Src promotes delta opioid receptor (DOR) desensitization by interfering with receptor recycling

Abstract An important limitation in the clinical use of opiates is progressive loss of analgesic efficacy over time. Development of analgesic tolerance is tightly linked to receptor desensitization. In the case of delta opioid receptors (DOR), desensitization is especially swift because receptors are rapidly internalized and are poorly recycled to the membrane. In the present study, we investigated whether Src activity contributed to this sorting pattern and to functional desensitization of DORs. A first series of experiments demonstrated that agonist binding activates Src and destabilizes a constitutive complex formed by the spontaneous association of DORs with the kinase. Src contribution to DOR desensitization was then established by showing that pre-treatment with Src inhibitor PP2 (20 μM; 1 hr) or transfection of a dominant negative Src mutant preserved DOR signalling following sustained exposure to an agonist. This protection was afforded without interfering with endocytosis, but suboptimal internalization interfered with PP2 ability to preserve DOR signalling, suggesting a post-endocytic site of action for the kinase. This assumption was confirmed by demonstrating that Src inhibition by PP2 or its silencing by siRNA increased membrane recovery of internalized DORs and was further corroborated by showing that inhibition of recycling by monensin or dominant negative Rab11 (Rab11S25N) abolished the ability of Src blockers to prevent desensitization. Finally, Src inhibitors accelerated recovery of DOR-Gαl3 coupling after desensitization. Taken together, these results indicate that Src dynamically regulates DOR recycling and by doing so contributes to desensitization of these receptors.


Cell culture and transfection
Experiments were carried out in HEK293 cells stably expressing murine DORs or its truncated mutant [24,25], with the exception of experimental series requiring co-transfection of wild-type DORs either with dominant negative c-Src (DNM-Src: K295R/Y527F) [26] or dominant negative Rab11 (DNM-Rab11: Rab11S25N) [27]. Cells stably expressing wild-type DORs or truncated DOR mutants (DOR344T) were transfected with 6 g/100 mm petri dish of corresponding DNA using lipofectamine (Invitrogen, Burlington, ON, Canada) as transfection agent, followed by selection with G418 (500 g/ml).  (15 g) or the empty vector (pcDNA3) using polyethylenimine as transfection agent. Receptor expression was verified by ELISA so as to obtain similar membrane expression across transfection conditions. Cells were grown and maintained in complete DMEM containing 10% (v/v) foetal bovine serum, 1000 units/ml penicillin, 1 mg/ml streptomycin and 5 mM glutamine, in a humidified atmosphere of 5% CO2 at 37ЊC.

Src knock-down by siRNA
A pool of four desalted, deprotected siRNA oligonucleotide duplexes specifically targeted to human Src (GenBank NM_005417) and bearing UU overhangs were purchased from Dharmacon Research (Lafayette, CO, USA) and introduced into DOR-expressing cells performed with DharmaFECT1 according to manufacturer's specifications. In order to achieve optimal Src, knockdown cells were transfected with increasing concentrations of siRNA (100, 50 and 25 nM) and Src expression subsequently verified by Western blot analysis 48,64 and 72 hrs after transfection. Non-targeting DNA in equivalent concentrations was used to control for off-target changes in Src expression. 25 nM siRNA and 48 hrs recovery post-transfection produced maximal Src knock-down, and were therefore chosen for the study.

Immunopurification of FLAG-tagged receptors
This procedure was adapted from a previously described method [23] and was used to assess receptor interaction with endogenous Src or G␣ subunits. Briefly, cells were incubated overnight in serum-free medium and the day of the experiment were exposed to PP2 (20 M) or vehicle (DMSO) for 1 hr prior to treatment with DPDPE (1 M) for the indicated periods of time. In experiments in which PTX was used the toxin (100 ng/mL) was introduced 16 hrs prior to the experiment. After agonist stimulation, the reaction was stopped on ice by washing cells with cold PBS. Cells were then suspended in lysis buffer (5 mM Tris, 3 mM MgCl2, 2 mM EDTA, 1 mM NaF, 1mM Na3VO4, 5 g/ml leupeptine, 5 g/ml soybean trypsin inhibitor and 10 g/ml benzamidine) and homogenized using an ultraturax (IKA, Wilmington, NC, USA). Following a short centrifugation at 1500 rpm, the supernatant was centrifuged at 18,500 rpm for 20 min. and the resultant pellet was resuspended in lysis buffer for a second round of centrifugation (18,500 rpm; 20 min.). The pellet obtained was then solubilized in 0.5% n-dodecyl-maltoside, 25 mM Tris pH 7.4, 140 mM NaCl, 2 mM EDTA, 1 mM NaF, 1 mM Na3VO4, 5 g/mL leupeptine, 5 g/ml soybean trypsin inhibitor and 10 g/mL benzamidine. Following agitation at 4ЊC for 2 hrs, the solubilized fraction was centrifuged at 10,000 rpm for 60 min., and the receptor was immunopurified from the supernatant fraction using an anti-FLAG M2 antibody resin. 20 l of antibody-coupled resin equilibrated in solubilization buffer and supplemented with 0.1% bovine serum albumin (w/v) were used to purify the receptor overnight at 4ЊC under gentle agitation. The next morning the resin was pelleted, washed twice with 500 l of solubilization buffer and four times with 500 l of modified solubilization buffer (containing 0.1% instead of 0.5% n-dodecyl-maltoside (w/v)). The receptor was then eluted by incubating the resin for 10 min. at 4ЊC with 100 l of modified solubilization buffer containing of a FLAG peptide (150 g/ml). This elution was repeated three times, and the eluates were combined and concentrated by membrane filtration over Microcon-30 concentrators (Millipore). SDS sample buffer was then added and samples were used for SDS-PAGE.

SDS-PAGE and Western blotting
SDS-PAGE was performed as previously described [23]  cAMP accumulation assays cAMP accumulation assays were carried out according to a previously described protocol [23]. Desensitization of DORs (DES) was achieved by 30-

Internalization assays
Measurement of surface-expressed FLAG-tagged DORs and quantification of receptor internalization was assessed using an ELISA method adapted from [28,29]. Cells were seeded at a density of 10 5

Data analysis
Statistical analysis and curve fitting were done using Prism 4 (GraphPad, San Diego, CA, USA).

DORs and Src form a constitutive complex that dissociates upon receptor activation
We have previously shown that Src activity regulates the extent and duration of DOR responsiveness to different ligands [23]. To start to examine the nature of this regulation, we first investigated functional and physical interactions between the two proteins. As previously observed, the agonist DPDPE (1 M; 5 min.) stimulated Src activity [23], an effect that could be blocked by pre-exposure to PTX (Fig. 1A). Physical interaction between Src and the receptor was next monitored by immunopurifying FLAG-tagged DORs and performing Western blot analysis to measure the total amount of kinase recovered. As shown in Fig. 1B, Src copurified with the receptor in the absence of ligand, suggesting a spontaneous association of both proteins. Addition of DPDPE (1 M) to the incubation medium induced a rapid destabilization of the complex, as indicated by more than 30% reduction in the amount of Src recovered within the first 5 min. of agonist exposure (Fig. 1B). This rapid destabilization was followed by a much slower dissociation that progressed during the remaining 25 min. of agonist treatment. Maximal dissociation of the DOR-Src complex was not modified by PP2 (Fig. 1C) but was blocked by PTX ( Fig. 1D) indicating that disruption of the complex was dependent upon activation of the G-protein but not of Src.  Fig. 2A), desensitization was no longer observed since DORs exposed to DPDPE in the presence of PP2 maintained similar signalling efficacy as non-desensitized receptors ( Fig. 2A, right panel). [26] (Fig. 2B). Transfections were optimized to achieve similar membrane expression of DORs as in the previous experimental series, and DNM-Src levels were titrated to obtain complete inhibition of endogenous Src activity (Inset Fig. 2B). Results confirmed that in cells overexpressing DNM-Src, signalling efficacy of desensitized DORs (DPDPE; 1 M; 30 min.) did not differ from that of the corresponding controls (Fig. 2B, right panel). In contrast, in cells where DORs were co-transfected with the empty vector (pcDNA3) agonist pre-treatment significantly reduced subsequent ability of DPDPE to inhibit cAMP production (Fig. 2B, left panel).

Src inhibitors prevent DOR desensitization without modifying internalization, but maximal protection requires normal sequestration
Internalization contributes to rapid desensitization of DORs [30] and Src activity has been implicated in the sequestration of various GPCRs [31,32]. Thus, it was of interest to determine whether each of the experimental approaches that reduced DOR desensitization by blocking Src activity had any effect on DOR internalization. To do so, Src inhibition was achieved as in the previous experiments using PP2 or DNM-Src and DOR internalization by DPDPE was assessed by monitoring loss of membrane receptors at different treatment intervals. In cells stably expressing wild-type DORs, DPDPE-dependent internalization was in reasonable agreement with previous reports [33,34], displaying a half-life of 14 min. and a maximal sequestration of 87% Ϯ 3 of surface receptors within the first 2 hrs of agonist exposure. In transiently transfected cells expressing DOR/pcDNA3, a half-life of 19 min. was observed with a maximal sequestration of 64% Ϯ 3 of surface receptors. As shown in Fig. 3, neither of the experimental conditions that afforded protection from desensitization interfered with DOR endocytosis.
Having established that Src inhibitors protect against desensitization without influencing internalization, the next series of experiments was ran to explore whether the converse was also true, i.e. if changes in internalization influenced the ability of Src blockers to counter desensitization. DOR internalization was modified by two alternative approaches: (i ) using an internalization-deficient DOR mutant (DOR344T) that lacks C-terminal Ser/Thr residues [35,36] and (ii ) by incubating cells expressing wild-type DORs in hypertonic medium (0.4 M sucrose), a procedure that interferes with normal clathrin polymerization [37]. Changes in DOR internalization appear in Fig. 4A, which shows that agonist-dependent internalization was reduced by 30% in truncated DORs, while sucrose abolished sequestration for the first 10 min. of agonist exposure and reduced it by more than 60% for the remainder of treatment.  4C). As is apparent from the figures, the protective effect of PP2 was progressively reduced by treatments that increasingly interfered with internalization. In particular, the ability of PP2 to maintain DPDPE signalling in desensitized mutants became apparent at higher agonist concentrations than in wild-types (P ϭ 0.02; n ϭ 4; Fig.  4B) while in presence of sucrose PP2 protective actions were not only right shifted but also reduced in magnitude.

Src contributes to receptor desensitization by interfering with recycling
The fact that optimal sequestration was necessary for Src inhibitors to counter agonist-dependent desensitization suggested that the kinase negatively influenced DOR responsiveness via regulation of a post-endocytic event. Previous studies have demonstrated that a small proportion of internalized DORs are recycled to the membrane, while the bulk of them are rapidly trapped in an endocytic compartment from where they are slowly targeted for lysosomal degradation [19,38]. Since the functional consequence of this sorting pattern is a reduction in receptor signalling [39,40], we reasoned that Src inhibitors could counter desensitization by redirecting internalized receptors to the recycling pathway. To assess this possibility, we determined how Src silencing by siRNA or its pharmacological blockade by PP2 influenced membrane recovery of internalized DORs (Fig. 5). In experiments assessing the effect of the pharmacological inhibitor, control and PP2-treated cells both displayed internalization of approximately 65% surface receptors following 30 min. exposure to DPDPE. Consistent with previous reports [15,41], agonist removal allowed 36 Ϯ 3% of internalized receptors to recycle back to the membrane of control cells. Most interestingly, a similar PP2 pre-treatment as the one that protected DORs from desensitization (20 M; 1 hr) increased basal membrane recovery by almost 25%, implying that after Src inhibition 45% of internalized receptors were recycling back to the membrane (Fig. 5A).
We next examined whether knock-down of the Src gene would similarly enhance DOR recycling. For this purpose, cells stably expressing DORs were transfected with on-target or off-target siRNA for human Src. Forty-eight hours after transfection with a pool of four siRNA duplexes specifically directed against human Src (concentration range 25-100 nM), expression of the protein was reduced by 90-95% (Fig. 5B) (Fig. 5C), reproducing results obtained with pharmacological inhibitors and directly implicating Src in the regulation of DOR recycling.
Results obtained with pharmacological inhibition or gene silencing of Src confirm this tyrosine kinase as a regulator of post-endocytic sorting, but they do not indicate whether the small increase in recycling might have any consequences concerning desensitization. So we reasoned that if the increase in recycling were to play a significant role in the protective effect of Src blockers, then interfering with recycling would prevent the protective effect exerted by Src inhibitors. Thus, we determined whether protective action of PP2 could be affected by interfering with recycling. In a first series of experiments, we tested the effect of monensin, a ionophore that blocks receptor recycling by trapping internalized receptors within endosomes [38,42]. Results showed that at a concentration that reduced DOR recycling (50 M; Inset Fig. 6A), monensin abolished PP2-mediated protection from desensitization (Fig. 6A)

. To confirm this observation, cells were transfected with a mutant form of Rab11 (DNM-Rab11) in which substitution of Ser 25 by Asn interferes
with GTP-binding capacity of this small G protein and inhibits recycling [27,43]. Similar to monensin, a decrease in DOR recycling by DNM-Rab11 (Inset Fig. 6B) abolished the protective effect of Src-blockers upon desensitization (Fig. 6B), confirming that recycling is essential for Src modulation of DOR signalling efficacy.

Src activity interferes with the recovery of DOR-G␣ interaction after desensitization
Functional desensitization involves uncoupling of the receptor from the G protein [44] while resensitization requires recovery of a physical and functional interaction between the two proteins [40,45]. Recycling is thought to contribute to this recovery by

Fig. 3 Src blockers protect from desensitization without modifying DOR internalization. Internalization of surface receptors was measured following incubation with DPDPE (1 µM) for the indicated periods of time. Presence of DORs at cell surface was measured by ELISA as explained in 'experimental procedures'. Results are expressed as a loss of surface receptors (percentage of surface receptors observed before internalization) and represent mean ± S.E.M. of seven independent experiments carried out in triplicate. (A) DOR internalization was assessed in cells stably expressing wild-type DORs, following exposure to PP2 (20 mM; 1 hr) or vehicle. (B) DOR internalization was assessed in transiently transfected cells expressing DOR/pcDNA3 or DOR/DNM-Src.
allowing internalized receptors to return to the membrane, and making them available for a new cycle of interaction with their signalling partners [46][47][48]. Thus, if membrane recycling of DORs is enhanced by interfering with Src function (Fig. 5), Src blockade would be expected to improve recovery of DOR interaction with the ␣ subunit. In order to examine this issue, FLAG-tagged DORs were immunopurified and the amount of G␣ recovered with the receptor was determined by Western blot analysis. As shown in Fig. 7A, ␣i3 spontaneously associated with DORs and was susceptible to modulation by the agonist [49]. In particular, exposure to DPDPE for increasing time periods progressively reduced the amount of G␣i3 recovered with the receptor (Fig. 7A), but the interaction recovered upon agonist removal (Fig. 7B). Surprisingly, PP2 did not modify DOR-␣i3 uncoupling during exposure to DPDPE (1 M), but the Src blocker accelerated the recovery of DOR-␣i3 interaction after agonist removal (Fig. 7B). [15,19]. This particular sorting phenotype is encoded in the receptor's primary sequence, which predetermines non-covalent interactions between DORs and lysosomal sorting proteins [18,22,50]. By showing that its encoded sorting pattern may be modified by changes in Src activity, our results indicate that post-endocytic sorting of DORs is not irreversibly determined by the primary sequence.

Results obtained in this study indicate that the non-receptor tyrosine kinase Src inhibits DOR recycling and by doing so contributes to the desensitization of this receptor. It is well established that only a discrete portion of internalized DORs normally recycles back to the membrane while the great majority of them is targeted for lysosomal degradation
Functional significance of DOR regulation by Src is emphasized by the remarkable ability of PP2 and DNM-Src to eliminate Fig. 3 and results Table 1 Signalling and desensitization in wild-type DORs, truncated DORs and wild-type DORs exposed to sucrose cAMP accumulation assays were performed in cells stably expressing wild-type DORs that were exposed to sucrose (0.4M) or not (CTL), as well as in cells stably expressing similar amount of truncated DOR mutants (DOR344T). Results are the mean ± S.E.M. from at least three experiments carried out in triplicate. For each condition, Emax values represent the percentage of change with respect to cAMP production in the absence of ligand. *Two-way ANOVA CTL versus sucrose: P = 0.0007, n = 3 The possibility that Src inhibition could enhance membrane targeting of newly synthesized receptors is unlikely since membrane recovery of DORs was assessed in cells whose protein synthesis had been blocked by cycloheximide. On the other hand, the idea that Src contributes to DOR desensitization by regulating their post-endocytic sorting is supported by two observations. First, PP2 ability to prevent desensitization of an internalization-deficient DOR mutant was lower than the ability of this blocker to prevent desensitization of normally internalizing receptors. Second, in cells in which pre-incubation with sucrose produced a more   dramatic decrease in receptor internalization than truncation of the C-terminus, reduction in PP2 protective actions was even more remarkable. Indeed, sucrose produced a short period of complete inhibition of internalization (10 min.) followed by a progressive recover to attain internalization of almost 40% of surface receptors at the end of 2 hrs of agonist exposure. This observation is in agreement with previous reports showing that DORs are internalized through clathrin-dependent and -independent pathways [36], and sequestration via the latter most likely provided the source of receptors necessary for maintaining residual protection by PP2 in sucrose-treated cells. Sucrose also reduced DOR responsiveness to acute agonist exposure (Table 1), an observation that is not surprising given that hypertonic medium interferes with constitutive DOR internalization [34], which is necessary for maintaining a steady-state level of active membrane receptors [51,52]. Interestingly, despite its ability to enhance recycling, PP2 did not modify DOR internalization in steady state assays. Since dynamin phosphorylation by Src has been shown to contribute to internalization of other G-protein coupled receptors [31], one possible explanation for the lack of effect of Src blockers on internalization is that the latter inhibited internalization to the same extent as they enhanced recycling. Another possibility is that steady state internalization assays are not sensitive enough to detect a small increase in recycling. Independent of these alternatives, the functional relevance of the observed increase in recycling was established in experiments in which monensin or Rab11 interfered with the ability of Src blockers to prevent DOR desensitization.

EC50 (nM) Emax (% of inhibition) EC50 (nM) Emax (% of inhibition)
Apart from its negative impact on DOR ability to maintain agonist-dependent signalling, Src had a deleterious effect on physical coupling of the receptor to the ␣ subunit. This was particularly evident when evaluating the effect of Src inhibitor PP2 on the recovery of DOR-␣i3 interaction after desensitization. In contrast, given the ability of Src inhibitors to protect DORs from agonist-dependent desensitization it was surprising to find out that PP2 failed to prevent DOR uncoupling from ␣i3. This apparent contradiction could be explained by the mechanism underlying desensitization. Indeed, Tran and coworkers [48] have recently shown that GPCRs remain phosphorylated in the presence of agonist and have also established that resensitization does not require receptor dephosphorylation. These observations led them to propose that resensitization relies on ␤-arrestin dissociation and not on removal of phosphate groups. Within this model, resensitization of DOR signalling by recycling would entail membrane reinsertion of receptors that are ␤-arrestinfree but remain phosphorylated. Being free of ␤-arrestin allows recycled receptors to interact once again with the G protein. However, their phosphorylation status makes them highly prone to re-binding ␤-arrestin [53] and compromises the stability of DOR interaction with ␣i3 subunits. Hence, it is possible that by enhancing recycling Src blockers would promote the formation of DOR-␣i3 complexes capable of sustaining signalling but that are too weak to endure sample preparation for immunopurification.
DOR interaction with lysosomal sorting proteins like GASP1 and SNX-1 takes place few amino acids downstream of the NPXnY domain [18,22,50]. The tyrosine residue within this sequence has been identified as the major target for DOR phosphorylation by Src [54]. Hence, modifications in the phosphorylation status of the NPXnY are expected to influence receptor interaction with either of the lysosomal sorting proteins, providing a biochemical substrate for Src modulation of DOR recycling. Src activity may also be directed towards sorting proteins themselves, a possibility that is consistent with results from the present study showing that agonist binding destabilizes the constitutive association between DORs and the kinase. A plausible substrate for free Src is Hrs (Hepatocyte growth factor-regulated tyrosine kinase substrate) [55], which has been recently shown to participate in GPCR recycling and resensitization [56,57]. A major goal for future study is to determine if and how Src regulates Hrs activity and DOR interaction with sorting proteins.
Immunopurification of unstimulated DORs allowed to recover both Src and ␣i3 subunits, indicating that the three proteins form part of a constitutive complex. The ability to form spontaneous multimeric arrays containing receptors and their signalling partners is being increasingly recognized as a general characteristic of GPCRs [58,59], and this property has been recently confirmed for DORs [60]. However, spontaneous association between Src and DORs distinguishes them from other GPCRs whose interaction with the kinase only takes place upon receptor activation [32,[61][62][63]. As a matter of fact, DOR activation rapidly disrupts its association with Src, an observation that is highly reminiscent of the way agonist binding causes calmodulin to be released from this receptor [64]. Src release from DORs was concomitant with its activation and both events were sensitive to PTX, suggesting that organization within a constitutive complex provides physical proximity to ensure rapid Src activation by G␣i/o proteins. Within this context, a possible scenario for Src activation would entail the following series of events: agonist binding to the receptor induces conformational changes that modify protein-protein interactions within the DOR-Src-G protein complex leading to G␣i/o activation, Src stimulation and loosening of DOR interaction with the kinase. Released from the sterical constraints imposed by its association with the complex, Src is now able to phosphorylate its substrates, which may include sorting proteins and/or DORs themselves [23]. In addition to modifying agonist-mediated responses incubation with PTX also increased basal Src activity, but this effect was not accompanied by any visible changes in complex stability. Src is a target for PKA [65] and the observed increase in basal Src stimulation could be related to an increase in basal cAMP levels following G␣i/o protein inactivation by PTX. However, it should be noted that Src activation by PTX was consistently lower than its activation by DPDPE (≅ 1/3), excluding the possibility that the absence of agonist-mediated responses could be linked to pre-existing maximal Src activity.
In conclusion, this study showed that Src modulates postendocytic sorting of DORs, contributing to rapid desensitization of these receptors. The idea that DOR trafficking may be dynamically regulated raises the possibility of pharmacologically altering its sorting pattern to enhance recycling and counter desensitization. Analgesic tolerance to opiates is intrinsically linked to desensitization and any intervention that could prevent this adaptive response should improve clinical efficacy of these powerful analgesics.