Residency time of agonists does not affect the stability of GPCR–arrestin complexes

The interaction of arrestins with G‐protein coupled receptors (GPCRs) desensitizes agonist‐dependent receptor responses and often leads to receptor internalization. GPCRs that internalize without arrestin have been classified as “class A” GPCRs whereas “class B” GPCRs co‐internalize with arrestin into endosomes. The interaction of arrestins with GPCRs requires both agonist activation and receptor phosphorylation. Here, we ask the question whether agonists with very slow off‐rates can cause the formation of particularly stable receptor–arrestin complexes.


| INTRODUCTION
The arrestins are a small family of four cytosolic proteins that specifically bind to activated and phosphorylated G-protein coupled receptors (GPCRs) to prevent further G-protein coupling by sterically blocking the G-protein binding site. Furthermore, they also act as adaptors for the endocytic machinery and promote the internalization of activated receptors (Ferguson, 2001). Based on the apparent stability of arrestin-receptor complexes, GPCRs are classified into two groups (Oakley, Laporte, Holt, Caron, & Barak, 2000). Class A receptors such as the β 2 -adrenoceptor or μ opioid receptors interact only transiently with arrestins resulting in the dissociation of arrestin during the endocytic process. In contrast, class B receptors like the vasopressin V 2 receptor form very stable complexes with arrestins that co-internalize into endosomes. Notably, agonists for class A receptors typically have fast off-rates whereas most agonists for class B receptors are peptides which dissociate slowly from the receptor under physiological conditions. However, it has been found that a sufficient number of phosphate residues in the correct spatial arrangement in the C-terminal tail of a receptor is required for high-affinity binding of arrestins (Mayer et al., 2019) and therefore determines which class a receptor belongs to. While the significance of phosphorylation sites for class A or B behaviour of a GPCR has been studied intensively (Latorraca et al., 2020;Mayer et al., 2019;Oakley, Laporte, Holt, Barak, & Caron, 1999;Zhou et al., 2017;Zindel et al., 2015Zindel et al., , 2016, it is unknown whether the stability of arrestin-receptor complexes also depends on agonist affinity. While it was previously shown that arrestin binding increases agonist affinity to a receptor (Gurevich, Pals-Rylaarsdam, Benovic, Hosey, & Onorato, 1997), it is still unclear if agonists with prolonged receptor occupancies might stabilize the binding of arrestin at the receptor more than agonists with fast offrates and whether arrestin actually dissociates from agonist-occupied receptors without previous agonist dissociation. Therefore, we compared the stability of arrestin-receptor complexes induced by several ligands with different off-rates by employing two different methods.
Firstly, Foerster resonance energy transfer (FRET) was used to assess the interaction between arrestin-3 and receptor upon agonist application and withdrawal. In addition, the steady-state apparent affinity of arrestin-3 to receptors in the continuous presence of agonist was observed by dual-colour fluorescence recovery after photobleaching (FRAP). Furthermore, we imaged the co-internalization of arrestin-3 with activated receptors to find out whether agonist off-rates determine the class A or class B behaviour of GPCRs.

| Foerster resonance energy transfer (FRET)
FRET between fluorescently tagged arrestin and receptors was measured as described previously (Krasel, Bünemann, Lorenz, & Lohse, 2005). HEK293T cells were transiently transfected with YFPlabelled receptors (0.7 μg), mTurquoise-labelled arrestin-3 (0.9 μg) and non-fluorescent GRK2 (0.4 μg). Measurements were either performed on an inverted eclipse Ti Nikon (Düsseldorf, Germany) microscope as described by Zindel et al. (2015) or on an inverted Zeiss (Oberkochen, Germany) Axiovert 100 microscope as described by Wolters, Krasel, Brockmann, and Bünemann (2015). In both cases, the illumination frequency was set to 2 Hz and the illumination time to 30-60 ms, depending on the brightness of the cells. During all measurements, cells were continuously superfused with FRET buffer (137-mM NaCl, 5.4-mM KCl, 2-mM CaCl 2 , 1-mM MgCl 2 and 10-mM HEPES, pH 7.3) or buffer containing agonist in different concentrations using a pressure-driven fast-switching eight-channel valve-controlled pressurized perfusion system (VC3-8xP Series; ALA Scientific Instruments, Farmingdale, NY, USA). Data were corrected for background fluorescence, spillover of CFP into the YFP channel and direct YFP excitation. The resulting emission ratios F YFP /F CFP were normalized to baseline and plotted as mean ± SEM. For quantification, kinetics were quantified by a monophasic exponential fit of the full (μ receptor) washout phase or the first 200 s upon agonist withdrawal (β 2 -adrenoceptor).

| Fluorescence recovery after photobleaching (FRAP)
FRAP was used to assess the stability of the receptor-arrestin interaction similar to the protocol described previously (Zindel et al., 2015(Zindel et al., , 2016. For this type of experiment, it is important that the receptor is present in excess over the arrestin because otherwise arrestin mobility will only be partially altered. HEK293T cells were transfected with mTurquoise-tagged arrestin3, GRK2 and receptors labelled at the N-terminus with SYFP2. Receptors were immobilized by crosslinking them with a polyclonal anti-GFP antibody (Rockland Cat# 600-101-215, RRID:AB_218182; Rockland, Gilbertsville, PA) at a dilution of 1:100 for 30 min at 37 C. Subsequently, cells were stimulated with agonist and imaged using a Leica TCS SP5 scanning confocal microscope equipped with a Lambda Blue 63Â/1.4NA oil immersion lens (Leica, Wetzlar, Germany). Image size was 256 Â 256 pixels, and scan speed was 1000 Hz. mTurquoise was bleached with a 405-nm diode laser and SYFP2 with the 514-nm line of an argon laser. A 3 Â 1 μm area was bleached, and the fluorescence recovery was

| Confocal microscopy
HEK293T cells were transiently transfected with mCherry-tagged receptors (0.6 μg), GFP-tagged arrestin-3 (1.0 μg) and non-fluorescent GRK2 (0.4 μg). Cells were treated with agonists for 30 min at 37 C and immediately examined for receptor-arrestin colocalization by confocal microscopy (Leica TCS SP5, Lambda Blue 63Â/1.4NA oil immersion lens). GFP was excited at 488 nm with an argon laser and emission was collected at 500-540 nm. mCherry was excited at 543 nm with a helium/neon laser, and mCherry emission was collected at 600-680 nm. Pearson's colocalization coefficient was calculated from individual images using ImageJ 1.47 and the Colocalization Test plugin as described previously (Zindel et al., 2015).

| Statistics
The data and statistical analysis in this study complies with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2018). Results are represented as means ± SEM from at least three independent transfections of n (at least five) individual randomly selected cells. No statistical methods were used to predetermine group size. Experiments were designed with equal group sizes, but sometimes, data from individual cells had to be excluded; for example, if the data were too noisy to allow a reliable curve fit as explained above. Otherwise, all outliers were included in the data analysis and presentation. Statistical analysis was performed using GraphPad Prism 8.3. Unpaired Student's t tests were used for comparison of two groups. To compare more than two groups and to test each condition against the mean of every other, one-way ANOVAs followed by Tukey's post hoc tests were used when the standard deviations of each group were not significantly different, whereas Brown-Forsythe tests followed by Dunnett's T3 multiple comparisons test were used for significantly different standard deviations. Post hoc tests were run only performed if F achieved P < 0.05 and there was no significant variance inhomogeneity. Differences were considered statistically significant for P < 0.05.

| Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, and are permanently archived in the Concise Guide to PHARMACOLOGY 2021/22 (Alexander, Christopoulos et al., 2021;Alexander, Fabbro, et al., 2021).

| RESULTS
In this study, we compared two different methods for the assessment of GPCR-arrestin interactions in single living cells, FRET and FRAP, using two receptors, the β 2 -adrenoceptor and the μ receptor.
Interactions between β 2 -adrenoceptor-YFP and arrestin-3-mTurquoise in the presence of co-transfected GRK2 were mea-  which were quantified by mono-exponential curve fits of the first 200 s of the washout phase. Arrestin-3 dissociated significantly faster from the β 2 -adrenoceptor after washout of adrenaline compared to isoprenaline as previously reported (Krasel, Bünemann, Lorenz, & Lohse, 2005). In contrast, the washout of BI-167107 did not lead to any dissociation of arrestin from the β 2 -adrenoceptor which can be explained by the long dissociation half-life of the ligand of approximately 30 h (Rasmussen et al., 2011). To enable statistical comparison between the exponential decays of adrenaline, isoprenaline and the apparent non-dissociation of BI-167107, the area under the curve (AUC) of traces normalized to the maximum was calculated ( Figure 1c). As the AUC values for BI-167107 were markedly higher than for adrenaline or isoprenaline, the FRET measurements demonstrated that arrestin-3 dissociation from the β 2 -adrenoceptor depends on the specific agonist. These data suggest that the dissociation kinetics of arrestin in FRET assays are more suited as a proxy for agonist off-rates from the receptor as discussed by Krasel, Bünemann, Lorenz, & Lohse (2005). We have used agonist off-rates before as a measure of the apparent affinity of arrestin to a receptor; however, in these experiments, we did not compare different agonists but always used the same agonist (isoprenaline) and examined different receptor phosphorylation patterns (Zindel et al., 2015).
We also measured agonist-induced interactions between β 2adrenoceptor-YFP and arrestin-2-mTurquoise in the presence of GRK2 ( Figure S1a). The FRET amplitude was notably smaller than with arrestin-3-mTurquoise, consistent with a lower affinity of arrestin-2 to class A receptors. While isoprenaline washout led to a dissociation of the β 2 -adrenoceptor-arrestin-2 complex, BI-167107 could not be washed out, analogous to the results with arrestin-3.
Furthermore, we investigated the agonist-induced interaction between β 2 -adrenoceptor-YFP and arrestin-3-mTurquoise in the absence of GRK2 ( Figure S1b). As expected, the binding of arrestin-3 was slower in the absence of GRK2 than in the presence of GRK2, but the final amplitude and the dissociation rate of the complex upon isoprenaline washout were identical to those in the presence of GRK2. This reiterates that the rate-limiting step for the formation of the β 2 -adrenoceptor-arrestin complex is β 2 -adrenoceptor phosphorylation, as previously shown (Krasel, Bünemann, Lorenz, & Lohse, 2005).
There was no significant difference in complex dissociation rates upon agonist washout ( Figure S1c). F I G U R E 1 Interaction of arrestin-3 with the β 2 -adrenoceptor. (a) Arrestin-3 recruitment to the β 2 -adrenoceptor induced by isoprenaline, adrenaline or BI-17018 was measured by FRET as described in Section 2. (b) Dissociation kinetics upon withdrawal of isoprenaline (Iso) or adrenaline (Adr) agonist were quantified by fitting the first 200 s of the washout phase with a monophasic exponential decay whereas the very slow off-rate for BI-167107 did not permit an exponential curve fit. Statistical analyses were performed using an unpaired Student's t test (*P < 0.05). (c) Additionally, dissociation kinetics were quantified using the area under the curve (AUC) of traces for the first 200 s of the washout phase normalized to the maximum to compare linear and exponential off-rates. Statistical analyses were performed using a one-way ANOVA followed by Tukey's multiple comparisons test (*P < 0.05). Data are shown as means ± SEM with n = 9 for isoprenaline or adrenaline and n = 8 for BI-167107. (d) The stability of β 2 -adrenoceptor-arrestin-3 complexes was analysed by dual-colour FRAP as described in Section 2. Traces were normalized to pre-bleach fluorescence intensities (100%) and represent means ± SEM with n = 13 for isoprenaline and BI-167107 and n = 17 for adrenaline. Filled symbols show the fluorescence intensities of arrestin-3 (arr3) whereas open symbols show the fluorescence intensities of the β 2 -adrenoceptor (β 2 AR). (e) Time constants for the recovery of β 2 -adrenoceptor and arrestin-3 were obtained by mono-exponential curve fits of the post-bleach values. Statistical analyses were performed using a one-way ANOVA followed by using a Brown-Forsythe test followed by Dunnett's T3 multiple comparisons test (n.s. if P ≥ 0.05). (f ) Cellular trafficking of arrestin-3 with the β 2 -adrenoceptor was analysed by confocal microscopy. HEK293T cells transiently expressing β 2 -adrenoceptor-mCherry, Arr3-GFP and GRK2 were stimulated with the indicated agonists for 30 min at 37 C before imaging. Colocalized β 2 -adrenoceptor (β 2 AR, magenta) and arrestin-3 (green) are observed as white in the overlay image. Scale bars represent 10 μm. (g) Colocalization of arrestin-3 and β 2 -adrenoceptor was quantified by calculating Pearson's correlation coefficients. Statistical analyses were performed using a Brown-Forsythe test followed by Dunnett's T3 multiple comparisons test (n.s. if P ≥ 0.05). Data are shown as means ± SEM (n = 25) Figure S2 shows representative raw data from the FRAP experiments.
As the lateral mobility of the crosslinked receptors was restricted, the recovery of the receptor fluorescence was reduced to about 20% for all agonists (Figures 1 and S2, right, yellow circles). Mono-exponential curve fits of the post-bleach values showed no significant differences in the kinetics of the arrestin-3 recovery regardless of the applied agonist ( Figure 1e). This shows that BI-167107 did not cause the formation of more stable arrestin-3-β 2 -adrenoceptor complexes than the other two full agonists. Thus, the slow dissociation in the FRET experiments ( Figure 1b) can rather be attributed to stable agonist binding than to stable arrestin binding. This was further supported by confocal imaging which revealed that stimulation of the FRAP experiments with arrestin2 also did not reveal significant differences between isoprenaline and BI-167107 ( Figure S3a,b). The recovery rates were apparently faster for arrestin-2 than for arrestin-3 (compare Figure 1e with Figure S3b), confirming the lower affinity of arrestin-2 to the β 2 -adrenoceptor.
To test this assumption on another receptor, similar experiments were also performed with the μ opioid receptor. In the FRET assay, the observed peak amplitudes upon agonist stimulation were remarkably smaller for the partial agonists morphine and SR17018 compared to DAMGO and etorphine (Figure 2a). The arrestin recruitment also seemed to be slower for SR17018 than for DAMGO or etorphine, consistent with its slower phosphorylation kinetics (Gillis et al., 2020). Normalization of the traces to the maximum revealed that the dissociation kinetics for DAMGO and morphine were quite similar, whereas SR17018 and etorphine showed considerably slower off-kinetics (Figure 2b) which was also confirmed by lower time constants (Figure 2c). However, the FRET experiments again did not correlate with the stability of arrestin complexes in continuous presence of agonist in dual-colour FRAP measurements (Figure 2d). In these experiments, we initially omitted treatment with morphine as this very rarely leads to visible arrestin translocation to the plasma membrane (Zhang et al., 1998). The recovery kinetics of fluorescent arrestins into the bleached area did not show statistical differences between the applied agonists ( Figure 2e), similar to the β 2 -adrenoceptor. Furthermore, none of the μ receptor agonists could induce co-internalization of arrestin-3 with the receptors (Figure 2f). Again, morphine was omitted in these experiments as it does not cause μ receptor internalization in HEK293 cells (Keith et al., 1996). Quantification of the colocalization of arrestin-3 with the μ receptor using the Pearson's correlation coefficient did not reveal any agonist-specific difference (Figure 2g) as seen before for the β 2 -adrenoceptor. Taken together, ligands with longer binding times do not stabilize receptor-arrestin complexes and therefore do not induce co-internalization of arrestin-3 with the receptor.
The experiments with the μ receptor described so far were all conducted in the presence of co-transfected GRK2. To investigate whether the FRAP assay can detect differences in receptor phosphorylation, we tried to conduct FRAP assays with DAMGO and morphine in the absence of co-transfected GRK2. Whereas DAMGO causes a full phosphorylation of the μ receptor leading to fairly robust μ receptor-arrestin interaction, morphine causes only a phosphorylation of the μ receptor at Ser375 (Just et al., 2013) leading to a very weak μ receptor-arrestin interaction (McPherson et al., 2010;Zhang et al., 1998). As a negative control, we also included untreated cells in the assay; no receptor-arrestin interaction should occur under these conditions, and therefore, a very fast fluorescence recovery was expected. Even though we could not see any visible arrestin-3 translocation to the membrane in our confocal images, the FRAP kinetics indeed showed a slight delay of fluorescence recovery in morphine-treated cells compared to untreated cells (Figure 3a). It appears from the raw data that the arrestin in untreated or morphine-treated cells is less efficiently bleached than in DAMGO-treated cells (Figure 3a). However, we believe that this is caused by a very fast recovery that occurs during the bleach. Our confocal microscope takes about 1.7 s from the start of the bleach to the start of the acquisition of the first post-bleach image. Since the recovery is so fast, it was impossible to fit mono-exponential kinetics to the individual morphine and untreated data, even if constraints were taken into account. Therefore, we quantitated the area under the curve within the first 10 s after bleaching and could demonstrate significant differences between DAMGO, morphine and untreated cells (Figure 3b). These results are to our knowledge the first that demonstrate ligand-dependent changes in arrestin affinity to a class A GPCR in cells.

| DISCUSSION
In this study, we assessed the interaction between arrestin-3 and both the β 2 -adrenoceptor and the μ receptor upon application of various agonists by two different methods. Ligands with prolonged off-rates significantly slowed the dissociation kinetics of arrestin-3 from the receptor upon agonist withdrawal in FRET measurements as expected from previous experiments (Krasel, Bünemann, Lorenz, & Lohse, 2005).
However, the stability of arrestin-receptor complexes in the continuous presence of these agonists (as assessed by FRAP measurements) did not differ at all. Furthermore, the ligand off-rates at the β 2adrenoceptor or μ receptor did not affect the co-trafficking of F I G U R E 2 Interaction of arrestin-3 with the μ opioid receptor. (a) Arrestin-3 recruitment to the μ receptor induced by DAMGO, etorphine, SR17018 or morphine was measured by FRET as described in Section 2. (b) Traces were normalized to the maximum peak to facilitate direct comparison of the dissociation kinetics upon agonist withdrawal. (c) Kinetics were quantified by a monophasic exponential fit. Statistical analyses were performed using a Brown-Forsythe test followed by Dunnett's T3 multiple comparisons test (n.s. if P ≥ 0.05, *P < 0.05). Data represent means ± SEM with n = 6 for DAMGO or SR17018, n = 9 for morphine and n = 11 for etorphine. (d) The stability of μ receptor-arrestin-3 complexes was analysed by dual-colour FRAP as described in Section 2. Traces were normalized to pre-bleach fluorescence intensities (100%) and are shown as means ± SEM with n = 36 for DAMGO or etorphine and n = 33 for SR17018. Filled symbols show the fluorescence intensities of arrestin-3 whereas open symbols show the fluorescence intensities of the μ receptor (MOR). (e) Time constants for the recovery of μ receptor and arrestin-3 were obtained by mono-exponential curve fits of the post-bleach values. Statistical analyses were performed using a one-way ANOVA followed by Tukey's multiple comparisons test (n.s. if P ≥ 0.05). (f) Cellular trafficking of arrestin-3 with the μ receptor was analysed by confocal microscopy. HEK293T cells transiently expressing μ receptor-mCherry, Arr3-GFP and GRK2 were stimulated with the indicated agonists for 30 min at 37 C before imaging. Colocalized μ receptor (MOR, magenta) and arrestin-3 (green) are observed as white spots in the overlay image. Scale bars represent 10 μm. (g) Colocalization of arrestin-3 and μ receptor was quantified by calculating Pearson's correlation coefficients. Statistical analyses were performed using a one-way ANOVA followed by Tukey's multiple comparisons test (n.s. if P ≥ 0.05). Data are shown as means ± SEM (n = 28) arrestin-3 as none of the agonists induced co-internalization of arrestin with these class A receptors.
In FRET measurements, the dissociation of arrestin from the receptor is induced by the dissociation of the ligand upon agonist washout which forces the receptor back to its inactive state. Arrestin senses this conformational change so that its dissociation kinetics from the receptor depends on the ligand off-rate (Krasel, Bünemann, Lorenz, & Lohse, 2005). During FRAP experiments, the agonist is continuously present and therefore the receptor remains in an active conformation. This enabled the assessment of the apparent affinity of arrestin to activated GPCRs without the interference of ligand dissociation kinetics. Therefore, we conclude that our dual-colour FRAP assay is more suitable for this analysis than the FRET assay. On the other hand, FRET but not FRAP assays should be able to sense a transition from the "core" to the "tail" conformation of a GPCR-arrestin complex (Shukla et al., 2014). Surprisingly, we could use the FRAP assay to assess the relative apparent affinity of morphine for the μ receptor even though morphine causes very little visible membrane recruitment of arrestins (Zhang et al., 1998). These results again demonstrate that agonist off-rate is irrelevant for arrestin affinity as DAMGO and morphine display roughly the same off-rate in the arrestin FRET assay (Figure 2b) but clearly the morphine-arrestin complex is less stable (Figure 3); this is probably also reflected by the lower amplitude that can be achieved in the FRET assay (Figure 2c).
An open question that is not clarified by our experiments is the role of agonist efficacy for the stability of the receptor-arrestin complex. In other words, is the lower stability of the morphine-arrestin complex only caused by altered phosphorylation (Just et al., 2013), or is it also caused by lower agonist efficacy?
For some agonist-receptor pairs, the kinetics of receptor dephosphorylation upon agonist washout has been investigated (Doll et al., 2012;Fritzwanker, Schulz, & Kliewer, 2021;Krasel, Bünemann, Lorenz, & Lohse, 2005). For all these pairs, arrestin dissociation is faster than receptor dephosphorylation. This is particularly apparent for the SR17018-stimulated μ receptor which shows little dephosphorylation within 1 h of agonist washout (Fritzwanker, Schulz, & Kliewer, 2021). We are not aware of other publications correlating the lifetime of GPCR-arrestin complexes with the kinetics of GPCR dephosphorylation.
Finally, our results demonstrate that a class A GPCR like the β 2 -adrenoceptor or the μ receptor cannot be converted to a class B receptor by using orthosteric agonists with prolonged off-rates.
In contrast, it has been shown for the CCR7 which is a class B GPCR that upon stimulation with an agonist that leads to reduced receptor phosphorylation, arrestin-3 can no longer co-internalize with the CCR7 (Zidar, Violin, Whalen, & Lefkowitz, 2009). Numerous studies have shown that a sufficient number of phosphate residues in an appropriate pattern in the C-terminal tail is required for a receptor to bind arrestin with higher affinity (Dwivedi-Agnihotri et al., 2020;Latorraca et al., 2020;Mayer et al., 2019;Zhou et al., 2017;Zindel et al., 2015Zindel et al., , 2016. Assuming that a full agonist causes maximal phosphorylation of a GPCR, we cannot envisage how orthosteric agonists could increase the number of phosphorylated residues. Allosteric compounds, in contrast, may generate entirely novel receptor conformations that bind arrestins with higher affinity. We did not observe differences in the stability of receptor-arrestin complexes when GRK2 was overexpressed, probably because this "blurs" the phosphorylation pattern; this has been demonstrated at least for the μ receptor. When we did not overexpress GRK2, we could observe differences at the μ receptor between morphine and DAMGO which cause different phosphorylation patterns in the absence of overexpressed GRKs (Just et al., 2013).
In our study, we used the novel μ receptor ligand SR17018 (Schmid et al., 2017) that has been reported to be G-protein-biased. In our hands, this compound is able to recruit arrestin to the μ receptor under conditions of GRK overexpression (see also Gillis et al., 2020) and shows apparent arrestin affinity comparable to the full agonists DAMGO and etorphine. Compared to other agonists at the μ receptor, recruitment is delayed which suggests a slowed phosphorylation F I G U R E 3 Stability of μ receptor (μ R)-arrestin complexes induced by morphine. (a) FRAP measurements were conducted as described in Section 2. Traces normalized to pre-bleach fluorescence intensities (100%) are shown as means ± SEM with n = 18 for DAMGO, n = 19 for morphine and n = 17 for untreated. Filled symbols show the fluorescence intensities of arrestin-3 whereas open symbols show the fluorescence intensities of the μ receptor. (b) Recovery kinetics of arrestin-3 were quantified using the AUC of the first 10 s after photobleaching. Statistical analyses were performed using Brown-Forsythe tests followed by Dunnett's T3 post hoc tests (*P < 0.05) kinetics of the μ receptor as described recently (Gillis et al., 2020). We also used etorphine which we have previously described as causing quasi-irreversible arrestin binding (McPherson et al., 2010). However, in the experiments described here, we could observe arrestin dissociation from the μ receptor upon etorphine washout. The main difference to our previous experiments is that we used a tenfold lower concentration of etorphine in the present study. It is possible that the lack of dissociation in our previous experiments was caused by a residual low concentration of etorphine that was not washed away by the perfusion system. The dissociation kinetics of the μ receptor-arrestin complex triggered by washout of DAMGO and morphine are in the same range as those determined with a μ receptor biosensor in vitro (Livingston, Mahoney, Manglik, Sunahara, & Traynor, 2018)  The physiological, let alone clinical, significance of class A versus class B GPCR behaviour has not been investigated. If there is a relevant difference, our study shows that class A behaviour of a GPCR cannot simply be converted into class B behaviour by increasing the residency time of an orthosteric agonist.
Taken together, our study reveals that the ligand off-rate does not influence arrestin-receptor interactions or the co-trafficking of arrestin-3 with GPCRs and points to the importance of receptor phosphorylation for both of these processes. In addition, we demonstrate that dual-colour FRAP measurements are more suitable than FRET experiments to assess arrestin-receptor interactions induced by different ligands.