-arrestin for ERK1/2 signaling.

Figure 6: CB1R dwell times control β-arrestin signalling. (a) HEK293 cells expressing SEP-CB1R were preincubated with 30μM dyngo-4a and imaged under TIRF before and after bath application of 10μM WIN55212-2. Representative kymograph shows prolonged SEP-CB1R dwell times in the presence of dyngo-4a. (b) HEK293 cells expressing SEP-CB1R were pre-incubated with 30μM dyngo-4a before exposure to agonists and compared with no dyngo-4a treatment. (c) Graph provides the quantified ERK1/2 phosphorylation induced by 10 μM of each compound shown in b. Data are expressed as a percentage of the level of phosphorylation at 5 min for each compound without dyngo-4a pre-treatment. (d) HEK293 cells expressing SEP-CB1Rs with and without dyngo-4a pretreatment were exposed to 10μM 2-AG as indicated with PTX pre-treatment. (e) HEK293 cells co-expressing SEP-CB1R and β-arrestin-1 siRNAs with and without dyngo-4a pretreatment were exposed to 10μM 2-AG as indicated. (f) Quantified time course in d,e showing ERK1/2 phosphorylation levels induced by 10μM 2-AG for 5, 15, 30, 45 and 60 min for cells either pre-treated with PTX or co-transfected with β-arrestin-1 siRNA. (g) HEK293 cells expressing SEP-CB1R were treated with PTX (or no PTX as a control), and then incubated with 30μM dyngo-4a before subsequent exposure to WIN 55,212-2. (h) Graph provides the quantified ERK1/2 phosphorylation induced by 10μM WIN 55,212-2 in the absence of PTX treatment shown in g. (i) HEK293 cells expressing SEP-CB1R with or without dominant-negative dynamin 2 K44A were exposed to agonists. (j) Graph provides the quantified ERK1/2 phosphorylation induced by 10 μM of each compound shown in i. All data are expressed as a percentage of the level of phosphorylation at 5 min for 2-AG without dyngo-4a pre-treatment and represent the mean±s.e.m. of at least three independent experiments. Full size image

Finally, to determine whether the increase in ERK1/2 phosphorylation by 2-AG in the presence of dyngo-4a was mediated by β-arrestins, cells were incubated with PTX or co-transfected with β-arrestin-1 siRNA. Figure 6d shows that preincubation with PTX inhibited 2-AG-induced ERK1/2 phosphorylation at 5 min but had no effect during the later time points between 15 and 60 min. In contrast, silencing of β-arrestin-1 expression resulted in a substantial decrease in the level of phosphorylation induced by 2-AG at the later time points between 15 and 60 min without affecting its phosphorylation at the 5-min time point (Fig. 6e,f). To investigate whether prolonging endocytic dwell times with dyngo-4a treatment results in an increase in ERK1/2 phosphorylation in the absence of Gi protein, we treated PTX-pretreated HEK293 cells expressing SEP-CB1R with dyngo-4a. WIN 55,212-2 treatment failed to show any increase in ERK1/2 phosphorylation in the presence of dyngo-4a and PTX compared with those without PTX treatment (Fig. 6g,h). These data suggest that WIN 55,212-2 promotes the CB1 conformation that couples to Gi but not to β-arrestin

To further confirm the correlation between longer endocytic dwell times and extended β-arrestin signalling induced by dyngo-4a, we expressed a dynamin 2-dominant-negative mutant. Expression of the dominant-negative dynamin also substantially extended ERK1/2 phosphorylation, similarly to dyngo-4a treatment, upon 2-AG treatment (Fig. 6i top and Fig. 6j). In contrast, WIN 55,212-2 treatment showed essentially no increase in ERK1/2 phosphorylation upon co-expression of the dynamin 2-dominant-negative mutant (Fig. 6i bottom), also comparable to dyngo-4a treatment. Taken together, these results show that prolonging CB1R dwell times by the dynamin inhibitor dyngo-4a or expression of the dominant-negative dynamin 2 significantly increased 2-AG-induced activation of ERK1/2 mediated by β-arrestin-1.