Pyk2 uncouples metabotropic glutamate receptor G protein signaling but facilitates ERK1/2 activation
© Nicodemo et al. 2010
Received: 29 December 2009
Accepted: 21 January 2010
Published: 21 January 2010
Group I metabotropic glutamate receptors (mGluRs) are coupled via Gαq/11 to the activation of phospholipase Cβ, which hydrolyzes membrane phospholipids to form inositol 1,4,5 trisphosphate and diacylglycerol. This results in the release of Ca2+ from intracellular stores and the activation of protein kinase C. The activation of Group I mGluRs also results in ERK1/2 phosphorylation. We show here, that the proline-rich tyrosine kinase 2 (Pyk2) interacts with both mGluR1 and mGluR5 and is precipitated with both receptors from rat brain. Pyk2 also interacts with GST-fusion proteins corresponding to the second intracellular loop and the distal carboxyl-terminal tail domains of mGluR1a. Pyk2 colocalizes with mGluR1a at the plasma membrane in human embryonic kidney (HEK293) cells and with endogenous mGluR5 in cortical neurons. Pyk2 overexpression in HEK293 results in attenuated basal and agonist-stimulated inositol phosphate formation in mGluR1 expressing cells and involves a mechanism whereby Pyk2 displaces Gαq/11 from the receptor. The activation of endogenous mGluR1 in primary mouse cortical neuron stimulates ERK1/2 phosphorylation. Treatments that prevent Pyk2 phosphorylation in cortical neurons, and the overexpression of Pyk2 dominant-negative and catalytically inactive Pyk2 mutants in HEK293 cells, prevent ERK1/2 phosphorylation. The Pyk2 mediated activation of ERK1/2 phosphorylation is also Src-, calmodulin- and protein kinase C-dependent. Our data reveal that Pyk2 couples the activation mGluRs to the mitogen-activated protein kinase pathway even though it attenuates mGluR1-dependent G protein signaling.
analysis of variance
days in vitro
calcineurin inhibitor protein
African green monkey cells
extracellular signal-regulated kinase
G protein-coupled receptor
G protein-coupled receptor kinase
glutathione S transferase
- HEK29 cell:
Human embryonic kidney cell
- InsP3 :
metabotropic glutamate receptor
mitogen-activated protein kinase
protein kinase C
proline-rich tyrosine kinase 2
Glutamate is the predominant excitatory neurotransmitter in the brain and regulates physiological responses that include the development, survival, and death of neurons as well as neural plasticity [1–6]. The receptors that respond to glutamate can be classified into two types: ionotropic and metabotropic. Ionotropic glutamate receptors are comprised of cation-specific ion channels that can be subclassified into NMDA, AMPA and kainate receptors. In contrast, metabotropic glutamate receptors (mGluRs) are coupled to the activation of heterotrimeric G proteins and the regulation of intracellular second messenger levels. They are subcategorized into three groups based on sequence similarity and G protein specificity . Group I mGluRs (mGluR1 and mGluR5) are coupled to Gαq/11 and the activation of phospholipase Cβ which in turn stimulates the formation of the intracellular second messengers, inositol 1,4,5-triphosphate (InsP3) and diacylglycerol. InsP3 stimulates the release of Ca2+ from intracellular stores and both Ca2+ and diacylglycerol regulate the activation of protein kinase C (PKC).
Agonist-activated G protein-coupled receptors (GPCRs) function as guanine nucleotide exchange factors facilitating the exchange of GDP for GTP on the Gα subunit. Thus, allowing the dissociation of the Gα and Gβγ subunits, which function to regulate different effector enzymes and ion channels . The activity of Group I mGluRs is regulated by protein kinases that uncouple the receptors from heterotrimeric G proteins via both phosphorylation-dependent- and phosphorylation-independent mechanisms [8, 9]. Both PKC and G protein-coupled receptor kinase 4 (GRK4) uncouple Group I mGluR signaling by phosphorylating serine and threonine residues localized to the intracellular loop and carboxyl terminal tail domains of the receptors [10–14]. In contrast, the attenuation of both mGluR1 and mGluR5 signaling by GRK2 is mediated by a phosphorylation-independent mechanism that involves the interaction of the GRK2 amino terminal regulator of G protein signaling homology domain with Gαq/11 resulting the displacement of the heterotrimeric G protein from the receptor [9, 15]. Recently, we have demonstrated that optineurin and calcineurin inhibitory protein (CAIN) can also contribute to the phosphorylation-independent attenuation of Group I mGluR signaling by a mechanism that also involves Gαq/11 displacement from the receptor [16, 17].
A number of studies have also implicated tyrosine kinases in the regulation of Group I mGluR signaling. For example, Canepari et al  have reported that inhibitors of tyrosine phosphorylation enhance mGluR1-mediated cation-permeable ion channel conductances at parallel fibers that synapse on Purkinje neurons in the cerebellum of the rat. In contrast, G protein-dependent inward currents in midbrain dopaminergic neurons activated by mGluR1 are antagonized by tyrosine kinase inhibitors . A potential candidate tyrosine kinase that is involved in the regulation of Group I mGluR signaling is proline-rich tyrosine kinase 2 (Pyk2), which is found in a multimolecular complex with both mGluR1 and the NMDA receptor . Heidinger and coworkers  have also demonstrated that mGluR1-mediated upregulation of NMDA receptor currents in cortical neurons involves a Ca2+-, calmodulin- and Src-dependent activation of Pyk2. We show here that Pyk2 can be co-immunoprecipitated with both mGluR1 and mGluR5 from rat brain lysates and that Pyk2 interacts directly with Group I mGluRs via their second intracellular loop and carboxyl-terminal tail domains. We also find that Pyk2 expression attenuates mGluR1a signaling by disrupting receptor/Gαq/11 interactions, but functions to couple mGluR1 to the activation of extracellular regulated kinase 1/2 (ERK1/2) in cortical neurons.
Human embryonic kidney (HEK293) cells and COS-7 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Tissue culture reagents were purchased from Invitrogen (Burlington, ON, Canada). Isopropyl-β-D-thiogalactopyranoside (IPTG) was obtained from Bioshop Canada Inc (Mississauga, ON, Canada). Quisqualate, DHPG, MPEP and LY367385 were purchased from Tocris Cookson Inc. (Ellisville, MO, USA). [3H]myo-inositol was acquired from PerkinElmer Life Sciences (Woodbridge, ON). The Dowex 1-X8 (formate form) resin with 200-400 mesh was purchased from Bio-Rad. Horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG secondary antibodies, protein G-Sepharose beads, enhanced chemiluminescence (ECL), and mouse monoclonal anti-hemagglutinin (HA) (12CA5) antibody were purchased from GE Healthcare (Oakville, ON Canada). Anti-GST mouse monoclonal antibody and anti-Gαq/11 rabbit polyclonal antibody were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Anti-mGluR5 rabbit polyclonal antibody was obtained from Upstate (Lake Placid, NY, USA). Anti Pyk2/CAKβ mouse monoclonal antibody was from BD Transduction Laboratories (Mississauga, ON, Canada). Rabbit polyclonal phospho-p44/44 MAP kinase (Thr202/Tyr402), p44/44 MAP kinase, phospho-Pyk2 (Tyr402) antibody, phospho-Pyk2 (Tyr457), Pyk2, phosphor-Src (Tyr 416) and Src antibodies were all obtained from Cell Signaling Technology. (Pickering, ON, Canada). Lipofectamine Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 568 goat anti-rabbit IgG were purchased from Invitrogen/Molecular Probes (Burlington, ON, Canada). siRNAs were purchased from Dharmacon RNAi Technologies (Lafayette, CO, USA). Hi-Perfect Transfection Reagent was purchased from Qiagen (Misssauga, ON, Canada). All other biochemical reagents were purchased from Sigma (Oakville, ON, Canada), Fisher Scientific (Ottawa, ON, Canada) and VWR Scientific (Mississauga, ON, Canada).
The cDNA for the human Pyk2 was first amplified by PCR from the human universal Quick Clone™ library and tagged with a hemagglutanin (HA) epitope tag (BD Biosciences/Clontech, Mississauga, ON, Canada). The PCR product generated was digested with SalI/XbaI and subcloned into a mammalian expression HA vector. All other cDNA constructs have been described previously [12, 17].
Cell Culture and transfection
HEK293 cells were maintained in Eagle's minimal essential medium supplemented with fetal bovine serum (8% v/v) and gentamicin (5 μg/ml). African green monkey (COS7) cells were grown at 37°C in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum (8% v/v) and gentamicin (5 μg/ml). Cells were transfected using a modified calcium phosphate method as described previously . Following transfection (18 h), the cells were incubated with fresh medium and allowed to recover for 24 h for coimmunoprecipitation studies. Otherwise, they were allowed to recover for 6-8 h and reseeded into either 12-well dishes coated with collagen or containing collagen-coated coverslips, or into 24-well dishes and then grown an additional 18 h prior to experimentation.
Primary neuronal cultures were prepared from the cortex of embryonic day 15 CD1 mouse embryos. Cells were plated on either 100 mm dishes or 15 mm glass coverslips coated with poly-L-ornithine (Invitrogen) in Neurobasal media with B-27 (Gibco) and N2 (Gibco) supplements, 2 mM glutamax, 50 μg/ml penicillin, 50 mg/ml streptomycin. Cells were incubated at 37°C and 5% CO2 in a humidified incubator and cultured for up to 21 days in vitro with media replenishment every 3 days. Rat primary cortical cultures were cultured as described for primary mouse cultures and were prepared from the cortex of embryonic day 20 Wistar rat embryos. All animal procedures were approved by the University of Western Ontario Animal Care Committee.
After a growth period of 4 days, primary mouse cortical neurons growing in 6-well or 12-well plates were transfected with 100 nM of Pyk2 siRNA (GAACATGGCTGACCTCATATT) using Hi-Perfect Transfection Reagent according to the manuscfacturer's specifications (Qiagen, Mississauga, ON). After two days, the cells were transfected again in the exact same manner. The next day, the neurobasal media was aspirated, and the cells were washed twice with HBSS and left overnight in HBSS supplemented with bovine serum albumin Fraction V. For COS7 cells Pyk2 siRNA (GGACGAGGACTATTACAAA) was transfected with Lipofectamine 2000 following manufacturer's instructions. Scrambled siRNA (Non-Targeting siRNA #1) was purchased from Dharmacon. Experiments were performed 48 or 72 hours after transfection and knock down of proteins was confirmed by western blot.
Coimmunoprecipitation experiments were performed using 500-1000 μg of total cell lysate protein solubilized from HEK293 cells transiently transfected with the various cDNA constructs as described in the Figure Legends. The cells were solubilized in lysis buffer (25 mM HEPES, pH 7.5, 300 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA and 0.1% Triton X-100) containing protease inhibitors. Phosphorylated Pyk2 was examined in the presence of 1 mM sodium vanadate, 100 μM GDP, 2 mM MgSO4, 30 μM aluminum chloride, and 5 mM sudium fluoride. FLAG-mGluR1a was immunoprecipitated with FLAG Sepharose beads from cell lysates by overnight rotation at 4°C. For the coimmunoprecipitation of endogenous proteins from whole-brain extracts, adult rat brains were employed. Tissue was dissected and homogenized on ice in lysis buffer containing protease inhibitors. The particulate fraction was removed by centrifugation and 500-1000 μg of supernatant protein was incubated with polyclonal anti-mGluR1a or anti-mGluR5a rabbit antibodies and protein G-sepharose beads by overnight rotation at 4°C. For HEK293 and rat brain co-immunoprecipitations, the beads were washed 4-6 times with lysis buffer, and proteins were eluted in SDS loading buffer by boiling for 3-5 min. Eluted samples were separated by SDS-PAGE, followed by electroblotting onto nitrocellulose membranes for immunoblotting. Immunoblots were visualized by chemiluminescence using an ECL kit. Intensities of immunoblot signals were determined with the 4.0.2 version of the data acquisition and analysis software from Scion Corp. (Frederick, MD, USA).
Confocal microscopy and Immunostaining
Confocal microscopy was performed using a Zeiss LSM-510 META laser scanning confocal microscope equipped with a Zeiss 63X, 1.4 numerical aperture, oil immersion lens (North York, ON, Canada). HEK293 cells expressing both HA-Pyk2 and FLAG-mGluR1a as well as primary rat cortical neurons were seeded on 15 mm glass coverslips. HEK293 cells were serum starved for 30 min at 37°C in HBSS (116 mM NaCl, 20 mM HEPES, 11 mM glucose, 5 mM NaHCO3, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, pH 7.4). HEK293 cells were prelabeled with Alexa Fluor 568-conjugated anti-FLAG polyclonal rabbit antibody. Cells were then either kept on ice or stimulated with 100 μM quisqualate for 30 min at 37°C. Subsequently, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.05% Triton X-100, and labeled with Alexa Fluor 568-conjugated anti-HA mouse monoclonal antibody. Primary rat cortical neurons were fixed with 4% paraformaldehyde and permeabilized with 0.05% Triton X-100, and then stained for endogenous mGluR5a and Pyk2 with rabbit polyclonal anti-mGluR5a antibody and mouse monoclonal anti-Pyk2/CAKβ antibody. Coverslips were mounted in IMMU-MOUNT (Thermo Shandon, Pittsburgh, PA) onto glass slides and allowed to air dry before viewing. Colocalization studies were performed using dual excitation (488, 543 nm) and emission (band pass 505-530 nm and long pass 560 nm for Alexa Fluor 488 and 568, respectively) filter sets. The specificity of labeling and absence of signal crossover were established by examination of single-labeled samples.
GST-mGluR1a fusion protein purification and pull down assays
GST-mGluR1a peptides were generated by growing recombinant Escherichia coli BL21 bacteria at 37°C to an A600 of 0.65-0.8. Bacterial cultures were then induced for 3 hrs with 1 mM IPTG. After induction, cultures were pelleted, resuspended in PBS containing protease inhibitors and lysed by mild sonication. The bacterial lysates were cleared of cellular debris by centrifugation and then incubated with to glutathione-Sepharose 4B resin overnight at 4°C. GST-mGluR1a peptides bound to the matrix were washed extensively in PBS and then resuspended in lysis buffer containing protease inhibitors. COS-7 cell lysates overexpressing HA-Pyk2 were prepared and cleared of cellular debris by centrifugation. 400 μg of total cell lysate protein was used in each pull-down assay. One μg of matrix-bound mGluR1a peptides were incubated together with COS7 cell lysates and mixed overnight on an orbital shaker at 4°C. The matrix-bound protein complexes were washed extensively in lysis buffer and then eluted off the matrix in SDS loading buffer by boiling for 3-5 mins. Eluted samples were analyzed by SDS-PAGE and Western blotting with anti-Pyk2.
Inositol Phosphate Formation
Inositol lipids were radiolabeled by incubating HEK293 cells overnight with 1 μCi/ml [3H]myo-inositol in Dulbecco's modified Eagle's medium. Unincorporated [3H]myo-inositol was removed by washing the HEK293 cells with HBSS. HEK293 cells were preincubated for 1 h in HBSS at 37°C and then preincubated in 500 μl of the same buffer containing 10 mM LiCl for an additional 10 min at 37°C. HEK293 cells were subsequently treated in either the absence or the presence of increasing concentrations (0-30 μM) of quisqualate for 30 min at 37°C. The reaction was stopped on ice by adding 500 μl of 0.8 M perchloric acid and then neutralized with 400 μl of 0.72 M KOH, 0.6 M KHCO3. The total [3H]myo-inositol incorporated into the cells was determined by counting the radioactivity present in 50 μl of the cell lysate. Total inositol phosphate was purified from the cell extracts by anion exchange chromatography using Dowex 1-X8 (formate form) 200-400 mesh anion exchange resin. [3H]Inositol phosphate formation was determined by liquid scintillation using a Beckman LS 6500 scintillation system.
Agonist-independent FLAG-mGluR1a internalization was measured by flow cytrometry. Cell surface epitope-tagged receptors were prelabeled with primary anti-mouse FLAG antibody (1:500) on ice for 45 min. Cells were then warmed to 37°C in the absence of agonist for the times indicated in the Figure Legends. Cells were then transferred back on ice and labeled with secondary FITC-conjugated anti-mouse IgG antibody (1:500 dilution) for 45 min. Under these conditions, receptors are able to undergo only a single round of internalization. Receptor internalization is defined as the fraction of total cell receptors lost from the cell surface and thus is not available for labeling with the secondary antibody.
ERK1/2 and Pyk2 immunoblots
100 μg samples of cell lysate proteins solubilized from either HEK293 cells, COS7 cells or mouse primary cortical neurons were subjected to were subjected to SDS-PAGE, followed by electroblotting onto nitrocellulose membranes for immunoblotting with antibodies described in the Figure Legends. The primary antibodies used in the experiments were phospho-p44/44 MAP kinase (Thr202/Tyr402) antibody (diluted 1:1000), p44/44 MAP kinase antibody (1:1000), phospho-Pyk2 (Tyr402) antibody (1:1000), phospho-Pyk2 (Tyr457) antibody (1:1000), Pyk2 antibody (1:1000), rabbit anti-Flag antibody (1:1000). Antibody reactive bands were visualized and quantified as described above.
The means ± S.E.M. are shown for values obtained for the number of independent experiments indicated in the figure legends. GraphPad Prism software (Graph Pad, San Diego, CA) was used to analyze data for statistical significance, as well as to analyze and fit dose-response and time course data. The statistical significance was determined by either one way analysis of variance or t-test.
Interaction between Pyk2 and mGluR1
Colocalization of Pyk2 and mGluR1
Pyk2-mediated attenuation of mGluR1a signaling
mGluR1 mediates ERK1/2 phosphorylation by a Src/Pyk2-dependent pathway
In the present study, we provide evidence that the non-receptor tyrosine kinase Pyk2 interacts with and regulates mGluR1a signaling and desensitization. Pyk2 associates with the second intracellular loop domain and the carboxyl-terminal tail domains of mGluR1a, and similar to what has been described for GRK2, optineurin and CAIN, Pyk2 functions to disrupt Gαq/11 interactions with the receptor resulting in the attenuation of IP formation [9, 16, 17]. However, Pyk2 also facilitates mGluR1a-mediated activation of the ERK1/2 pathway, an effect that also requires the activity of Src, calmodulin and PKC. Thus, we describe a new mechanism by which Pyk2 attenuates mGluR1a by disrupting mGluR1a/Gαq/11 interactions, while simultaneously coupling the receptor to the activation of the mitogen-activated protein kinase (MAPK) signaling pathway.
Pyk2 is a non-receptor tyrosine kinase that is known to be activated by stimuli that either increase intracellular Ca2+ concentrations or activate PKC in response to stress signals, such as UV light, hyperosmotic shock and tumor necrosis factor-α . Pyk2 has also been shown to be involved in G protein-coupled-receptor signaling. For example, Felsch et al.  reported that Pyk2 phosphorylates the potassium channel Kv1.2 in response to the activation of m1 muscarinic acetylcholine receptor. In vascular smooth muscle cells, angiotensin II stimulates the association of Janus kinase 2 with the angiotensin II type1 receptor via a mechanism that involves increases in intracellular Ca2+ concentrations and the activation of both PKCδ and Pyk2 . Similarly, Heidinger et al.  showed that Pyk2 is involved in the mGluR1-mediated phopshorylation of NR2A/B subunits of the NMDA receptor in cortical neurons. Moreover, in HEK293 cells both the α1B- or α2A-adrenergic receptors stimulate ERK1/2 phosphorylation via a Ca2+-, calmodulin-, Src- and Pyk2-dependent pathway . In the present study, we provide evidence that Pyk2 interacts with mGluR1a and plays a role in of the mGluR1a-dependent activation of ERK1/2 phosphorylation via a mechanism that also requires calmodulin, Src, and PKC activity. Moreover, Pyk2 is associated with mGluR1a in the absence of agonist treatment and the mGluR1a/Pyk2 protein complex dissociates in response to quisqualate treatment for 20 min. The physiological consequence of this dissociation is unclear, but it is possible that the loss of Pyk2 from the receptor may occur as the result GRK2 binding to the receptor, which like mGluR5 might be required for agonist-stimulated mGluR1a endocytosis . Consistent with this hypothesis and the observation that Pyk2 dissociates from the receptor in response to agonist activation, we do not observe Pyk2 internalization with mGluR1a.
Pyk2 has previously been demonstrated to be autophosphorylated on Tyr402 in response to the activation of the G protein-coupled lysophosphatidic acid receptor resulting in the creation of a docking site for the Src SH2 domain . The recruitment of Src to Pyk2 subsequently leads to Pyk2 phosphorylation at both Tyr579 and Tyr580 and results in enhanced Pyk2 kinase activity [28–30]. The overexpression of Pyk2 is also reported to result in increased basal ERK1/2 phosphorylation [28, 29, 31]. However, we find that in HEK293 cells basal ERK1/2 phosphorylation is increased only when mGluR1a is overexpressed with Pyk2. Thus, despite the fact that Pyk2 reduces basal mGluR1a G protein coupling, Pyk2 overexpression results in an increase in basal ERK1/2 phosphorylation. In contrast, mGluR1a-mediated ERK1/2 phosphorylation is reduced following the expression of dominant-negative Pyk2-Y402F and catalytically inactive Pyk2-K457A mutants. Thus, we conclude that Pyk2 is essential for ERK1/2 phosphorylation in response to mGluR1a activation.
Both mGluR1 and mGluR5 are expressed in cortical tissue and primary cortical neurons can be positively stained for the expression of both mGluR1a and mGluR5 proteins . However, in primary mouse cortical neurons ERK1/2 phosphorylation is selectively activated by mGluR1, as ERK1/2 phosphorylation in response to DHPG is not inhibited by a mGluR5-specific antagonist. This is similar to what was observed by Heidinger and colleagues , who demonstrated that the phosphorylation of NMDA receptors by Pyk2 is selectively mediated by the activation of mGluR1 and not mGluR5 in cortical neurons. It is likely that mGluR5 will also activate ERK1/2 phosphorylation in other neuronal cell types for several reasons. First, Pyk2 can be co-immunoprecipitated from rat brain with mGluR5. Second, mGluR5-mediated nociception in the spinal cord involves the activation of ERK1/2 and we have recently demonstrated that mGluR5-dependent ERK1/2 phosphorylation is increased in mutant huntingtin protein knockin mice [32, 33]. Finally, MacDonald and coworkers have demonstrated that mGluR5-dependent activation of Pyk2 stimulates NMDA and AMPA receptor currents in hippocampal neurons .
It has previously been reported that Group I mGluRs increase the extent of Pyk2 phosphorylation in mouse neuronal cortical cultures via a mechanism that is both calmodulin- and Src-dependent, but that is independent of PKC activity . We find that Pyk2-mediated activation of ERK1/2 phosphorylation is both calmodulin- and Src-dependent, but it also involves a mechanism that requires PKC activation. Calmodulin binds to both mGluR1 and mGluR5 in a Ca2+-dependent manner and can be regulated by PKC-mediated phosphorylation of the receptor within the calmodulin binding domain [35–37]. The identification of Pyk2 and CaM as Group I mGluR interacting proteins suggests the possibility that Pyk2, Src and calmodulin may exist as a preformed complex that is scaffolded on the intracellular face of mGluR1. Thus, in response to PKC-mediated phosphorylation of mGluR1a, the complex may be released from the receptor to activate ERK1/2 phosphorylation.
We also find that siRNA treatment of cortical neurons results in a significant reduction in Pyk2 protein expression, but unexpectedly this also results in increased basal ERK1/2 phosphorylation. The increase in ERK1/2 phosphorylation is associated with increased phosphorylation of the remaining fraction of Pyk2 protein expressed in the neuronal cultures. The depletion of Pyk2 expression in COS7 cells also leads to increased basal Pyk2 phosphorylation, but mGluR1a expression is required for this Pyk2-mediated increase in ERK/1/2 phosphorylation. The mechanism by which a loss of Pyk2 expression leads to alterations in the phosphorylation of the residual pool of Pyk2 and increased ERK1/2 phosphorylation is unclear. However, it appears to require mGluR1 expression suggesting that constitutive mGluR1 activity underlies the phenomenon and that Pyk2 plays a role in regulating basal mGluR1 activity.
In summary, we show here that Pyk2 interacts with Group I mGluR1a via the intracellular loop2 and the carboxyl-terminal tail domains of mGluR1a and functions to uncouple the receptor from Gαq/11 protein, while facilitating ERK1/2 phosphorylation. The direct association of Pyk2 with mGluR1a appears to coordinate the formation of a protein complex that includes Src bound to Pyk2 and potentially calmodulin bound to the carboxyl-terminal tail of the receptor. The formation of this complex may be important for the spatial temporal regulation of Pyk2 activity at post-synaptic densities that is expected to be required for the efficient regulation of NMDA and AMPA receptor function as well as the regulation of other ERK1/2-dependent activities in synaptic transmission. The observation that several signaling proteins, such as Pyk2, optineurin CAIN and GRK2 regulate mGluR signaling via their interaction with the interface provided by the second intracellular loop suggests that peptides which mimic this interface may be useful for the modulation of Group I mGluR signaling.
MP was the recipients of a Canadian Hypertension Society/CIHR fellowship and a Heart and Stroke Foundation of Canada Fellowship. FMR was the recipient of a Heart and Stroke Foundation of Canada Fellowship. SSGF is the recipient of a Canada Research Chair in Molecular Neurobiology and is a Heart and Stroke Foundation of Ontario Career Investigator. This work was supported by CIHR grants MA-15506 and CIHR grant MOP-62738 to SSGF.
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