Brain-specific Gαz interacts with Src tyrosine kinase to regulate Mu-opioid receptor-NMDAR signaling pathway

Cellular Signalling 21 (2009) 1444–1454

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Cellular Signalling j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c e l l s i g

Brain-specific Gαz interacts with Src tyrosine kinase to regulate Mu-opioid receptor-NMDAR signaling pathway Pilar Sánchez-Blázquez a,b, María Rodríguez-Muñoz b, Elena de la Torre-Madrid a,b, Javier Garzón a,b,⁎ a b

Instituto Cajal CSIC. Doctor Arce 37, 28002 Madrid, Spain Centro de Investigación Biomédica en Red de Salud Mental, CIBERSAM. ISCIII, Madrid, Spain

a r t i c l e

i n f o

Article history: Received 10 March 2009 Received in revised form 6 May 2009 Accepted 6 May 2009 Available online 13 May 2009 Keywords: Mu-opioid receptor NMDA receptor Src regulation Gz protein Chaperone PKCγ

a b s t r a c t There is a certain cross-talk in the nervous system between N-methyl-D-aspartate receptors (NMDARs) and Mu-opioid receptors (MORs). While NMDARs participate in the desensitization of MORs, these in turn modulate NMDAR-mediated glutamate responses. The G protein coupled receptors (GPCRs) activate NMDARs via Src although the role of Gα subunits in this process is not well defined. We have found that in the absence of MOR activation, the brain specific Gαz subunit binds to and stabilizes Src in its inactive form. The administration of morphine provokes the phosphorylation of specific cytosolic tyrosine residues in NMDAR2A subunits. This was achieved by PKCγ disrupting this Gαz–Src complex, enabling Src to be activated (pTyr416) by binding to GαiGTP proteins. These changes increased the activation of the calcium/ calmodulin-dependent protein kinase II (CaMKII), thereby promoting MOR desensitization. This regulatory pathway is disrupted by inhibiting PKC, preventing MOR-activated Gαi2 subunits from gaining control over Src. Thus, in neural cells the Gαz subunits exert a negative control on Src function reducing the activating influence of MORs on this tyrosine kinase. This MOR-triggered signaling pathway recruits PKCγ and Gαi subunits to activate Src tyrosine kinase, resulting in the potentiation of NMDAR function. Most relevant, this mechanism which operates in neural cells is essential for the development of tolerance to the analgesic effects of morphine. © 2009 Elsevier Inc. All rights reserved.

1. Introduction There is convincing evidence that MOR signaling can be modulated by the NMDA/nitric oxide cascade [1] and indeed, the development of morphine-induced antinociceptive desensitization has been related to the activity of glutamate NMDARs [2–4]. The interaction between these receptors is bidirectional since MOR signaling in the brainstem increases the activity of NMDARs [5], while in the periaqueductal grey matter (PAG) opioids promote the activation of CaMKII through the facilitation of NMDAR [6]. The distribution of MORs and NMDARs is closely related in many regions of the CNS. Indeed, they co-localize in patches within spiny neurons of the caudate-putamen nucleus, in the spinal cord dorsal horn (particularly within lamina II), in the shells of the nucleus accumbens, and in neurons of the solitary tract nucleus. In particular, the PAG is densely innervated by glutamatergic projections from the forebrain, and MORs and NMDARs co-localize in these PAG neurons (reviewed in [7]). In the nervous system certain GPCRs can promote the activation of the Src tyrosine kinase. In fact, GPCRs potentiate NMDAR-mediated

⁎ Corresponding author. Neurofarmacología, Instituto Cajal, Avda Dr Arce 37, 28002 Madrid, Spain. Tel.: +34 91 5854733; fax: +34 91 5854754. E-mail address: [email protected] (J. Garzón). 0898-6568/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2009.05.003

glutamate responses through the activation of PKC [6], but only if the non-receptor tyrosine kinase Src interacts with and phosphorylates the NR2 subunits of the NMDAR [8,9]. The activation of these GPCRs seems to be coupled to NR2 tyrosine-phosphorylation and it requires PKC, intracellular Ca2+ release and Src activation [10]. NMDAR activity is governed by a balance between tyrosine phosphorylation and dephosphorylation [11], and here Src is critical for the up regulation of NMDARs [12]. The Src family of kinases (SFKs) behaves as effectors for a series of signaling proteins that interact directly with specific protein domains in these SFKs, exerting non-catalytic influences on their behaviour. Among others, WASP, caveolin and RACK1 all inhibit SFK activity, whereas proteins that disrupt their inhibitory intramolecular interactions, displacing the SH2 or SH3 domains, promote their activation [13,14]. Other regulators of Src, such as Gαs, Gαi and H-Ras bind to its catalytic domain, although they may also interact with other regions of the kinase [15,16]. Indeed, a positive effect of Gαi on Src was observed even in the presence of p-Tyr527, a phosphorylated residue that inactivates Src by binding to the SH2 domain and restricting the access of the catalytic domain to its substrates [15]. By contrast, small GTPases such as H-Ras bind to and inhibit Src function [16]. These observations indicate the possibility of MORs to regulate Src to enhance the NMDAR function. The neural MORs regulate both Gi/o proteins as well as the pertussis toxin-insensitive Gz proteins [17,18].

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However, it is not known whether these G proteins could play a role in the MOR-mediated regulation of Src. Although H-Ras is not directly regulated by GPCRs, the Gαz subunit displays certain characteristics of the small GTPases. Like Ras, its endogenous GTPase is extremely inefficient and it requires the assistance of specific GTPase activating proteins to increase its rate of hydrolysis, for example that provided by the Rz subfamily of RGS proteins [19,20]. In the present study we sought to analyze the effects of Gαi and Gαz subunits on the tyrosine kinase activity of Src both in vitro and ex vivo, as well as how PKC could influence this regulation. We found that Gαz stabilized Src in its inactive form even when Tyr527 was not phosphorylated. Following MOR activation by morphine, PKC disrupted this complex and Src then underwent activating autophosphorylation on Tyr416 at GαiGTP subunits. This was followed by phosphorylation of NMDAR NR2A subunits and the activation of CaMKII. Thus, the concerted action of MOR-activated PKC and Gαi/z subunits regulates Src, producing an increase in NMDAR and CaMKII activity that contributes to MOR desensitization. Notably, the neural specific Gz protein behaves as a pTyr527-independent inactivating chaperone for Src.

2. Materials and methods 2.1. Tyrosine kinase activity of Src Increasing amounts of the recombinant full length human Src kinase (Cell Signaling #7775) and 1.5 µM of the substrate peptide (Cell Signaling #1366) were incubated at room temperature for 30 min in a 50 µL reaction volume (60 mM HEPES–NaOH [pH 7.5], 3 mM MgCl2, 3 mM MnCl2, 3 µM Na-orthovanadate, 1.2 mM DTT and 20 µM ATP). The reaction was stopped by adding 50 µL EDTA (50 mM [pH 8]), and then 25 µL of each reaction was added to 75 µL of dH2O and transferred to a 96-well streptavidin coated plate (PerkinElmer #AAAND-0005). Dose response curves of kinase activity were generated using Phospho-Tyrosine Mouse mAb to assay the phosphorylation of the peptide substrate (P-Tyr-100, Cell Signaling #9411). The HRP-linked anti-mouse IgG secondary antibody (Cell Signaling #7076) was detected with the Chemiluminescent HRP substrate (Millipore Iberica S.A., Madrid, Spain #WBKLS0100). Chemiluminescence was visualized with a Peltier cooled CCD camera at − 35° C (with a high signal-to-noise ratio and a wide dynamic range of 3.4 OD) in a ChemiImager IS-5500 (Alpha Innotech, San Leandro, California) and the data was analyzed by densitometry (AlphaEase v3.2.2). Changes in kinase activity were generated by adding increasing concentrations of H-Ras (Calbiochem #553325) or Gα subunits to the reaction. The following Gα subunits were used: Gαi2 subunits (Calbiochem #371796); Gαo subunits (Calbiochem #371790); Gαz subunits [21]. Prior to performing the kinase assays about 3 µM of the Gα subunits or H-Ras was incubated with 10 µM GTPγS (Sigma #G8634) or GDP (Sigma #G7127) for 15 min at 25° C. Ultrafree-MC filter units (10,000 NMWL, Millipore) were used to eliminate unbound nucleotide and to recover the GαGTPγS/GDP and H-RasGTPγS/GDP in the appropriate buffer before they were incorporated into the reaction mixture. A set of samples was used to analyze the activating autophosphorylation of Src in immunoblots. In these assays, 250 ng (20 nM final concentration) of the Src kinase was incubated in a 150 µL reaction volume containing: 60 mM HEPES–NaOH [pH 7.5], 3 mM MgCl2, 3 mM MnCl2, 3 µM Na-orthovanadate, 1.2 mM DTT and 20 µM ATP. The reaction was carried out at room temperature and the accumulation of pTyr416 Src was determined at different intervals. The effect of different concentrations of Gα subunits or PKCγ (68 ng= 5 nM) on the accumulation of pTyr416 Src was also evaluated. These reactions were terminated after 10 min and the samples were analyzed by Western blotting.

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2.2. Surface plasmon resonance analysis of the interaction between GαzGDP and Src Interactions were determined using a BIACORE X (GE Healthcare). Src (50 µg/mL) was coupled to channel two of a CM5 sensor chip (GE, BR-1000-14) by amine coupling at pH 7.0 (GE, BR-1000-50), whereas channel one acted as the blank. The sensor surface was equilibrated with HBS-EP buffer (GE, BR-1001-88) and the sensograms were collected at 25 °C with a flow rate of 10 µL/min after passing GαzGDP (75 µL) over the sensor surface. The CM5 sensor chip was regenerated after each cycle with two 15 µL pulses of 10 mM glycine given at a 30 s interval (pH 2.5, GE, BR-1003-56). Increasing analyte concentrations were studied and the results were plotted using the BIAevaluation software (v 4.1). 2.3. Membrane preparation from neural cells and pull-down assays Experimental tissue was obtained from male albino CD1 mice weighing 22–27 g (Charles River, Barcelona, Spain). For the immunoprecipitation studies, the neural structures from about 6 to 8 mice were pooled for each interval post-opioid administration. Typically, the study was repeated two or three times but on samples obtained from different mice that had received an identical opioid treatment and that had been killed at the same interval post-opioid administration. The methods used to prepare the neural membrane fraction enriched in synaptosomes have been described elsewhere [22,23]. The affinity purified IgGs against the helical domain of the Gαi2 and Gαz subunits, the extracellular domains of the MOR and against Src were labelled with biotin (Pierce #21217 and 21339) and the target proteins were then immunoprecipitated from solubilized membranes as described previously [22]. In the assays to determine the morphine-induced serine phosphorylation of Src, the interactions between proteins were disrupted prior to performing immunoprecipitation. Thus, the synaptosomal membranes (P2) were heated for 10 min at 100° C in a buffer containing 40 mM Tris–HCl (pH 7.5), 2% 2-mercaptoethanol, and 1% SDS. This mixture was then cooled to room temperature, the SDS concentration was reduced 7-fold by adding octylthioglucoside to a final concentration of 20 mM, and the Src was immunoprecipitated with specific anti-Src antibodies. Subsequently, the phosphoserines in the PKC phosphorylation motifs were detected with a mouse monoclonal antibody (IgM, 1:1000; Calbiochem, clone 1C8 525281). 2.4. Detection of signaling proteins The P2 membranes or the proteins obtained in pull-down assays were resolved by SDS/polyacrylamide gel electrophoresis. The proteins were transferred to 0.45 µm polyvinylidene difluoride membranes (PVDF; GE Healthcare Bio-Sciences) and probed with the selected antibodies in DecaProbe chambers (PR 150, Hoefer-GE Healthcare Bio-Sciences). The antibodies used included affinity purified rabbit IgGs directed against: Gαi2 subunits or Gαz subunits [1:1000, [24]; MOR [1:3000, [25], Tyr416 phospho-Src (1:1000 Cell Signaling 2101); Tyr527 phospho-Src (1:1000, Cell Signaling 2105); NMDAR2A (1:1000, ab14596); NMDAR2A phospho Y1325 (1:300, ab16646); PSD95 (1:1000, ab18258); Phospho-Ca2+/calmodulindependent protein kinase IIα (CaMKII Thr286, 1:2000, Cell Signaling 3361); and the heat shock protein 90 (hsp90, 1:1000 Santa Cruz sc 1055). We also used affinity purified mouse IgGs against Src (L4A1, 1:1000, Cell Signaling 2110) and CaMKIIα (1:3000, BD Transduction labs 611292). Equal loading of the material from the post-opioid intervals studied was verified by determining the target protein in parallel blots generated with the same immunoprecipitated samples used to study coprecipitation. When the variation of these signals with respect to that of the control group (no opioid) was higher than 15%, those of the coprecipitated proteins were adjusted.

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2.5. Animals Male albino CD-1 mice weighing 22–25 g were housed and used strictly in accordance with the European Community guidelines for the Care and Use of Laboratory Animals (Council Directive 86/609/ EEC). The animals were kept at 22 °C under a 12 h light/dark cycle (lights on from 8 a.m. to 8 p.m.), and food and water were provided ad libitum.

2.6. Downregulation of Gαz and Gαi2 expression Synthetic antisense oligodeoxynucleotides (ODN) were used to reduce the expression of the murine Gαz (NM-010311) and Gαi2 subunits (NM-008138). The efficacy of ODN-induced knockdown of Gαi2/z and their lack of effect on the expression of related proteins has been described elsewhere [21,24].

3. Results Neurons predominantly express a variant of Src that differs from that found in cells of non-neuronal origin, and that contains a six-amino acid insert [26]. This insert affects the position of the critical tyrosine residues implicated in its activation and inactivation, which are Tyr418 and Tyr529 in the 541 aa neural Src isoform 1 (NM_009271), and Tyr424 and Tyr535 in the 535 aa Src isoform 2 (NM_001025395). For the sake of simplicity, we will use the standard terminology described for chicken Src and we will refer to these positions as Tyr416 and Tyr527, respectively.

3.1. Regulation of the Src tyrosine kinase by Gαz and Gαi2 subunits We first explored the capacity of distinct MOR-regulated Gα subunits to modify the tyrosine kinase activity of Src in vitro. Src phosphorylated the peptide substrate used here (core sequence EGIYDVP) with an ED50 of 1 nM (Sigmaplot), a concentration at which activating pTyr416 autophosphorylation was promoted in the initial minutes of incubation (Fig. 1A). Hence, this concentration of 1 nM Src was used to study the effect of various Gα subunits and that of H-Ras on its tyrosine kinase activity. In accordance with previous reports [16], H-Ras reduced the kinase activity of Src and its GTPγS form was more active than H-RasGDP. While no effects of Gαi2 and Gαo were observed in their GDP state, GαzGDP did diminish the autophosphorylation of Src Tyr416 and hence, its kinase activity (Fig. 1B). By contrast, Gαi2GTPγS but not GαzGTPγS or GαoGTPγS enhanced the activity of Src (Fig. 1C). These data are in accordance with the earlier demonstration that GαiGTPγS enhanced the tyrosine kinase activity of Src by increasing its affinity for the substrate without altering its avidity for ATP or the Vmax [15]. This enhancement was dose-dependent, with an apparent ED50 of 20 nM, and it reached a maximum of almost three-fold the basal Src kinase activity. Significantly, the Gαi2/z forms that displayed no direct effect on the basal kinase activity of Src interfered with the regulation exerted by the active forms. Thus, GαzGTPγS reduced the Gαi2GTPγS-mediated stimulation of Src in a dose-dependent manner (Fig. 2A). As expected, the GDP form of the Gαz subunit reduced the maximum activity of Src to about one/third of the basal activity, and it also diminished the capacity of Gαi2GTPγS to activate Src (Fig. 2B). In addition, Gαi2GDP antagonized the inhibitory effect exerted by GαzGDP on Src activity (Fig. 2C). Interestingly, GαzGDP did not impair the tyrosine kinase activity of activated Src (pTyr416; Fig. 2D). We analyzed the interaction between recombinant Gαz and Src proteins by Plasmon surface resonance and the data indicated that their direct interaction could certainly regulate Src activity (Fig. 2E).

3.2. MOR activation transfers Src from under the control of Gαz towards that of Gαi2 subunits, where it undergoes autophosphorylation on Tyr416 The icv administration of morphine to mice produces a dose and time-dependent antinociceptive effect that peaks 30 min later. The ED50 (icv morphine nmol/mouse) determined in the tail-flick test is about 3.06 (95% confidence limits: — 2.11–4.43). The analgesia produced by administration of ED80 morphine (10 nmol) ceases after 3 h and it is completely absent on the following day [3]. We selected this dose of morphine because it produces a robust MOR-mediated activation of both Gi and Gz proteins [21]. The supraspinal analgesic effects of icv-injected opioids occur mainly through their respective binding to MORs in the midbrain [27,28]. Opioids act on the periaqueductal grey matter (PAG) to rostral ventromedial medulla (RVM) connection, where certain neurons carrying these receptors in the RVM project down to the substantia gelatinosa in the dorsal horn of the spinal cord and reduce the intensity of the ascending nociceptive signals [29,30]. Therefore, to compare the analgesic effects with molecular events, we examined the discrete neural structure, the PAG. Notwithstanding, the ex vivo study was also conducted in synaptosomal membranes derived from the cerebral cortex and striatum. Since the results were always comparable, only those results corresponding to PAG membranes are shown in the figures. Icv morphine administration produced a gradual reduction in the co-precipitation of Src with Gαz subunits. This change was clearly evident 30 min after morphine administration and although it recovered partially, it was still detected after 24 h (Fig. 3A). The Src that co-precipitated with Gαz subunits contained the inactivating pTyr527 and very little of the activating pTyr416. After morphine injection, there was a simultaneous decrease in the Src associated with Gαz and in the pTyr527 detected. The reduction in the association of Src with Gαz subunits was concomitant with increases in that of Src with Gαi2 subunits. Significantly, the autophosphorylation of Tyr416 was only evident in the Src associated with the Gαi2 subunits (Fig. 3A). The capacity of Gαz to interfere with the activation of Src at Gαi subunits was observed in brain membranes from mice in which these Gα subunits were depleted. The knockdown of Gαz greatly increased the association of Gαi2 subunits with pTyr416. In contrast, there was no significant change in the association of Gαz subunits with pTyr416 when it was precipitated from Gαi2 depleted mice (Fig. 3B). These data provide further evidence that both Gα subunits display certain affinity for Src binding, and that while Gαi2 binds to and activates Src, Gαz stabilizes the kinase in its inactive form. The morphine-induced Gαmediated regulation of Src could affect the activity of NMDARs. Indeed, Src co-precipitated with Gαz and Gαi2 subunits, NMDAR NR2A subunits and with the postsynaptic marker PSD95 protein (Fig. 4). Morphine diminished the association of Src with Gαz and augmented that with Gαi2, PSD95 and the NR2 subunits. This increased association with PSD95 and NR2 subunits reverted to basal levels after the interval in which the analgesic effect of morphine disappeared, a pattern that paralleled the presence of Src pTyr416. 3.3. PKC facilitates the activation of Src at Gαi2 subunits Since the inactive Src was mostly bound to Gαz in the absence of morphine, we considered the possibility that the transfer and activation of Src at Gαi2 subunits required the participation of third party proteins. Given that PKC is implicated in the activation of Src via GPCRs (see Introduction), we analyzed whether the Src co-precipitated with Gαi subunits was serine phosphorylated. In the absence of morphine, some serine phosphorylation of Src was detected (Fig. 5A). Icv-injection of the opioid greatly increased the pSer in Src, which diminished thereafter as the effects of the opioid disappeared (Fig. 5B). The Src that coprecipitated with Gαz contained little pSer both before and during the time-course of morphine antinociception, whereas the Src associated

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Fig. 1. In vitro regulation of Src tyrosine kinase activity by Gα subunits and by H-Ras. (A) Src titration: increasing amounts of recombinant full length Src were incubated with 1.5 µM of biotinylated substrate peptide for 30 min and the extent of its tyrosine phosphorylation was measured by densitometry. Time-course: 1 nM Src was incubated with 1.5 µM peptide substrate and the tyrosine kinase activity was determined at different intervals. The insert shows the presence of the activating autophosphorylation of Src at Tyr416. The effect of GDP (B) or GTPγS (C) Gα subunits and H-Ras on Src kinase activity was studied. The data are expressed relative to the Src activity observed for the control group (attributed an arbitrary value of 1). The bar histogram represents the mean ± SEM of duplicate samples for three independent experiments. ⁎ Significantly different from the control reaction that contained no Gα subunit or H-Ras (ANOVA-Student-Newman-Keuls test, P b 0.05). Images from representative streptavidin coated wells are shown. A parallel set of samples were used to analyze the activating autophosphorylation of Src. The reaction was carried out at room temperature and the accumulation of pTyr416 Src was examined in Western blots.

with Gαi2 displayed significant pSer (Fig. 5B). The exposure of the recombinant Src to PKCγ in vitro prevented Gαz from reducing the activating autophosphorylation of Src at pTyr416 (Fig. 5C). PKC also prevented GαzGTPγS from reducing the stimulating activity of

Gαi2GTPγS on Src (not shown). Thus, by phosphorylating specific residues in Src, PKC weakens its inactivating interaction with Gαz and it facilitates the binding of Src to Gαi2 subunits, where it becomes activated.

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Fig. 2. Gαi2 and Gαz are mutual antagonists in the regulation of Src activity. Gαi2GTPγS increased Src activity at nM concentrations. Addition of GαzGTPγS, which by itself promoted no change on the Src activity (A), or GαzGDP, which decreases this activity (B), significantly reduced the enhanced activity of Gαi2GTPγS on Src (⁎ ANOVA-Student-Newman-Keuls test; P b 0.05). (C) GαzGDP at nM concentrations reduced the activity of Src. Gαi2GDP, which by itself does not affect Src, opposes the reduction in Src activity induced by GαzGDP. (D) When GαzGDP was added 10 min after commencing the Src-mediated enzyme reaction (open circles), an interval at which most of the Src is autophosphorylated (see Fig. 1A), no reduction in tyrosine kinase activity was observed. Images from representative streptavidin coated wells are shown. The data are the mean ± SEM of duplicate samples from three independent experiments and they are expressed relative to the Src activity observed in the absence of the Gα subunit (attributed an arbitrary value of 1). (E) Surface Plasmon Resonance analysis of the interaction between GαzGDP and Src. The Src was coupled to a CM5 sensor chip. The sensorgrams were collected at 25° C with a flow rate of 10 µL/min, passing GαzGDP concentrations from 0.5 to 10 µg/mL (75 µL) over the sensor surface.

The administration of the PKC inhibitors Gö7874 or Calphostin C in vivo prevented the morphine-induced serine phosphorylation of Src, as well as the transfer of this kinase from Gαz to Gαi2 subunits.

Moreover, Src accumulated much less pTyr416 in the presence of Gö7874 (Fig. 6A). Notably, when the PKC inhibitor was administered 24 h after morphine the Src that associated with Gαi2 diminished

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Fig. 3. The icv-injection of morphine transfers the control of the Src kinase from the Gαz subunits to that of Gαi2 subunits, where it autophosphorylates Tyr416. (A) Groups of 6 to 8 mice were each icv-injected with 10 nmol morphine and sacrificed at different intervals post-opioid administration. The co-precipitation of pTyr416 Src and pTyr527 Src with Gαz/i2 subunits was then studied in PAG membranes. The assays were repeated two or three times on preparations obtained from independent groups of mice, and they are shown as the mean ± SEM of the densitometry values. The densitometry data are expressed relative to the levels observed for the control group that received no morphine (0 m; attributed an arbitrary value of 1). Equal loading was verified and where necessary the data were adjusted to the signals obtained for the immunodetection of the Gα subunits. Representative images are shown. (B) The knockdown of Gαz greatly increased the activation of Src (pY416) at morphine-activated Gαi subunits. By contrast, there was no significant change in the association of Gαz subunits with pTyr416 in Gαi2 depleted mice. Immunoprecipitation (IP) was carried out 3 h after administering 10 nmol morphine to the control and the Gα-depleted groups.

further (see Figs. 3A and 4), while that associated with Gαz subunits increased (Fig. 6B). Thus, long after the effects promoted by the administration of morphine had ceased, residual activation of PKC appeared to be responsible for the incomplete return of the Src from Gαi2 to Gαz subunits.

3.4. MOR-mediated potentiation of NMDAR In the post-synapses, one of the main targets of GPCR-regulated Src is the cytoplasmic tail of the NR2A and NR2B subunits [12]. Thus, we tracked the relationship between Src activation and its association

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of Src, Gi proteins and also of other signaling elements whose expression is practically restricted to neural tissue, Gz proteins and the serine threonine kinase PKCγ [19,31]. In this pathway, the Gz proteins exert a negative regulation of Src tyrosine kinase activity which is independent of pY527. Thus, the Gαz subunit acts as a chaperone by binding to and maintaining Src in its inactive state. Upon MOR activation the neural specific PKCγ, which is recruited to this receptor [32], disrupts

Fig. 4. Interaction between Src tyrosine kinase, Gα subunits and the NMDAR complex. Membranes obtained at different times after icv administration of 10 nmol morphine were solubilized under non-denaturing conditions and immunoprecipitated with antiSrc. The co-precipitation of the associated proteins was analyzed at the post-opioid intervals indicated (for details see Fig. 3).

with NR2 subunits and PSD95 proteins (Fig. 4). These changes were correlated with the increased Tyr1325 phosphorylation of NR2A subunits, as well as with the NMDAR-mediated activation of CaMKII (Fig. 7). Inhibition of PKC in vivo prevented the tyrosine phosphorylation of the NR2A subunits and the subsequent activation of CaMKII by autophosphorylation of Thr286. 4. Discussion This study describes new links of the chain of events that connects morphine-activated MORs with the development of analgesic tolerance via potentiation of NMDARs and CaMKII. This process requires

Fig. 5. The activated Src at Gi2 subunits is serine phosphorylated. (A) Phosphorylation of Src in the neural membrane (before morphine challenge). (B) Influence of morphine on the serine phosphorylation of Src. The synaptosomal membranes were solubilized under non-denaturing conditions and incubated with IgGs directed against Src, or the Gαz or Gαi2 subunits. To reduce the risk of interference with signals from proteins other than Src, the serine phosphorylation of this tyrosine kinase was studied by its immunoprecipitation after releasing the associated proteins by SDS solubilization (denaturing conditions; see Materials and methods). (C) Src was incubated for 15 min at room temperature in the presence or absence of 5 nM recombinant PKCγ (Cell Signaling #7596). Afterwards, 5 µM Gö7874 was added to all the samples and the tyrosine kinase reaction was assayed for 20 min after addition of the peptide substrate and the Gαz subunits. Images are shown from streptavidin coated wells representative of these experiments. Parallel samples were used to analyze the activating autophosphorylation of Src. The reaction was carried out at room temperature and the accumulation of pTyr416 Src was examined in Western blot.

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Fig. 6. PKC facilitates the transfer of Src from Gαz to morphine-activated Gαi2α subunits. (A) Inhibition of PKC disrupts the morphine-induced activating binding of Src with Gαi2 subunits. Mice that had received Gö7874 (30 min) or Calphostin C (60 min) before the opioid was administered were sacrificed at different intervals post-morphine and the synaptosomal membranes were obtained. The samples were solubilized and incubated with IgGs directed against Gαz or Gαi2 subunits. The co-precipitation of pSer Src, Src and pTyr416 Src was then studied in Western blots of these membranes. (B) In this protocol the mice received Gö7874 24 h after the icv injection of 10 nmol morphine, and the association of Src with immunoprecipitated Gαi2 and Gαz was determined. For further details see Fig. 3.

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Fig. 7. Morphine promotes the tyrosine phosphorylation of NR2A subunits and the activation of CaMKII: the effect of PKC inhibition. The mice were icv administered saline or 1 nmol of the PKC inhibitor Gö7874 (indicated by arrows) 30 min prior to the administration of 10 nmol morphine. Subsequently, the animals were divided into groups of 6 mice and they were sacrificed at various intervals post-injection. The PAG membranes from these mice were analyzed with antibodies directed against pTyr1325 NR2A and pThr286 CaMKII. The bars are the mean ± SEM from three assays and the data was normalized to the control group that received no morphine (0 m; attributed an arbitrary value of 1). A loading control was run simultaneously (bottom line in each panel).

this Gαz–Src complex, and the activated GαiGTP subunits then facilitate Src autophosphorylation at Y416. This regulation is specific and other G proteins such as Go, Gq or G12 do not affect Src activity in this way ([15]; present study). The role played by Gαz on Src function is reminiscent of the activity of the 90 kDa heat-shock protein (hsp90). This chaperone binds to Src during its intracellular trafficking and while in this complex, Src cannot act as a kinase. Thus, this association must be disrupted in order for Src to display tyrosine kinase activity, and the ser/thr phosphorylation of the hsp90–Src complex facilitates the release of the kinase from the chaperone [33]. However, we did not find any hsp90 associated with Src in brain synaptosomes, indicating that here it is the Gαz subunit that silences Src activity until it is required. The activation of GPCRs regulates different cellular effectors involved in the production of second messengers, as well as modulating the activity of transcription factors and gene expression that may affect the integrity of the cytoskeleton. GPCRs activate Stat3 via the Gi family of proteins and this process appears to be mediated by Src [34]. Thus, understanding the molecular mechanisms behind the activation of Src by the MOR is clearly relevant not only for opioid tolerance but also for a variety of distinct physiological processes. Interestingly, Src has also been implicated in delta-opioid receptor desensitization by interfering with receptor recycling [35]. However, morphine promotes almost no internalization of neural MORs [23]. Thus, Src contributes to MOR desensitization by a different mechanism, the potentiation of the NMDAR-CaMKII route. The in vitro studies indicated that the GDP and GTP forms of Gαi/z subunits can interact with Src, although only Gαi2GTP and GαzGDP promoted direct changes in the activity of the kinase. Nevertheless, in neural membranes these interactions are precisely regulated by PKCγ and only a few of the possible interactions can occur. Thus, in the absence of MOR activation the binding of Gαz to Src prevails over that of Gαi2, indicating that in contrast to the situation in vitro, there is no free competition between these proteins for inactive Src. The interaction of these Gi/Gz proteins with third party

proteins explains these observations. When activation of the MORs has ceased, the GTP form of the Gαi2 subunit decreases in favour of the GDP form and then, Gαi2GDP rapidly binds to the Gβγ dimers and separates from Src. By contrast, the Gαz subunit is maintained apart from the Gβγ dimer for longer periods than that of the Gαi2, and this favours the gradual increase in Src binding to free Gαz subunits. Therefore, the relocation of the Src from the Gαi protein towards the control of Gαz is facilitated by the low intrinsic GTPase activity of Gαz, which delays the formation of the GDP form needed to re-associate with the Gβγ dimers [19]. In addition, PKC prevents deactivation of GαzGTP by the RGS-Rz proteins by acting on its serine16 [36]. Moreover, this pSer16 prevents GαzGTP from binding to the Gβγ dimers even when it spontaneously adopts the GDP form. Most relevant, this regulation by PKC is not evident for Gαi subunits [36]. Gαz displays low affinity for PKC-phosphorylated Src and therefore, the action of ser/thr protein phosphatases to reverse the action of PKC on the tyrosine kinase is required to foment the formation of the Gαz–Src complex. Once this association is made possible, activated GαzGTP can reduce the stimulating action of Gαi2GTP on Src, binding to this tyrosine kinase but without altering its capacity to autophosphorylate Y416. However, in ex vivo assays low levels of pY416 Src was recovered in a complex with Gαz, raising doubts over the presence of its GTP form in this complex. GαzGDP does not inhibit the activity of Src but rather it prevents its activation. Thus, the stabilization of Src at GαzGDP requires its previous inactivation by a PTP, probably STEP, removing pY416 Src. Therefore, Gαz subunits and H-Ras differ in the guanine nucleotide they require to stabilize Src in its inactive form, GDP and GTP respectively. Therefore, the activation of MORs up-regulates NMDAR currents by means of the concerted activity of the Src non-receptor tyrosine kinase, and the serine/threonine kinase PKC [9]. Indeed, morphine recruits the neural specific PKCγ to the HINT1-RGSZ signaling module, which is attached to the MOR C terminus. This recruitment of PKCγ requires free zinc ions, which are generated by the NMDAR-nNOS cascade, further

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NMDARs. Src phosphorylates specific residues in the cytosolic tails of the NR2A and NR2B subunits [12,37], and this modification produces an increase in the permeation of Ca2+ ions towards the cytosolic side of the postsynapse. These increased Ca2+ fluxes recruit calmodulin (CaM), promoting the formation of the Ca2+-CaM complexes required to propagate the signals originated by glutamate/glycine at the NMDARs (e.g., by activation of CaMKII). This action increases CaMKII activation, which in turn exerts a negative control on MOR signaling (Fig. 8). The inhibition of PKC brings about a low tolerance to morphine analgesic effects [7]. This can now be explained by the essential role of PKCγ to connect MOR activation with the potentiation of NMDAR currents. In the absence of active PKCγ, the GαiGTP subunits exhibit only a weak capacity to disrupt the association of Gαz with Src. Moreover, by acting on Src, PKCγ not only weakens its interaction with GαzGDP but it also prevents regulation by activated GαzGTP, increasing the probability of Src binding to Gαi2GTP. Thus, PKCγ reduces the possible competition between Gαz and Gαi2 for the control of Src, and it thereby facilitates the transfer of Src from GαzGDP to Gαi2GTP subunits where it becomes activated to phosphorylate the NR2A subunits. Notably, Gαz and Gαi also exhibit marked differences in the regulation of other signaling pathways. Whereas activated Gαi/o subunits inhibit both type V and VI adenylyl cyclase (AC), Gαz only interacts with and inhibits type V AC [38]. Similarly, inactivated Gαo/i2GDP binds to and sequesters Rap1GAP, thereby increasing the amount of activated Rap1GTP [39]. However, GαzGTP diminishes Rap1 signaling by recruiting its regulator, Rap1GAP, from the cytosol to the plasma membrane where it can effectively down-regulate Rap1 signaling [40]. The GTP form of Gαz and Gαi interacts with the eyes absent transcription cofactor, Eya2, and prevents its translocation to the nucleus. However, the much lower intrinsic GTPase activity of Gαz with respect to that of Gαi promotes the cytosolic sequestering of Eya2, where Gαi competes for its release [41]. Since Gαz subunits stabilize Src in its inactive state, the absence of this negative regulation would lead to rapid and sustained up regulation of NMDARs via MORactivated Gαi subunits. Indeed, morphine produces a much more rapid and profound desensitization of MORs in Gαz knockout mice than in wild type mice [42,43]. CaMKII is certainly important in the desensitization of morphine analgesia. The NMDAR antagonist, MK801, suppresses the morphine-induced increases in CaMKII activation, the autophosphorylation of CaMKII Thr286, and it reduces the development of tolerance ([44], and references therein). 5. Conclusions

Fig. 8. Model describing the activation of Src mediated by morphine and MOR-activated PKCγ and GαiGTP subunits. In the resting state, inactivated Src (not phosphorylated at Tyr416) is under the control of GαzGDP subunits in the NR1 environment (A). On MOR activation by morphine (B, 1), Src and PKCγ collaborate to translate this information to the NMDARs. The MORs generate GαiGTP subunits and free Gβγ dimers which activate PLCβ (B, 2). The increased levels of DAG and calcium ions promote the activation of PKCγ (B, 3). The PKCγ acts on the chaperone (inactivating) complex GαzGDP–Src (B, 4) and it facilitates their separation (C, 1). Src binds to the MOR-activated GαiGTP subunits (C, 2) and undergoes activating autophosphorylation on Tyr416 (C, 3). Finally, this MOR-triggered signaling pathway leads to the phosphorylation of tyrosine residues on NR2A/B subunits that are required to up regulate the NMDAR currents (C, 4, 5). As a result, CaMKII becomes activated (C, 6, 7) and it translocates to the MOR environment to negatively regulate the function of this receptor (C, 8).

indicating the proximity of both receptors in the postsynapse [32]. PKCγ can thereafter participate in the up regulation of the NMDARs by an indirect effect on NR2A/B subunits via Src. PKCγ acts upstream of Src in this pathway to up regulate the Ca2+ currents produced by

The nervous tissue has developed a specific mechanism to connect the activation of MORs with that of Src, via PKC and Gi/z proteins. The brain specific Gαz subunit binds and stabilizes the inactive form of Src until MOR activated PKCγ disrupts this complex, enabling Src to be activated at GαiGTP subunits. Moreover, this novel regulatory mechanism connects the activation of MORs with an enhancement of NMDAR function, which in turn correlates with CaMKII activation and the desensitization of the MORs. These results provide the rational to understand the positive effects of NMDAR antagonist and of PKC inhibitors to prevent and even rescue the analgesic effects of morphine from tolerance. Acknowledgements María Rodríguez-Muñoz is a member of the Centro de Investigación Biomedica en Red CIBERSAM. Elena de la Torre-Madrid is a predoctoral fellow funded by the Spanish Ministerio de Educación y Ciencia (FPI). We would like to thank Beatriz Fraile and Carmelo Aguado for their excellent technical support. These studies were supported by the Spanish Ministerio de Ciencia e Innovación (SAF200603193) and Instituto de Salud Carlos III (PI08-0417).

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