Research ArticlePharmacology

Quantitative Encoding of the Effect of a Partial Agonist on Individual Opioid Receptors by Multisite Phosphorylation and Threshold Detection

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Science Signaling  09 Aug 2011:
Vol. 4, Issue 185, pp. ra52
DOI: 10.1126/scisignal.2001748

Abstract

In comparison to endogenous ligands of seven-transmembrane receptors, which typically act as full agonists, many drugs act as partial agonists. Partial agonism is best described as a “macroscopic” property that is manifest at the level of physiological systems or cell populations; however, whether partial agonists also encode discrete regulatory information at the “microscopic” level of individual receptors is not known. Here, we addressed this question by focusing on morphine, a partial agonist drug for μ-type opioid peptide receptors (MORs), and by combining quantitative mass spectrometry with cell biological analysis to investigate the reduced efficacy of morphine, compared to that of a peptide full agonist, in promoting receptor endocytosis. We showed that these chemically distinct ligands produced a complex and qualitatively similar mixture of phosphorylated opioid receptor forms in intact cells. Quantitatively, however, the different agonists promoted disproportionate multisite phosphorylation of a specific serine and threonine motif, and we found that modification at more than one residue was essential for the efficient recruitment of the adaptor protein β-arrestin that mediated subsequent endocytosis of MORs. Thus, quantitative encoding of agonist-selective endocytosis at the level of individual opioid receptors was based on the conserved biochemical principles of multisite phosphorylation and threshold detection.

Introduction

Morphine has been used for its therapeutic and euphoric properties since before the time of Hippocrates, and it remains among the most important drugs in modern medicine (1). Morphine is a plant-derived alkaloid that acts as a partial agonist of μ-type opioid receptors (MORs), seven-transmembrane receptors (7TMRs) whose endogenous agonists are opioid neuropeptides, such as enkephalin. The discovery that morphine and opioid peptides activate the same receptors contributed to the molecular mimicry hypothesis of drug action (2). Despite compelling evidence that morphine mediates its main biological effects through MORs (3), it differs substantially from opioid peptide agonists in various receptor-linked regulatory processes. A long-standing question is precisely how such “agonist selectivity” of opioid action is determined (410). 7TMRs comprise the largest family of signaling receptors and therapeutic drug targets (11, 12), and many 7TMR-acting drugs are traditionally considered to be partial agonists (8). Accordingly, the question of how the regulatory effects of morphine are discriminated is relevant not only to opioid biology but also to the cellular basis of drug action more generally.

Morphine is classified as a partial agonist of MORs because, even when present at saturating concentrations that achieve full receptor occupancy, it drives various receptor-mediated signaling and regulatory processes less strongly than do peptide full agonists (13, 14). A clear example is the regulated endocytosis of MORs, which internalize within minutes after activation by opioid peptide agonists and some highly efficacious nonpeptide drugs; however, even high doses of morphine promote MOR internalization inefficiently in cultured cell models (1518) and in various neuronal populations (1925). Regulated endocytosis of MORs, by initiating subsequent receptor trafficking through specific recycling or degradative membrane pathways, is thought to be a critical event in determining the responsiveness of cells to opioids (5, 26). Moreover, differential endocytic regulation of MORs affects physiological measures of opioid tolerance and dependence in rodent models, thus suggesting its relevance to neuroadaptive processes that limit the clinical utility of opioid drugs in vivo (2731).

The mechanistic basis of the reduced endocytic activity of morphine is reasonably well understood when considered at the “macroscopic” level of the overall cellular complement of MORs. Regulated endocytosis of MORs is mediated by clathrin-coated pits (17). MORs engage this conserved endocytic machinery by recruiting β-arrestins (also called nonvisual arrestins), which function as regulated endocytic adaptors to drive the accumulation of various 7TMRs into coated pits (32, 33), a process that is promoted by agonist-induced phosphorylation of the receptors (21, 22, 34). Morphine stimulates overall phosphorylation of the MOR relatively inefficiently when compared to that stimulated by highly efficacious agonists (14, 16, 34, 35). Further, morphine-induced endocytosis of MORs is enhanced by experimental manipulations that increase agonist-induced receptor phosphorylation (21, 22, 36), including phosphorylation specifically of the MOR cytoplasmic tail (C-tail) (37). Thus, when examined at the macroscopic level of the overall MOR population, the determination of agonist-selective endocytic activity involves differential phosphorylation of the MOR C-tail.

What is not known is whether the reduced endocytic activity of morphine is also specified at a “microscopic” level, that is, at the level of individual receptors. A traditional view of partial agonism suggests that agonist-selective differences in MOR internalization are determined entirely at the macroscopic level on the basis of reduced phosphorylation of the overall receptor population (14, 38). An alternative possibility, consistent with emerging concepts of ligand bias or functional selectivity (6, 8, 10), is that agonist-selective endocytosis of MORs might also involve some sort of biochemical encoding through differential phosphorylation occurring at the level of individual receptors. If so, one might expect agonists that differ in their relative endocytic efficacies to disproportionately produce a particular phosphorylated form of the C-tail, which in turn could control the ability of MORs to engage the endocytic machinery.

Determining whether such receptor-intrinsic discrimination occurs is fundamental to understanding the cellular basis of opioid drug action, as well as that of partial agonism more generally, and hinges on two key experimental questions: First, do morphine and opioid peptides drive differential MOR phosphorylation in a strictly proportional manner, or are there particular phosphorylated receptor forms whose production is favored disproportionately by one of these agonists? Second, if disproportional phosphorylation of MORs does occur, are there particular phosphorylated species that “encode” agonist-selective differences in how the receptor engages with the endocytic machinery? These key questions, although simple to pose in principle, have proven difficult to answer in practice. Indeed, previous efforts to do so, based on correlative approaches or on the analysis of MOR phosphorylation averaged across the overall cellular receptor population, have failed to reach a consensus (14, 3740). Here, we combined quantitative mass spectrometry (MS) with detailed cell biological analysis to resolve discrete phosphorylated MOR species and their effects on receptor engagement of the arrestin and clathrin endocytic machinery, thus addressing both experimental questions directly.

Results

Phosphopeptide mapping of MOR phosphorylation in intact cells by MALDI MS

The reduced endocytic efficacy of morphine relative to that of the highly efficacious enkephalin analog [d-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) was clearly established in experiments involving human embryonic kidney (HEK) 293 cells expressing FLAG epitope–tagged MORs (15, 17). Differences in the endocytic effects of various opioids established in this cell model have proven to be relevant to the differential regulation that occurs in native neurons (19, 20, 23, 25, 28). Thus, we focused on HEK 293 cells as a model for investigating agonist effects on MOR phosphorylation by MS after rapid immunopurification of receptors (Fig. 1A). We efficiently isolated FLAG-tagged MORs from cell extracts and found that they exhibited electrophoretic mobility (Fig. 1B) consistent with that of the full-length receptor glycoprotein produced in cultured cells and native brain tissue (17, 41).

Fig. 1

Overview of MS analyses. (A) Experimental scheme for processing MORs for analysis by MS with or without phosphoenrichment by IMAC. HEK 293 cells expressing wild-type (WT) MOR were treated with morphine (10 μM) or DAMGO (10 μM) or were left untreated (No Drug) for 20 min at 37°C. (B) Receptors were purified from cell pellets, subjected to SDS-PAGE, and detected with SYPRO Ruby total protein stain. (C) Amino acid sequence of the C-tail of MOR. The potential phosphorylation sites are highlighted in blue. Abbreviations for the amino acids are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; H, His; I, Ile; K, Lys; L, Leu; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; and Y, Tyr. (D) MS of MOR tryptic peptides. Full coverage of the unphosphorylated C-tail was achieved: [349–365], m/z = 1967.92 (calc m/z = 1967.92); [372–382], m/z = 1226.58 (calc m/z = 1226.58); [383–398], m/z = 1776.88 (calc m/z = 1776.88). (E) MS of phosphopeptides after enrichment by IMAC revealed phosphorylation of MOR in regions 1 and 2: region 1, [349–365]+1Pi, m/z = 2047.86 (calc m/z = 2047.88); [349–365]+2Pi, m/z = 2127.86 (calc m/z = 2127.85); region 2, [368–382]+1Pi, m/z = 1805.79 (calc m/z = 1805.79); [366–382]+1Pi, m/z = 2074.97(calc m/z = 2074.98); [366–382]+2Pi, m/z = 2154.93 (calc m/z = 2154.95); [366–382]+3Pi, m/z = 2234.91(calc m/z = 2234.91). We did not observe phosphorylation within region 3. The results shown are from a single experiment; all species shown were detected in multiple receptor preparations and digestions (n > 3 in all cases).

To focus specifically on differences in agonist efficacy rather than affinity (10), we applied each agonist at a saturating concentration (10 μM) sufficient to drive maximal receptor occupancy (42). Phosphorylation and endocytic trafficking of MORs through the recycling pathway occur dynamically and iteratively in the presence of opioid peptide (43), and considerable agonist selectivity of net MOR internalization can be observed in HEK 293 cells within 20 min of agonist application (17); accordingly, we chose this time point for our initial comparisons. We first surveyed tryptic phosphopeptides covering the entire MOR C-tail (Fig. 1C) by matrix-assisted laser desorption/ionization (MALDI) MS (Fig. 1D). Immobilized metal affinity chromatography (IMAC), which was used to enrich phosphorylated peptides from the mixture (44), revealed prominent signals representing phosphorylated forms of a proximal region of the C-tail (region 1, corresponding to the phosphorylated peptide EFCIPTSSTIEQQNSAR) as well as a middle region (region 2, corresponding to the phosphorylated peptide IRQNTREHPSTANTVDR) (Fig. 1E). In contrast, the distal portion of the C-tail (region 3, corresponding to the peptide TNHQLENLEAETAPLP, or DRTNHQLENLEAETAPLP, using endoproteinase AspN) was not detectably phosphorylated.

Quantitative analysis of C-tail phosphorylation by SILAC and LC-MS techniques

We quantified the effects of agonists on the abundance of phosphorylated species by liquid chromatography–mass spectrometry (LC-MS) and stable isotope labeling in cell culture (SILAC) techniques (45), using isotope-tagged arginines (Fig. 2A) to achieve complete coverage of the C-tail of MOR [Fig. 2B, see table S1 for the corresponding mass-to-charge ratio (m/z) values]. Additionally, this approach enabled us to ensure co-elution of isoptomers (peptides containing the three different isotopic labels) in the LC separation step that preceded peptide ionization (45). Two singly phosphorylated (+1Pi) forms of region 1 (peptides 1 and 2) were resolved by LC (Fig. 2C) and differed in whether phosphorylation was present at Ser363 (fig. S1, peptide 1) or in the 354TSST357 serine and threonine cluster (fig. S2, peptide 2). Calculation of the isotope ratios for peptide 1 indicated relative abundances near unity for comparisons between morphine and no drug (M/ND), between DAMGO and no drug (D/ND), and between DAMGO and morphine (D/M) (Fig. 2D and table S2), which was indicative of constitutive phosphorylation. By contrast, phosphorylation of peptide 2, which contained a single phosphorylation event in the 354TSST357 motif, was agonist-dependent (Fig. 2E), because its abundance was markedly increased by the exposure of cells to both morphine (M/ND isotope ratio = 4.6) and DAMGO (D/ND isotope ratio = 9.3). Single phosphorylation in this region was also moderately agonist-selective, because DAMGO increased the abundance of the phosphorylated peptide more effectively than did morphine (D/M = 2.0). We also detected dual (+2Pi) phosphorylation in region 1, and phosphopeptides containing this modification were isolated from cells under all treatment conditions (Fig. 2F, peptide 3). Production of this species was agonist-dependent (M/ND = 2.8 and D/ND = 2.9), but not agonist-selective (D/M = 1.0). In summary, none of the phosphorylated forms of region 1 was unique to a particular treatment, and the degree of agonist selectivity observed in the production of any particular species was moderate (D/M ≤2).

Fig. 2

LC-ESI MS analysis of phosphorylated MOR region 1 under three different conditions after 20 min of exposure to agonist. (A) Schematic diagram of the experiment. The use of stable isotope labeling of MOR enabled all three conditions (ND, No Drug with [12C,14N]Arg; M, +Morphine with [13C,14N]Arg; D, +DAMGO with [13C,15N]Arg) to be purified, handled, and analyzed simultaneously. Adjusted isotope ratios were calculated by summing signal count areas for two to three mass isotope peaks, adjusting for isotope overlap, and calibrating with the internal MOR reference peptide multipliers [1.1× for (M) and 1.4× for (D)]. (B) The peptide sequence of the MOR C-tail, specifying region 1 as containing phosphorylated peptides of the amino acid sequence EFCIPTSSTIEQQNSAR. (C) Through LC-ESI MS, we observed two separate elutions of singly phosphorylated EFCIPTSSTIEQQNSAR peptide, at 29.3 and 30.5 min. Peptide 1, which contained a phosphorylation site at Ser363, eluted at 29.3 min. Peptide 2, which contained a phosphorylation site within the 354TSST357 region, eluted at 30.5 min. (D) At an elution time of 29.3 min, we observed monoisotopic signals at m/z = 1024.5, 1027.5, and 1029.5, which corresponded to the [M+2H]+2 of peptide 1 under the three agonist conditions, ND, M, and D, respectively. Adjusted isotope ratios calculated for peptide 1 include M/ND = 1.1, D/ND = 1.0, and D/M = 0.9. (E) At an elution time of 30.5 min, we observed monoisotopic signals at m/z = 1024.4, 1027.5, and 1029.5, which corresponded to the [M+2H]+2 of peptide 2 under the three agonist conditions, ND, M, and D, respectively. Adjusted isotope ratios calculated for peptide 2 include M/ND = 4.6, D/ND = 9.3, and D/M = 2.0. (F) At an elution time of 31.0 min, peptide 3 was eluted as a single peak on the LC with monoisotopic signals observed at m/z = 1064.4, 1067.4, and 1069.4, which corresponded to the [M+2H]+2 of peptide 3 under the three agonist conditions, ND, M, and D, respectively. Adjusted isotope ratios calculated for peptide 3 include M/ND = 2.8, D/ND = 2.9, and D/M = 1.0. Isotope labeling at this time point was performed in three independent culture preparations. Results shown in Figs. 2 and 3 are from SILAC analysis of the same isotope-labeled preparation to facilitate direct comparison of the relative abundance ratios across the various phosphorylated species in the same cell population.

Region 2 of the C-tail was represented by two overlapping proteolytic fragments, corresponding to amino acid residues 368 to 382, referred to as [368–382], and residues 372 to 383, referred to as [372–383] (Fig. 3A), suggesting that phosphorylation of a fraction of receptors occurred next to the protease cleavage site (46). Tandem MS (MS/MS) analysis of the singly phosphorylated [368–382] species (peptide 4) verified that phosphorylation occurred specifically at Thr370 (fig. S3). The abundance of this species was moderately increased by both morphine (M/ND = 1.4) and DAMGO (D/ND = 2.5), with DAMGO having a stronger effect (D/M = 1.8) (Fig. 3B). Single phosphorylation was also detected in the 375STANT379 serine and threonine motif (Fig. 3C, peptide 5), revealing similar fragmentation patterns under the three different treatments (fig. S4). The extent of single phosphorylation in this motif was increased by morphine (M/ND = 1.6) and DAMGO (D/ND = 2.7), with DAMGO again having a somewhat stronger effect (D/M = 1.7). Dual phosphorylation in the fragment containing both Thr370 and the 375STANT379 serine and threonine motif (Fig. 3D, peptide 6) was also observed under all treatment conditions. Quantification indicated that the abundance of this species was markedly increased by both morphine (M/ND = 3.5) and DAMGO (D/ND = 9.2), with DAMGO again having a moderately stronger effect (D/M = 2.6). The only phosphorylated species that was absolutely agonist-dependent (that is, was not detectable in untreated cells) included dual phosphorylation within the 375STANT379 serine and threonine cluster (Fig. 3E, peptide 7). Furthermore, this phosphopeptide species was the most highly agonist-selective in that it was preferentially produced by DAMGO rather than by morphine (D/M = 4.3). Thus, higher-order phosphorylation in region 2, including the 375STANT379 serine and threonine cluster, differentiated between the effects of morphine and DAMGO more effectively than did any of the other observed phosphorylation events.

Fig. 3

LC-ESI MS of phosphorylated MOR region 2 under three different conditions after 20 min of exposure to agonist. (A) Through LC-ESI MS, we observed phosphorylated region 2 in two different overlapping sequences, QNTREHPSTANTVDR and EHPSTANTVDR. Adjusted isotope ratios were calculated by summing the signal count areas for two to three mass isotope peaks, adjusting for isotope overlap, and calibrating with the internal MOR reference peptide multipliers [1.1× for (M) and 1.4× for (D)]. (B) Peptide 4, which had a phosphorylation site at Thr370, eluted at 15.9 min. Monoisotopic signals were observed at m/z = 602.6, 606.6, and 609.3, which corresponded to the [M+3H]+3 of peptide 4 under the three agonist conditions, ND, M, and D, respectively. Adjusted isotope ratios calculated for peptide 4 include M/ND = 1.4, D/ND = 2.5, and D/M = 1.8. (C) At an elution time of 15.9 min, we observed monoisotopic signals at m/z = 653.8, 656.8, and 658.8, which corresponded to the [M+2H]+2 of peptide 5 under the three agonist conditions, ND, M, and D, respectively. Similar to peptide 4, adjusted isotope ratios calculated for peptide 5 include M/ND = 1.6, D/ND = 2.7, and D/M = 1.7. (D) At an elution time of 16.5 min, we observed monoisotopic signals at m/z = 629.3, 633.3, and 635.9, which corresponded to the [M+3H]+3 of peptide 6 under the three agonist conditions, ND, M, and D, respectively. Adjusted isotope ratios calculated for peptide 6 include M/ND = 3.5, D/ND = 9.2, and D/M = 2.6. (E) At an elution time of 16.5 min, we observed monoisotopic signals at m/z = 696.8 and 698.8, which corresponded to the [M+2H]+2 of peptide 7 under the two agonist conditions, M and D, respectively. No signal above noise was observed for the ND treatment of peptide 7 at m/z = 692.8; therefore, the adjusted isotope ratio calculated was D/M = 4.3.

Persistent agonist-selective phosphorylation in the 375STANT379 motif

We carried out a separate analysis to independently verify agonist-selective phosphorylation in region 2 of the MOR and to examine whether skewed production of higher-order phosphorylation in this region could also be observed after longer periods of exposure to agonist. We were particularly interested in a longer time point because the duration of therapeutic action of morphine typically exceeds 20 min and because agonist-selective trafficking is evident in cultured cells even after prolonged agonist exposure (16, 47). Thus, we carried out a separate labeling experiment to compare agonists after continuous application for 3 hours, approximating the clinical duration of morphine action in humans (47). To assure independent analysis, we focused on a distinct proteolytic fragment that included the 375STANT379 motif of interest, but that was generated by alternate cleavage at the proximal arginine residue in region 2 (Fig. 4A). Production of a single phosphorylation event (+1Pi) in this peptide (peptide 8) was moderately agonist-selective (D/M = 2.7) (Fig. 4B), and the degree of agonist selectivity increased progressively for the production of higher-order phosphorylated forms, peptides 9 and 10, which represented +2Pi and +3Pi species, respectively (Fig. 4, C and D). The triple (+3Pi) phosphorylated form was completely undetectable in untreated cells and exhibited pronounced agonist selectivity in its production, as shown by a relative abundance ratio (D/M) of 14.8-fold between morphine- and opioid peptide–treated cells (Fig. 4D and table S2). These results independently verified agonist-selective phosphorylation in region 2, showed that this phenomenon persisted with longer agonist exposure, and emphasized the degree to which distinct agonists can drive disproportional, higher-order phosphorylation of this region.

Fig. 4

Independent LC-ESI MS analysis of phosphorylated MOR region 2 [diagrammed in (A)] after 180 min of exposure to agonist. An independent series of peptides spanning region 2 were generated by variation of trypsin cleavage and represented single (+1Pi), double (+2Pi), and triple (+3Pi) receptor phosphorylation events in this region. Adjusted isotope ratios were calculated by summing signal count areas for two to three mass isotope peaks, adjusting for isotope overlap, and calibrating with the internal MOR reference peptide multipliers [1.5× for (M) and 3.3× for (D)]. (B) A singly phosphorylated (+1Pi) peptide was eluted at 13.4 min. Monoisotopic signals were observed at m/z = 692.3, 698.3, and 702.3, which corresponded to the [M+3H]+3 of peptide 8 under the three agonist conditions, ND, M, and D, respectively. Adjusted isotope ratios (the D/M value is also indicated): M/ND = 1.1, D/ND = 2.9, and D/M = 2.7. (C) A doubly phosphorylated (+2Pi) peptide was eluted at 14.4 min. Monoisotopic signals were observed at m/z = 725.0 and 729.0, which corresponded to the [M+3H]+3 of peptide 9 under the two agonist conditions, M and D, respectively. No signal above noise was observed for ND at m/z = 718.9. Adjusted isotope ratio: D/M = 4.5. (D) A triply phosphorylated (+3Pi) peptide was eluted at 14.6 min. Monoisotopic signals were observed at m/z = 751.6 and 755.6, which corresponded to the [M+3H]+3 of peptide 10 under the two agonist conditions, M and D, respectively. No signal above noise was observed for ND at m/z = 745.6. The adjusted isotope ratio was D/M = 14.8.

Multisite phosphorylation in the 375STANT379 motif regulates endocytosis of the MOR

We next investigated whether phosphorylation in either of the serine and threonine clusters (354TSST357 or 375STANT379) present in C-tail regions 1 and 2 was functionally relevant to the control of MOR endocytosis. The mutation of all phosphorylatable residues in the proximal cluster in region 1 (354AAAA357) had no detectable effect on steady-state receptor localization or on agonist-selective internalization of the receptor, as visualized by fluorescence microscopy; however, mutation of the more distal cluster in region 2 (375AAANA379) resulted in the visibly reduced ability of DAMGO to induce receptor internalization (Fig. 5A). Complementary flow cytometry experiments quantitatively verified the specificity and reproducibility of these results (Fig. 5B). Thus, MOR endocytosis was controlled specifically by phosphorylation within the C-tail containing the 375STANT379 serine and threonine cluster, which, notably, represents a sequence motif that is conserved in MORs across vertebrates (48).

Fig. 5

Effects of the mutations 354AAAA357 and 375AAANA379 on the regulated endocytosis of MORs. (A) HEK 293 cells stably transfected with plasmids encoding the indicated FLAG epitope–tagged MOR constructs were incubated in the absence of agonist (ND) or were exposed to morphine (M, 10 μM) of DAMGO (D, 10 μM) for 20 min at 37°C. Cells were then chemically fixed, and receptors were detected with Alexa Fluor 488–conjugated antibody against the FLAG tag (M1). (B) Quantification of endocytic effects by flow cytometry, based on agonist-induced reduction in surface receptor immunoreactivity, as described in Materials and Methods. In each experiment, and for each condition, triplicate analyses were performed with 10,000 cells for each analysis. For each receptor construct, at least two independently isolated cell clones were tested. Bars represent averaged internalization values across independent experiments (WT, n = 10; 354AAAA357, n = 4; and 375AAANA379, n = 4 experiments). Error bars represent the SEM, and P values were calculated by Student’s t test.

Because the SILAC-LC/MS data revealed the most pronounced agonist selectivity in the production of higher-order phosphorylation in the C-tail region that includes the 375STANT379 motif, we investigated whether the endocytic activity conferred by this motif specifically required its phosphorylation at more than one residue. To do so, we mutated each potential phosphorylation site individually and examined the effects on regulated endocytosis across multiple clones of stably transfected cell lines selected for similar surface receptor abundances in untreated cells. Mutation of any individual serine or threonine residue in the 375STANT379 motif retained the plasma membrane localization of receptors (Fig. 6A) and inhibited regulated endocytosis (Fig. 6B) to a similarly large degree as did mutation of all three serine and threonine residues combined. These results suggest that the full endocytic activity conferred by the 375STANT379 motif requires its phosphorylation at more than one residue.

Fig. 6

Fine-structure analysis of endocytic activity conferred by the 375STANT379 motif. (A) HEK 293 cells stably transfected with plasmids encoding the indicated FLAG epitope–tagged MOR constructs were analyzed as described for Fig. 5. (B) Flow cytometric quantification of mutational effects on DAMGO-induced endocytosis (WT, n = 10; S375A, n = 6; T376A, n = 4; T379A, n = 4; 375AAANA379, n = 4 experiments; each condition in triplicate). Values are expressed as means ± SEM, with P values calculated by Student’s t test.

β-Arrestin recruitment to clathrin-coated pits functions as a specific detector of higher-order phosphorylation involving the 375STANT379 motif

We next investigated the mechanism by which multisite phosphorylation is interpreted to produce differential endocytosis of MORs. We focused on arrestins because they associate preferentially with phosphorylated 7TMRs, including MORs, and act as regulated endocytic adaptors by binding both to the 7TMR and to the assembled coat structure of clathrin-coated pits (32, 33). Previous studies have established that regulated endocytosis of MORs is arrestin-dependent (22, 49, 50), and manipulations that increase the extent of phosphorylation of the MOR C-tail in intact cells enhance both the recruitment of arrestins and receptor endocytosis (21, 22, 37). However, it is not known whether agonist-selective engagement of this endocytic adaptor is determined simply by a proportional difference in net phosphorylation of the overall receptor pool or whether arrestins discriminate between activated MORs according to specific higher-order phosphorylation in the C-tail.

To address this question, we investigated the potential role of phosphorylation in the 375STANT379 motif in controlling the recruitment of β-arrestin-2 to discrete clathrin-coated pits. DAMGO-induced activation of MORs robustly recruits green fluorescent protein (GFP)–tagged β-arrestin-2 to coated pits, whereas morphine-induced recruitment is below the detection limit of this assay (22, 34, 51). To investigate this process with high spatiotemporal resolution, we thus focused on DAMGO and imaged a GFP-tagged version of β-arrestin-2 in living cells by total internal reflection fluorescence (TIRF) microscopy. Activation of wild-type MORs with DAMGO (10 μM) elicited rapid and pronounced recruitment of β-arrestin to diffraction-limited spots on the plasma membrane (Fig. 7A, top row), which were shown previously to represent discrete clathrin-coated pits containing receptor-arrestin complexes (43, 52). Mutation of all three of the phosphorylatable residues in the 375STANT379 motif (to generate the 375AAANA379 mutant) visibly inhibited the recruitment of β-arrestin (Fig. 7A, bottom row), implicating the 375STANT379 motif in regulating this critical endocytic adaptor. This represented an effective blockade, rather than a kinetic delay, because β-arrestin clustering was reduced in a continuous image series (Fig. 7B) spanning the typical surface residence time of individual coated pits (45, 47). This result was verified quantitatively by TIRF microscopic analysis across multiple fixed specimens and was specific to the 375STANT379 motif because mutation of all four residues in the 354TSST357 cluster did not prevent β-arrestin clustering (Fig. 7C). Mutation of any individual phosphorylatable residue in the 375STANT379 motif caused a pronounced inhibition of arrestin recruitment that was indistinguishable from that obtained by mutation of all three residues combined (Fig. 7C), fully mirroring the effect of the same mutations on MOR endocytosis (Fig. 6). Thus, arrestins appear to function as a “decoding” device for agonist-selective endocytosis of MORs by selectively recognizing multiphosphorylated receptor species involving the second serine and threonine cluster that are disproportionately produced by morphine relative to opioid peptide.

Fig. 7

Contribution of the 375STANT379 motif to receptor-mediated recruitment of β-arrestin to clathrin-coated pits. (A) HEK 293 cells coexpressing β-arrestin-2–GFP and the indicated receptor constructs were imaged live by TIRF microscopy. Representative frames from untreated cells or from cells 2 or 4 min after the addition of DAMGO (10 μM) are shown. For clarity of presentation, fluorescence intensity values were pseudo-colored with the gradient shown. (B) Magnified frames 12 s apart from the representative areas delineated by the boxes in (A). The time course illustrates the appearance of puncta of β-arrestin-2–GFP from the time of agonist addition through a plateau of maximum fluorescence intensity. (C) Quantification of arrestin localization in HEK 293 cells coexpressing β-arrestin-2–GFP and the indicated receptor construct, and fixed 7 min after bath application of DAMGO (10 μM). Values for each condition (expressed as mean ± SEM) represent the number of puncta visualized per unit surface area in multiple cells (WT, n = 27; S375A, n = 20; T376A, n = 24; T379A, n = 16; 375AAANA379, n = 50; and 354AAAA357, n = 17 cells; P values were calculated by Student’s t test and are indicated only for P < 0.05).

Discussion

Here, we applied MS-based analytical methods to resolve discrete phosphorylated forms of MORs isolated from intact cells. We used this approach to investigate the nature of agonist-selective regulation of opioid receptor endocytosis by morphine, a partial agonist drug, in comparison to that by DAMGO, a highly efficacious peptide full agonist. The cellular pool of MORs was found to exist as a heterogeneous mixture of phosphorylated species under all the conditions tested. Despite this complexity, our MS-based approach enabled the resolution and quantitative comparison of discrete phosphorylated MOR species produced in cells in the presence of saturating concentrations of each agonist. Morphine and DAMGO differed most markedly (up to ~15-fold) in driving higher-order phosphorylation in a region of the C-tail that contains a specific and conserved serine- and threonine-rich motif (375STANT379). By contrast, agonist-selective differences in phosphopeptide abundance were far less evident in lower-order phosphorylation of the same region or in phosphorylation detected elsewhere in the MOR C-tail. In those regions, morphine and DAMGO produced various phosphopeptide species in similar (D/M ~1) or only modestly different (D/M ≤2) relative abundances. Next, we established, through site-directed mutagenesis combined with quantitative analysis of receptor trafficking, that multiphosphorylation specifically involving the 375STANT379 motif was required for the efficient endocytosis of MORs. Finally, and consistent with the previously established endocytic adaptor function of β-arrestins, we found that multisite phosphorylation involving the 375STANT379 motif was specifically required for the efficient recruitment of GFP-tagged β-arrestin-2 to resolved clathrin-coated pits.

In essence, our results support the hypothesis that agonist-selective differences in MOR regulation are determined not only by net incorporation of phosphate into the receptor population as a whole, but also by individual receptors achieving a critical number of phosphorylated residues in a specific region of the C-tail. In addition, our results indicate that differential production of particular multiphosphorylated receptor species effectively encodes agonist-selective endocytic information at the level of discrete opioid receptors. Our results also indicate that this agonist-selective regulatory information is subsequently decoded by β-arrestins, which selectively recognize receptors that have achieved a critical multiphosphorylation threshold, thereby promoting efficient endocytosis by clustering [together with receptors (52)] in clathrin-coated pits. Similar principles of biochemical encoding and decoding by multisite phosphorylation and threshold detection are widely used in other cellular contexts to generate nonlinear or ultrasensitive responses (53, 54). In the case of MORs and arrestins, these principles evidently function to disproportionately amplify and transduce differences in the regulatory effects of discrete agonists and at the level of individual phosphorylated receptors.

In experiments with a phosphoselective antibody, Ser363 of MOR was found to undergo constitutive (agonist-independent) phosphorylation (40); our results confirm this observation with high precision (Fig. 2D). With a similar strategy, Thr370 phosphorylation was previously reported to be stimulated by DAMGO but not morphine, suggesting the hypothesis that these agonists produce qualitatively different phosphorylated receptor species (40). Our MS-based analysis not only verified that Thr370 phosphorylation was stimulated by DAMGO, but also identified Thr370 phosphorylation in morphine-treated cells at a moderately reduced relative abundance (Fig. 3B). This finding illustrates the analytical power of MS to achieve precise and sensitive interrogation of a complicated mixture of phosphorylated receptors and supports the fundamentally different hypothesis that agonist-selective phosphorylation of MORs is primarily quantitative rather than qualitative in nature. Further supporting this view, we observed a similar degree of agonist selectivity in the phosphorylation of the proximal 354TSST357 serine and threonine cluster. Our results are also consistent with the previous conclusions that Ser375 is phosphorylated in response to both morphine and DAMGO (40, 55, 56) and that Ser375 contributes to the regulation of MOR endocytosis (55, 56). Our results also suggest that Ser375 acts as part of a multisite motif, whose higher-order phosphorylation by distinct agonists occurs in a markedly disproportional manner. We propose that this disproportionality is the basis for biochemical encoding that is subsequently decoded by β-arrestins into agonist-selective endocytic activity. We believe that the present identification of quantitative, receptor-intrinsic encoding and decoding of agonist selectivity by multisite phosphorylation represents a conceptual advance in our present understanding of opioid drug action.

Our study examined MOR phosphorylation in intact cells and in the presence of a full complement of endogenous cellular kinases and phosphatases. A logical next step is to identify the individual kinases and phosphatases that contribute to the formation of particular agonist-selective phosphorylated receptor species under these native conditions by combining the present analytical methods with chemical inhibition or specific gene knockout or knockdown approaches. Likely candidates for phosphorylation of the 375STANT379 motif include members of the G protein–coupled receptor kinase (GRK) family, which are sensitive to the conformational state of their 7TMR substrates (57). Furthermore, increasing the abundance (21, 22) or activity (58) of GRK2 enhances morphine-induced endocytosis of MORs, and GRK2 affects the phosphorylation state of Ser375 in the 375STANT379 motif (56). Other candidate kinases include protein kinase C family members and mitogen-activated protein kinases, which have been implicated in the regulation of MOR signaling and trafficking (59, 60). The human kinome contains ~500 members, most of which have not been tested for their effects on MOR endocytosis; thus, in principle, there are many additional possibilities. Another logical extension of our approach is to examine other clinically important opioid drugs, such as fentanyl and methadone, which produce differential MOR regulatory effects in cultured cells and in vivo (14, 20, 61, 62). A third future direction is to explore other cellular regulatory consequences linked to MOR phosphorylation, such as functional desensitization of MOR signaling and control of the specificity of signal regulation, which appear to involve modification of cytoplasmic residues distinct from, or in addition to, those of the 375STANT379 motif (18, 49, 55, 63, 64).

To our knowledge, our results are the first to establish multisite phosphorylation as a receptor-intrinsic basis for discriminating a biologically relevant 7TMR regulatory mechanism at the level of individual MORs, in intact cells, and under conditions of saturating concentrations of ligand. However, we note that multisite phosphorylation has been shown previously by other methods to influence several other aspects of 7TMR regulation (65). Multisite phosphorylation of the light-activated 7TMR rhodopsin, in a conserved serine and threonine cluster in its C-tail, increases the temporal precision with which individual rhodopsin molecules become inactivated after photoisomerization of bound retinal, thereby enhancing reproducibility of the single-photon response (66). Differential phosphorylation of the β2-adrenergic receptor, in discrete locations (in the third cytoplasmic loop or the C-tail) and by different kinases, can distinguish functional regulation of the cellular receptor complement according to the overall degree of agonist occupancy (67, 68). Phosphorylation of muscarinic acetylcholine receptors in different regions of the third cytoplasmic loop contributes to differential regulation of the cellular receptor pool in distinct cell or tissue backgrounds (69). Finally, we note that many important 7TMR-active drugs are chemically distinct from endogenous ligands and are classified as partial agonists, and that agonist-selective production of higher-order phosphorylated species has also been observed for the β2-adrenergic receptor (70, 71). Thus, our proposed mechanism of encoding and decoding agonist-selective control may not be unique to morphine, MORs, or regulated endocytosis. Instead, we anticipate that it likely contributes to the cellular basis of partial agonism more broadly—and to manifestations of functional selectivity or ligand bias of 7TMRs as a class.

Materials and Methods

Materials

13C-labeled arginine (l-[13C]Arg:HCl) and 13C,15N-labeled arginine (l-[13C,15N]Arg:HCl) were from Cambridge Isotope Laboratories. Dulbecco’s modified Eagle’s medium (DMEM) and dialyzed fetal bovine serum (FBS) were from Invitrogen. DMEM deficient in l-arginine was from Athena ES. l-arginine (l-Arg:HCl) and n-dodecyl β-d-maltoside (DDM) were from Sigma. Sequencing-grade endoprotease AspN and modified bovine trypsin were purchased from Roche Applied Science. Handee Mini Spin Columns were from Pierce, and the microcon centrifugal filter devices and C18 zip tips were from Millipore. Reversed-phase packing, Oligo R3, and POROS 50 R2 were purchased from Applied Biosystems. All other chemicals, unless indicated otherwise, were obtained from Sigma.

Constructs

The complementary DNA (cDNA) encoding FLAG-tagged murine MOR1 was described previously (17). MOR mutants were generated by oligonucleotide-directed mutagenesis (QuikChange system, Stratagene) and verified by dideoxy sequencing (Elim Biopharm). The rat β-arrestin-2–GFP construct was described previously (72).

Cell culture, transfections, and isotope labeling

Stably transfected HEK 293 cells expressing N-terminally FLAG-tagged murine μ-opioid receptor (MOR1), generated as described previously (17), were propagated in DMEM deficient in l-arginine containing 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml) and supplemented with l-arginine, 13C-labeled arginine, or 13C,15N-labeled arginine to a final concentration of 0.398 mM. Cells were maintained in specific isotope conditions for a minimum of six doublings, with frequent medium changes. Uniform isotopic labeling of receptors was confirmed at >99% by MALDI-TOF (time of flight) MS analysis of arginine-containing peptides. The stably transfected cell clone was selected on the basis of fluorescence intensity. This clone exhibited robust receptor internalization in response to DAMGO and normal growth rate and morphology typically seen in transfected HEK 293 cells expressing FLAG-tagged MORs. For cell biological analysis of mutant receptors, HEK 293 cells were transfected with pcDNA3-based plasmids expressing the indicated FLAG-tagged MOR variants with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. For assays of transiently transfected cells, experiments were carried out 48 hours after transfection. For experiments with stably transfected cells, clones were selected in the presence of G418 (50 μg/ml, Invitrogen). Clones used in subsequent studies were chosen on the basis of their having similar amounts of receptor at the cell surface, as assessed by flow cytometry.

Agonist stimulation and cell harvest for MS samples

Cells were grown to ~70% confluence in 15-cm round cell culture dishes containing 20 ml of the appropriate culture medium. For non-SILAC experiments, six dishes of HEK 293 cells were used for each agonist condition [morphine (10 μM) or DAMGO (10 μM)]. For SILAC experiments, two dishes were used for each condition. Cells were grown in arginine-depleted medium, which was supplemented with regular arginine, [13C]arginine, or [13C,15N]arginine. Cells supplemented with regular arginine were used as the control. [13C]arginine-supplemented cells were treated with morphine, whereas [13C,15N]arginine-supplemented cells were treated with DAMGO. Cells in dishes were incubated with agonist for the indicated time period at 37°C, and the medium was removed and replaced with ice-cold phosphate-buffered saline (PBS) containing 1 mM EDTA and incubated on ice for 10 min. Intact cells were harvested by gentle aspiration while on ice. The cell suspension of each condition was centrifuged at 500g for 2 min at 4°C, and aspirated pellets were quickly frozen at −80°C.

Receptor purification

The protocol for the purification of FLAG-tagged MORs was adapted from a procedure described previously for the isolation of FLAG-tagged adrenergic receptors (71). For nonisotopically labeled experiments, each cell pellet from a specific agonist condition was purified separately. For isotopically labeled experiments, cell pellets from three different isotope agonist conditions to be compared were solubilized together in buffer A [20 mM tris-HCl (pH 7.4), 100 mM NaCl] and 1% n-dodecylmaltoside containing protease inhibitors (Complete cocktail, Roche), leupeptin (2.5 mg/ml), and phosphatase inhibitors (50 mM NaF, 0.1 mM sodium orthovanadate, and 80 mM glycerol-2-phosphate) and 10 μM naloxone to stabilize the solubilized receptor. Pellets were processed in a Dounce homogenizer and extracts were supplemented with 1 mM CaCl2 and centrifuged at 18,000 rpm in an SS34 centrifuge rotor (Sorvall) for 30 min at 4°C. Clarified supernatant was applied to M1-FLAG affinity resin equilibrated in low-salt washing buffer (buffer A + 0.1% n-dodecylmaltoside) supplemented with 1 mM CaCl2. The volume of resin used was 0.05 ml per milliliter of extract. The column was washed three times with three column volumes of low-salt washing buffer and then washed with five sets of alternating high-salt buffer [20 mM tris-HCl (pH 7.4), 500 mM NaCl, 0.1% n-dodecylmaltoside, and 1 mM CaCl2] and low-salt washing buffer. After an additional wash with three column volumes of low-salt washing buffer, the receptor was eluted with buffer A supplemented with 1 mM EDTA and FLAG peptide (200 μl/ml, Sigma). Receptor-containing fractions were identified by Bradford assay, combined with SDS–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, and resolved by SDS-PAGE on 4 to 20% polyacrylamide gels. SYPRO Ruby staining reagent (Molecular Probes) was applied according to the manufacturer’s instructions and imaged with a Typhoon 9400 (Amersham Biosciences) with excitation at 610 nm to detect the SYPRO signal. Images were processed and staining intensities were quantified with ImageQuant software (Amersham Biosciences).

Sample preparation for MS analysis of proteolytic digests

To separate FLAG peptide (DYKDDDDK) from the MOR, we performed gel filtration with Handee Mini Spin Columns filled with Sephadex G-50 fine resin, essentially as described previously (71). Briefly, purified receptor (~10 pmol) was loaded and eluted from the resin with 20 mM tris (pH 7.5) buffer containing 100 mM NaCl. Reduction, alkylation (by iodoacetamide), and in-solution proteolytic digests (with modified trypsin and AspN) of the MOR were accomplished through minor modifications of methods described previously (73). All samples were depalmitoylated on the microconcentrator membrane before enzymatic digestion by adding 1 M hydroxylamine (adjusted to pH ~8 with NaOH) and leaving samples in the dark for ~20 min at room temperature. Reduction of disulfides with tris(carboxyethyl)phosphine (TCEP, pH 7) was performed for ~20 min at room temperature instead of at 37°C to minimize receptor aggregation. IMAC procedures for phosphopeptide enrichment were performed according to the manufacturer’s protocol (Phosphopeptide kit, Pierce Biotechnology), except that an extra elution step with 1 M EDTA (pH 8) was included. Phosphopeptides were subsequently purified by a step-elution, reversed-phase procedure [Oligo R3: POROS R2 (1:2) loaded on top of zip tips] (71, 74). For liquid chromatography electrospray ionization (LC-ESI) MS procedures, samples were dried with a SpeedVac concentrator (Thermo Electron) and redissolved in 0.1% formic acid.

MALDI MS

Comprehensive analysis of available proteolytic peptides before and after IMAC enrichment was performed with the MALDI orthogonal time-of-flight instrument prOTOF 2000 (PerkinElmer Life Sciences) as previously described (75). The MALDI matrix used was 4-hydroxy-α-cyanocinnamic acid (4HCCA) mixed with a mixture of trifluoroacetic acid (0.1%), acetonitrile (33%), and water (67%). We used M-over-Z data software (Genomic Solutions) to prepare all of the MALDI-prOTOF 2000 spectra for figures.

Nano-LC-ESI quadrupole/quadrupole TOF MS

IMAC-enriched or nonenriched peptide digests were separated with a 75-mm × 15-cm reversed-phase C18 column (LC Packings) at a flow rate of 350 nl/min, running a 3 to 32% acetonitrile gradient in 0.1% formic acid over 1 hour on a nano high-pressure liquid chromatography (HPLC) apparatus equipped with an autosampler (Eksigent). The LC eluent was coupled to a Nanospray II source attached to a QSTAR Elite mass spectrometer running Analyst QS 2.0 software (Applied Biosystems). The mass spectrometer was set up to an MS survey scan for 500 ms followed by up to 12 MS/MS spectra at fixed m/z values corresponding to peptides observed from the MOR (200 ms per MS/MS). The Q1 precursor selection resolution was set to “low,” with a full-width half-maximum of ~5 daltons. Collision-induced dissociation energies were calculated with an empirically determined ramp based on the optimal collision energies for a standard set of peptides. ESI peptides were identified with Protein Prospector version 5.2 (http://prospector.ucsf.edu). For quantification on the QSTAR, extracted ion chromatograms were generated for selected fragment ions. The MS/MS data were manually submitted to A-score (http://ascore.med.harvard.edu/ascore.php) to assess the identified sites of phosphorylation for each peptide.

LC-ESI QTRAP MS

IMAC-enriched or nonenriched peptides were separated with a 75-mm × 15-cm reversed-phase C18 column at a flow rate of 350 nl/min, running a 3 to 32% acetonitrile gradient in 0.1% formic acid over 1 hour on a nano-HPLC system equipped with an autosampler. The LC eluent was coupled to a Nanospray II source attached to a QTRAP 4000 triple-quadrupole mass spectrometer running Analyst QS 1.4 software. Selective reaction monitoring (SRM) transitions were based on informative fragment ions observed in the quadrupole time-of-flight (QTOF) experiments. The collision energies were identical to those used on the QTOF. Individual SRM measurements were measured for 20 ms each. For precursor ion isolation, the quadrupole resolution was set to “low.”

Quantification of MS data

To examine agonist effects on MOR phosphorylation, we mixed light (that is, 12C and 14N) and two different heavy MOR cell preparations (representing three different treatment conditions) in equal amounts before protein purification to ensure identical handling. After separation and analysis with LC-MS, we calculated peptide abundance ratios from the MS signal intensity areas for each treatment condition. These signal intensity areas were calculated by first averaging the mass signals at each LC elution time. Next, the total number of signal counts of two to three monoisotopic peak tops per peptide was summed for each condition. Subsequently, these areas were adjusted by normalizing all of the MS signal counts with an internal unmodified MOR proteolytic peptide to account for small variations in mixing. The counts from the MS signal of the AspN MOR peptide, [164–176] (DRYIACHPVAL), fulfilled this role as the internal standard for quantitation experiments (both trypsin and AspN digests). To determine quantitative ratios when there was overlap between light and heavy or the different heavy (13C,14N versus 13C,15N) isotope distributions, we applied an isotopic correction factor, as described previously by Ong et al. (76).

Quantification of receptor internalization by flow cytometry

Endocytosis of FLAG-tagged MOR was assessed by flow cytometry with minor modifications to a previously described method (77). Briefly, cells were incubated in the absence or presence of the indicated agonist for 20 min at 37°C and then quickly chilled on ice. Cells were dissociated, and surface-accessible receptors were labeled with Alexa Fluor 488–conjugated M1 antibody; surface receptor immunoreactivity was measured with a FACScan flow cytometer (BD Biosciences). We collected 10,000 cells for each sample. Triplicate samples were analyzed for each condition in each experiment.

Visualization of receptor trafficking by fluorescence microscopy

FLAG-tagged MORs were visualized by an antibody-feeding assay as previously described (22). Briefly, HEK 293 cells that expressed the indicated MORs were plated on glass coverslips. Surface-accessible receptors were labeled in intact cells by addition to the culture medium of Alexa Fluor 488–conjugated M1 antibody and antibody against the FLAG epitope (2 μg/ml) for 30 min at 37°C, after which cells were incubated for an additional 20 min in the presence or absence of morphine (10 μM) or DAMGO (10 μM), as indicated. After a quick wash with PBS, cells were fixed in 4% formaldehyde in PBS for 15 min and then washed four more times with PBS before being mounted onto glass slides with Vectashield (Vector Laboratories). Epifluorescence microscopy was performed with an inverted Nikon Diaphot microscope equipped with a 60×/numerical aperture (NA) 1.4 objective, mercury arc lamp illumination, and standard dichroic filter sets (Omega Optical). Images were collected with a charge-coupled device (CCD) camera (Princeton Instruments) interfaced with a PC running Micro-Manager software (http://www.micro-manager.org).

TIRF microscopic imaging of β-arrestin-2 recruitment in HEK 293 cells

TIRF microscopy was performed with a system described previously (52) based on a Nikon TE-2000 inverted microscope equipped with a 60×/NA 1.49 objective. Evanescent illumination was generated by focusing a fiber-coupled 488-nm argon ion laser (Melles Griot) onto the outer back focal plane of the objective with a micrometer-guided illuminator (Nikon). Temperature was controlled at 37°C by a thermoelectric stage (Bioscience Tools) and objective warmer (Bioptechs). Time-lapse sequences were collected in living cells with a deep-cooled electron-multiplying CCD camera (Andor) operated in frame transfer mode and in the linear range of detection. Image analysis was performed with ImageJ software (http://imagej.nih.gov/ij/index.html). For analysis across multiple cells and experiments, specimens were fixed 7 min after agonist application with 4% formaldehyde freshly dissolved in PBS supplemented with 150 mM sucrose. Samples were washed in PBS after exposure to fixative for 10 min and TIRF imaging was performed in PBS.

Statistical analysis

Significant differences between experimental groups were analyzed by Student’s t test or by analysis of variance (ANOVA) with Bonferroni multiple comparison test, depending on whether two or more groups were being compared, and were calculated with Prism software (GraphPad), with P < 0.05 as the threshold for statistical significance.

Supplementary Materials

www.sciencesignaling.org/cgi/content/full/4/185/ra52/DC1

Fig. S1. LC-ESI MS/MS analysis of peptide 1.

Fig. S2. LC-ESI QTRAP analysis of peptides 1 and 2.

Fig. S3. LC-ESI MS/MS analysis of peptide 4.

Fig. S4. LC-ESI MS/MS analysis of peptide 5.

Table S1. Table of observed and calculated mass-to-charge values of phosphorylated tryptic digest products of MOR.

Table S2. Reference table of raw and adjusted signals.

References and Notes

  1. Acknowledgments: We thank S. Granier and B. Kobilka for valuable advice and generously sharing reagents at an early stage of this work. We thank M. Caron for providing the β-arrestin-2–GFP construct, A. Henry and G. Yudowski for instruction and assistance with TIRF microscopy, and J. Benovic, H. Bourne, D. Kenski, and M. J. Kreek for critical discussion. Funding: These studies were supported by research grants from the NIH (DA06511 and DA012864 to M.v.Z.) and National Center for Research Resources (P41RR001614 to A.L.B.). E.K.L. received support from an NIH National Research Service Award Fellowship (F32-DA020972-01). Author contributions: E.K.L. and M.T.-Z. performed most of the MS analyses, mutagenesis, and cellular function and imaging experiments; J.C.T. processed LC-MS samples and advised on their analysis; S.J.K. contributed to cellular imaging experiments and corroborated the effects of MOR mutation on regulated endocytosis in another cell culture system; A.N.K. and A.L.B. provided substantial advice and access to critical instrumentation required for the study; and E.K.L., M.T.-Z., and M.v.Z. conceived the project and wrote the manuscript. Competing interests: The authors declare that they have no competing financial interests.
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