Advertisement
Research ArticleArticle

Activated CaMKIIα Binds to the mGlu5 Metabotropic Glutamate Receptor and Modulates Calcium Mobilization

Christian R. Marks, Brian C. Shonesy, Xiaohan Wang, Jason R. Stephenson, Colleen M. Niswender and Roger J. Colbran
Molecular Pharmacology December 2018, 94 (6) 1352-1362; DOI: https://doi.org/10.1124/mol.118.113142

Abstract

Ca2+/calmodulin-dependent protein kinase II (CaMKII) and metabotropic glutamate receptor 5 (mGlu5) are critical signaling molecules in synaptic plasticity and learning/memory. Here, we demonstrate that mGlu5 is present in CaMKIIα complexes isolated from mouse forebrain. Further in vitro characterization showed that the membrane-proximal region of the C-terminal domain (CTD) of mGlu5a directly interacts with purified Thr286-autophosphorylated (activated) CaMKIIα. However, the binding of CaMKIIα to this CTD fragment is reduced by the addition of excess Ca2+/calmodulin or by additional CaMKIIα autophosphorylation at non-Thr286 sites. Furthermore, in vitro binding of CaMKIIα is dependent on a tribasic residue motif Lys-Arg-Arg (KRR) at residues 866–868 of the mGlu5a-CTD, and mutation of this motif decreases the coimmunoprecipitation of CaMKIIα with full-length mGlu5a expressed in heterologous cells by about 50%. The KRR motif is required for two novel functional effects of coexpressing constitutively active CaMKIIα with mGlu5a in heterologous cells. First, cell-surface biotinylation studies showed that CaMKIIα increases the surface expression of mGlu5a. Second, using Ca2+ fluorimetry and single-cell Ca2+ imaging, we found that CaMKIIα reduces the initial peak of mGlu5a-mediated Ca2+ mobilization by about 25% while doubling the relative duration of the Ca2+ signal. These findings provide new insights into the physical and functional coupling of these key regulators of postsynaptic signaling.

Introduction

The ability of excitatory glutamatergic synapses to undergo dynamic changes in strength, termed synaptic plasticity, is critical for many behaviors. It is well established that glutamate activation of diverse ionotropic and metabotropic receptors is critical for short-term and long-term control of many neuronal responses (Niswender and Conn, 2010), and that these responses require a complex and incompletely understood network of signaling proteins. Among the seven members of the metabotropic glutamate (mGlu) receptor family, mGlu1 and mGlu5 specifically couple through Gαq/11 to stimulate multiple signaling pathways, including phosphoinositide hydrolysis and mobilization of intracellular Ca2+ stores. These receptors have long been implicated in multiple forms of long-term depression that require new protein synthesis (Oliet et al., 1997; Palmer et al., 1997; Huber et al., 2001) or increased endocannabinoid signaling (Lüscher and Huber, 2010). Despite many similarities, mGlu1 and mGlu5 can be differentially regulated by various mechanisms and have been shown to have different neuronal roles. For instance, in hippocampus, mGlu1 increases the frequency of spontaneous inhibitory postsynaptic currents, whereas mGlu5 potentiates N-methyl-d-aspartate receptor currents (Mannaioni et al., 2001). In particular, mGlu5 has been specifically implicated in a number of neuropsychiatric disorders including addiction, schizophrenia, fragile X syndrome, obsessive compulsive disorder, and Alzheimer’s disease (Grueter et al., 2008; Michalon et al., 2012; Ronesi et al., 2012; Hu et al., 2014; Ade et al., 2016; Foster and Conn, 2017).

Like mGlu5, Ca2+/calmodulin (CaM)-dependent protein kinase II α (CaMKIIα) is a key signaling protein in dendritic spines. CaMKIIα is activated by Ca2+/CaM binding and undergoes autophosphorylation at Thr286 (Miller et al., 1988; Mukherji et al., 1994; Rich and Schulman, 1998; Yang and Schulman, 1999; Baucum et al., 2015). Thr286 autophosphorylation increases the affinity for Ca2+/CaM and stabilizes the kinase in a constitutively active conformation. This constitutive activity is essential for normal synaptic plasticity in many brain regions (Silva et al., 1992a,b; Giese et al., 1998; Zhou et al., 2007; Mockett et al., 2011; Coultrap et al., 2014; Shonesy et al., 2014; Jin et al., 2015) including mGlu1/5-dependent long-term depression in the hippocampus (Huber et al., 2001; Mockett et al., 2011). Interestingly, both CaMKIIα- and mGlu5 knockout (KO) mice display deficits in learning and memory and hippocampal synaptic plasticity (Jia et al., 1998; Huber et al., 2001; Simonyi et al., 2005). Although both mGlu5 and CaMKII are critical to many forms of plasticity, a functional link between the two has not been widely investigated.

The intracellular C-terminal domains (CTDs) of mGlu1 and mGlu5 have emerged as important loci for regulation by protein binding and phosphorylation (Enz, 2012; Mao and Wang, 2016). Although there are two splice variants of mGlu5 (mGlu5a and mGlu5b), most studies have focused on mGlu5a, and a few pharmacological differences between splice variants have been identified (Joly et al., 1995; Minakami et al., 1995; Romano et al., 1996). The mGlu5-CTD has been shown to bind to a number of different proteins including Ca2+/CaM and Homer to regulate cell-surface expression of the receptor (Roche et al., 1999; Saito et al., 2002; Lee et al., 2008; Choi et al., 2011). Protein kinase A (PKA) and protein kinase C (PKC) also regulate mGlu5 surface expression through phosphorylation of the CTD (Mao et al., 2008; Uematsu et al., 2015). It was recently reported that CaMKII can bind to the CTD and intracellular loop 2 of both mGlu1 and mGlu5 (Jin et al., 2013a,b; Raka et al., 2015) and that CaMKII modulates mGlu5 agonist-induced internalization and ERK1/2 activation (Raka et al., 2015). Here, we identify three basic residues (Lys866-Arg877-Arg888) in the membrane proximal region of the mGlu5a-CTD that are essential for a direct interaction with activated CaMKIIα, and provide novel insights into multiple factors that modulate the interaction. We also show that CaMKII binding to the CTD is important for the regulation of mGlu5 surface expression and Ca2+ mobilization. These data provide novel insights into the molecular basis and function of the mGlu5-CaMKII interaction that may be involved in synaptic plasticity.

Materials and Methods

DNA Constructs

The glutathione S-transferase (GST)-mGlu5a-CTD expression construct was created by polymerase chain reaction amplification of the region encoding residues 827–964 of mGlu5a (NP_058708.1) using forward primer 5′-CTGGAAGTTCTGTTCCAGGGGCCCGGATCCAAACCGGAGAGAAAT-3′ and reverse primer 5′-GCCGCAAGCTTGTCGACGGAGCTCGAATTCTTAGGTCCCAAAGCGCTT-3′ and inserting the product into BamHI/EcoR1 sites of pGEX6P using a sequence- and ligation-independent cloning protocol (Li and Elledge, 2012).

The pCGN plasmid to express wild-type (WT) mGlu5a with an N-terminal hemagglutinin (HA) tag was made by amplifying the entire rat mGlu5a coding sequence (forward primer: 5′-TGACGTGCCTGACTATGCCTCTAGAATGGTCCTTCTGTTGATCCT-3′; reverse primer: 5′-ACTCACCCTGAAGTTCTCAGGATCCTCACAACGATGAAGAACTCT-3′) and inserting the fragment into XbaI and BamHI restriction sites of the empty pCGN plasmid [a gift from Dr. Winship Herr, Université de Lausanne, Switzerland; Addgene (Cambridge, MA) plasmid ID 53308].

The K866RR868 mutation to AAA in mGlu5a was generated by site-directed mutagenesis of the pGEX6P or pCGN constructs (see above) using a Quick Change protocol (Agilent Technologies, Santa Clara, CA) with the following primers: forward, 5′-GGGTTTCCCCAGAGGAGCCGGCGGCGGCCCACAGGTTGACTAGGCTGCT-3′; and reverse, 5′-AGCAGCCTAGTCAACCTGTGGGCCGCCGCCGGCTCCTCTGGGGAAACCC-3′.

We used pcDNA3.1 constructs to express untagged and mApple-tagged WT-CaMKIIα and a constitutively active (CA) T286D/T305A/T306A triple mutant of CaMKIIα (CA-CaMKIIα), as previously described (Jiao et al., 2008; Jalan-Sakrikar et al., 2012; Stephenson et al., 2017). In the CA-CaMKIIα, the phospho-mimetic T286D mutation results in constitutive CaMKIIα activity and the phospho-null T305A/T306A mutations prevent CaMKIIα phosphorylation at these sites, which interferes with binding of Ca2+/CaM and α-actinin (Jalan-Sakrikar et al., 2012).

Recombinant Protein Purification

Expression and purification of recombinant mouse CaMKIIα has been described before (McNeill and Colbran, 1995). To express GST-tagged proteins, pGEX6P-1 plasmids were transformed into BL21(DE3) bacteria cells. Cells were grown in Lysogeny broth media at 37°C to reach an optical density of ∼0.6. Cells were cooled to room temperature, and isopropyl β-d-1-thiogalactopyranoside (0.2 mM) was then added to induce protein expression for 12–16 hours. Inducing protein expression at room temperature substantially reduced the protein degradation seen when proteins were expressed at 37°C. Expressed proteins were purified using Pierce Glutathione Agarose Beads (cat. no. 16101; Thermo Fisher Scientific, Waltham, MA) following manufacturer instructions. Eluted proteins were then dialyzed in 10 mM HEPES pH 7.5, 25 µM phenylmethane sulfonyl fluoride (PMSF), 62.5 µM benzamidine, 62.5 µM EDTA, and 0.1% Triton X-100 overnight with one buffer change.

CaMKIIα Autophosphorylation

Purified mouse CaMKIIα was autophosphorylated under two different conditions. Typically, CaMKIIα was incubated with 50 mM HEPES, pH 7.5, 10 mM Mg(CH3-COO)2, 0.5 mM CaCl2, 2 µM CaM, and 40 µM ATP on ice for 90 seconds before the addition of EDTA and EGTA (20 mM final) to terminate phosphorylation by chelation of Mg2+ and Ca2+. Similar conditions were previously shown to result in the selective autophosphorylation of Thr286 (McNeill and Colbran, 1995). Where indicated, identical autophosphorylation reactions were incubated for 10 minutes at 30°C to perform a more extensive phosphorylation at several additional sites (Baucum et al., 2015).

GST Pull-Down and CaM Binding Competition

Purified GST-mGlu5-CTD (1 µM) and CaMKIIα (62.5 nM; preautophosphorylated as indicated in the figure legends) were incubated at 4°C in GST pull-down buffer [50 mM Tris-HCl pH 7.5; 150 mM NaCl; 1% (v/v) Triton X-100] with either 2 mM EGTA or 2.5 mM CaCl2 plus 10 µM CaM, as indicated. An aliquot (5%) of each incubation was saved as an input sample. After 1 hour, prewashed Pierce Glutathione Agarose Beads (cat. no. 16101; Thermo Fisher Scientific) (15 µl of a 50:50 slurry) were added, and incubation was continued at 4°C for an additional 1 hour. The beads were then separated by centrifugation (2000g, 30 seconds) and washed three times with GST pull-down buffer containing either 2 mM EGTA or 2.5 mM CaCl2, respectively. Beads were then incubated at 4°C with GST pull-down buffer containing 20 mM glutathione, adjusted to pH 8.0, for 10 minutes. After centrifugation, eluted proteins were transferred to a new tube, mixed with 4× SDS-PAGE buffer and heated for 10 minutes at 90°C prior to SDS-PAGE and Western blot analysis.

Cell Culture, Transfection and Immunoprecipitation

HEK293A cells (cat. no. R70507; Thermo Fisher Scientific) were maintained in complete Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 2 mM l-glutamine, 20 mM HEPES, 0.1 mM nonessential amino acids, and 1 mM sodium pyruvate, at 37°C in a humidified incubator containing 5% CO2 and 95% O2. Vectors encoding mApple-CaMKIIα (WT or CA) and mGlu5a (3 µg DNA each) or empty vector controls (3 µg) were cotransfected into one 10-cm dish of 60%–70% confluent HEK293A cells using 3 µl/μg DNA of Fugene 6 (cat. no. E2691; Promega, Madison, WI). About 48 hours later, cells were lysed in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol (DTT), 0.5% NP40 (v/v), 0.5% deoxycholate (v/v), 0.2 mm PMSF, 1 mm benzamidine, 10 μg/ml leupeptin, 10 μm pepstatin, and 1 μm microcystin. Cell lysates were cleared by centrifugation (10 minutes at 12,000g), and a 30 μl sample of the input was saved for SDS-PAGE. The remaining supernatant was incubated at 4°C for 1 hour with rabbit anti-HA antibodies and 20 µl prewashed Dynabeads Protein A (50% v/v; cat. no. 10001D; Thermo Fisher Scientific). Beads were isolated magnetically, washed three times using lysis buffer, and eluted using 2× Laemmli sample buffer for 10 minutes at room temperature prior to SDS-PAGE and Western blotting.

Biotinylation and Cell Surface Expression

Transfected HEK293A cells (see above) were placed on ice, the media were gently removed, and the cells were immediately washed two times using ice-cold phosphate-buffered saline (PBS). Cells were then scraped into ice-cold PBS, transferred to a 1.5-ml tube, centrifuged at 4°C (500g; 3 minutes), and gently resuspended in 1 ml of cold PBS containing 2 mg of EZ-Link sulfo-NHS-SS-biotin (Thermo-Fisher). After gently rocking for 1 hour, excess reagent was quenched by the addition of 50 mM Tris HCl, pH 8.0, and cells were centrifuged and washed again in 1 ml of 50 mM Tris HCl. Cells were then suspended in 1 ml of ice-cold lysis buffer (25 mM Tris HCl, pH 7.4; 150 mM NaCl; 1% NP40; 0.5% sodium deoxycholate containing 0.2 mm PMSF; 1 mm benzamidine; 10 μg/ml leupeptin; and 10 μm pepstatin) and incubated on ice for 30 minutes. Insoluble material was removed by centrifugation (16,000g; 10 minutes, at 4°C), and a 30-μl aliquot of the supernatant was saved for an input sample for SDS-PAGE (Cho et al., 2014). The remaining supernatants were mixed for 1 hour at 4°C with magnetic NeutrAvidin beads (30 μl; 50% slurry; Thermo Fisher Scientific). The beads were separated magnetically and washed three times with lysis buffer. Biotinylated proteins were dissociated from the beads in SDS sample buffer containing 150 mM DTT for 10 minutes at room temperature. The biotinylated and total protein samples were analyzed by Western blotting for mGlu5.

Immunoblotting and Semiquantitative Analysis

Since heating samples results in aggregation of full-length mGlu5 protein, all samples that were blotted for the full-length receptor were incubated for 10 minutes at room temperature before SDS-PAGE. SDS-polyacrylamide gels were transferred to nylon-backed nitrocellulose membranes in 10 mM 3-(cyclohexylamino)propanesulfonic acid buffer. After blocking in Tween Tris-buffered saline [TTBS; 50 mm Tris-HCl, pH 7.5, 0.1% (v/v) Tween 20, 150 mm NaCl] containing 5% nonfat milk, membranes were incubated for either 2 hours at room temperature for purified protein studies or overnight at 4°C in HEK293A cell and brain lysate samples with primary antibodies diluted in TTBS with 5% milk. Membranes were washed five times in TTBS and incubated for 1 hour at room temperature with secondary antibodies conjugated to horseradish peroxidase (HRP) (Promega; or Santa Cruz Biotechnology, Dallas, TX), or infrared (IR) dyes (LI-COR Biosciences, Lincoln, NE) diluted in TTBS with 5% milk. Antibody signals were visualized via enzyme-linked chemiluminescence using the Western Lightning Plus-ECL enhanced chemiluminescent substrate (PerkinElmer, Waltham, MA) and visualized using Premium X-ray Film (Phenix Research Products, Candler, NC). Secondary antibodies conjugated to IR dyes (LI-COR Biosciences) were used for development with an Odyssey System (LI-COR Biosciences). Images were quantified using ImageJ software.

Antibodies

The following antibodies were used for immunoblotting at the indicated dilutions: total CaMKIIα (1:5000; cat. no. MA1-048; Thermo Fisher Scientific) and p-Thr286 CaMKIIα (1:3000; cat. no. sc-12886-R; Santa Cruz Biotechnology); mGlu5-specific antibody (1:3000; cat. no. AB5675; MilliporeSigma, Burlington, MA); rabbit anti-HA (5 μl for immunoprecipitation; cat. no. sc805; Santa Cruz Biotechnology); and goat-GST antibody (1:10,000; cat. no. ab181652; Abcam, Cambridge, UK).

Secondary Antibodies.

Secondary antibodies were as follows: HRP-conjugated anti-rabbit (1:3000; cat. no. W4011; Promega), HRP-conjugated anti-mouse (1:3000; cat. no. W4021; Promega), and HRP-conjugated anti-goat (1:3000; cat. no. sc-2056; Santa Cruz Biotechnology); IR dye–conjugated donkey anti-rabbit 800CW (1:10,000; cat. no. 926–32213; LI-COR Biosciences), and IR dye–conjugated donkey anti-mouse 680LT (1:10,000; cat. no. 926–68022; LI-COR Biosciences).

Mice

CaMKII-KO mice were generated in the Vanderbilt Transgenic Mouse Core as a by-product of published CRISPR/Cas9-mediated experiments directed at creating a knockin E183V mutation of CaMKIIα (Stephenson et al., 2017). We selected a founder containing a deletion of 11 base pairs (bp) (TGCTGAGGAAG) from exon 8, leading to a frame shift and early translational termination. Primers used to genotype the CaMKIIα KO mice are as follows: forward, 5′-GATACCTCTCCCCAGAAGGAC-3′, reverse, 5′-TGCAGTGGTAAGGAGTGGTG-3′ for WT; and forward, 5′-GGACAGTACAACCCCAGCTT-3′, and reverse, 5′-CCCGTACGGGTCCTTCCTCA-3′ for KO, generating a 206-bp band for WT, a 351-bp band for KO, and a 557-bp band for all mice. The CaMKIIα KO was confirmed by immunoblotting brain lysates. All mice were on a mixed B6D2 [C57BL/6J (B6) × DBA/2J (D2)] background and were housed (2–5 per cage) on a 12-hour light/dark cycle with food and water ad libitum. WT and KO experimental mice (littermates) were generated using an HETXHET breeding strategy. All animal procedures were approved by the Vanderbilt University Institutional Animal Care and Use Committee in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Mouse Brain Tissue Preparation and Immunoprecipitation

Both male and female mice (30-60 days old) were anesthetized with isoflurane and decapitated, and forebrains were quickly dissected. Half of a forebrain (cut along the mid-line) was homogenized using at least 20 strokes with a dounce homogenizer in 1.5 ml of an isotonic buffer containing 150 mM KCl, 50 mM Tris-HCl, 1 mm DTT, 1% (v/v) Triton X-100, 1% sodium deoxycholate, 0.2 mM PMSF, 1 mM benzamidine, 10 μg/ml leupeptin, 10 μM pepstatin, and 1 μM microcystin. The homogenate was rotated end over end at 4°C for 30 minutes and then centrifuged at 10,000g for 30 minutes to remove insoluble material. A 30-μl input sample was saved before CaMKIIα (MA1-048) antibody, and 20 μl of magnetic Protein G beads (cat. no. 10003D; Invitrogen) were added to 1 ml of homogenate and rotated end over end for 3–4 hours. Beads were separated magnetically and washed three times with homogenization buffer. Immunoprecipitated complexes were eluted using 2× Lamelli Sample Buffer containing 150 mM DTT for 10 minutes at room temperature and analyzed by immunoblotting.

Ca2+ Imaging in 96-Well Plates

A FlexStation II liquid handler/plate reader (Molecular Devices, Sunnyvale, CA) was used for intracellular Ca2+ measurements in HEK293A cells stably expressing low amounts of the rat mGlu5a receptor (293A-5aLOW cells), as previously described (Hammond et al., 2010; Gregory et al., 2012; Noetzel et al., 2012). The cells were maintained at 37°C in complete DMEM supplemented with 10% FBS, 2 mM l-glutamine, 20 mM HEPES pH 7.5, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, antibiotic/antimycotic solution (Thermo Fisher Scientific), and 500 μg/ml G418 in a humidified incubator containing 5% CO2/95% O2. For experiments, 10-cm dishes were transfected with 3 µg of mApple control vector (mApp) or 3 µg of mApp-CaMKIIα (WT or CA; see above). On the following day, cells were transferred to clear-bottomed, black-walled, poly-d-lysine–coated 96-well plates (BD BioCoat; BD Biosciences, San Jose, CA) (3 × 104 cells/well) in DMEM containing 10% dialyzed FBS, 20 mM HEPES, 1 mM sodium pyruvate, and incubated overnight at 37°C in 5% CO2. Approximately 24 hours later, medium was manually removed and replaced with Hanks’ balanced salt solution containing 20 mM HEPES, 2.5 mM probenecid, and 2 μM Fluo-4/acetoxymethyl ester (AM) dye (pH 7.4), and plates were incubated for 30 minutes (37°C, 5% CO2). This medium was manually removed and replaced with 40 μl of calcium assay buffer (Hanks’ balanced salt solution, 20 mM HEPES, and 2.5 mM probenecid, pH 7.4). Glutamate additions were performed after a 30-second baseline to construct concentration-response curves, and plates were monitored for a total of 120 seconds using an excitation wavelength of 488 nm, an emission wavelength of 525 nm, and a cutoff wavelength of 515 nm. Data were collected with SoftMax Pro (Molecular Devices) then transformed, and agonist concentration-response curves were fitted to a four-parameter logistic equation with GraphPad Prism (GraphPad Software, San Diego, CA). For area under the curve measurements, time parameters were set to measure the area under the curve between the time points of 10–60 seconds to capture the initial Ca2+ peak. Raw values were generated by SoftMax Pro and normalized to the maximum response of control cells.

Single Cell Ca2+ Imaging

HEK293A cells were transiently transfected to express full-length mGlu5a (WT or mutated as indicated: 0.3 µg DNA) and either mApple or mApple-CA-CaMKIIα (3 µg DNA). On the following day, transfected cells were plated in clear glass-bottomed, poly-d-lysine–coated 29 mM dishes (D29-10-1.5-N; Cellvis, Mountain View, CA) (5 × 104 cells) in DMEM containing 10% dialyzed FBS, 20 mM HEPES, 1 mM sodium pyruvate, and 1% penicillin/streptomycin (Thermo Fisher Scientific) and incubated overnight at 37°C in 5% CO2. On the day of the experiment, cells were incubated in media supplemented with 2 µM Fura-2/AM (Thermo Fisher Scientific) for 20 minutes at 37°C in 5% CO2, and then transferred to a Ca2+ imaging solution (150 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM glucose, and 10 mM HEPES; at pH 7.5 and ∼313 mOsm). After incubation for 20 minutes at 37°C in 5% CO2, fluorescence imaging was performed using a Nikon (Tokyo, Japan) Eclipse TE2000-U Microscope equipped with an epifluorescence illuminator (Sutter Instrument, Novato, CA), a charge-coupled device camera (model HQ2; Photometrics, Tucson, AZ), and Nikon Elements software. Cells were perfused at 37°C at a flow rate of 2 ml/min with Ca2+ imaging solution. First, the field of view was imaged using 568-nm excitation to detect cells expressing mApple. Then, ratios of emitted fluorescence (at 510 nm) from mApple-positive cells were measured after excitation at 340 and 380 nm (F340/F380); ratios were measured every 3 seconds for a 1-minute baseline period. Then the cells were treated with 100 µM glutamate (added to the Ca2+ imaging solution) for 10 minutes, during which time the Fura-2 F340/F380 ratios were collected every 3 seconds. Relative changes in Ca2+ levels of the mApple-expressing cells were analyzed using Nikon Elements software. The F340/F380 ratios of each cell were normalized to the first F340/F380 ratio acquired for that cell during the baseline period [fluorescence intensity ratio (F/F0) = (340/380 value)/(baseline 340/380 value)] and then analyzed using Clampfit software (Molecular Devices). Peak Ca2+ responses for all cells were aligned (at 45 seconds), a ROUT test was first used to identify outliers in the maximal ΔF/F0 values for all cells within each experimental group, and then ΔF/F0 values for each time point were averaged together for each dish of cells. The maximal Ca2+ response was defined as the average of all the peak ΔF/F0 values on an experimental day. An average trace for each day of experiments was generated to calculate the half-life of the Ca2+ signal; the decline of the average ΔF/F0 values in each dish were normalized to the average maximal Ca2+ response in that dish and then fitted to a nonlinear one-phase exponential decay fit constrained to y0 = 1 using GraphPad Prism version 6.0. We determined ΔF/F0 and half-lives in five independent experiments (transfections) on separate days (19–124 cells/condition per day) and tested for differences using a Student’s t test. All values are presented as the mean ± S.E.M.

Results

Mouse Forebrain Lysates Contain CaMKIIα-mGlu5 Complexes.

To confirm that mGlu5 specifically associates with CaMKII in the brain, we incubated forebrain lysates from WT or CaMKIIα-KO mice with a CaMKIIα-specific monoclonal antibody or a control IgG. The resulting immune complexes were isolated and then immunoblotted for mGlu5 and CaMKIIα. Note that the mGlu5 antibody used for these studies recognizes an epitope that is shared by the two known mGlu5 splice variants, mGlu5a and mGlu5b, which are differentially expressed during development (Minakami et al., 1995; Romano et al., 1996). Input samples from WT and CaMKIIα KO tissue prepared in parallel contain similar levels of the monomeric and dimeric forms of mGlu5 (Fig. 1), although the ratio of monomeric and dimeric species varied between independent experiments (data not shown). CaMKIIα complexes isolated from WT mouse forebrain contained both monomeric and dimeric forms of mGlu5, with the ratio of these forms reflecting variability in the ratio detected in the inputs. However, very little mGlu5 could be detected in IgG control complexes isolated from WT tissue, or in CaMKIIα complexes isolated from CaMKIIα KO tissue (Fig. 1). Thus, mGlu5 is a bona fide component of the CaMKIIα complexes present in mouse brain lysates.

Fig. 1.
Fig. 1.

Coimmunoprecipitation of mGlu5 with mouse forebrain CaMKIIα. Solubilized fractions from WT or CaMKIIα KO mouse forebrain were immunoprecipitated (IP) using CaMKIIα-specific (α) or control (IgG) antibodies, as indicated. Inputs and immune complexes were analyzed by immunoblotting: mGlu5 was detected only in immune complexes isolated from WT tissue using the CaMKIIα antibody. Open and closed arrowheads indicate dimeric and monomeric species of mGlu5. The figure is representative of three similar experiments.

CaMKIIα Directly Binds to the mGlu5 C-Terminal Domain.

Like most prior studies, we chose to use the mGlu5a splice variant for our molecular studies. It was previously reported that residues 827–964 of the mGlu5a-CTD bind to inactive CaMKIIα, but that CaMKII autophosphorylation disrupted the interaction (Jin et al., 2013b). To confirm this finding, we generated a GST-tagged mGlu5a-CTD construct containing residues 827–964 (GST-mGlu5a-CTD) for use in glutathione agarose cosedimentation experiments. Initial studies detected weak binding of inactive CaMKIIα to GST-mGlu5a-CTD that was not consistently above background binding to a GST negative control (data not shown). Therefore, we systematically tested interactions of GST-mGlu5a-CTD with CaMKIIα in various activation states.

Purified CaMKIIα was autophosphorylated in the presence of Ca2+/CaM in vitro at either 4°C or 30°C. Similar total levels of Thr286 autophosphorylation were detected by immunoblotting after incubation at either 4°C or 30°C (Fig. 2A), but the 30°C autophosphorylation reduced the electrophoretic mobility of CaMKIIα. These observations are consistent with those of prior studies showing that incubation at 4°C results in selective Thr286 autophosphorylation (McNeill and Colbran, 1995), whereas incubation at 30°C allows for extensive autophosphorylation at several other sites (Baucum et al., 2015).

Fig. 2.
Fig. 2.

CaMKII autophosphorylation at Thr286 enhances binding to the mGlu5 CTD. (A) Autophosphorylation (Autophos) of purified CaMKIIα. Purified CaMKIIα was incubated with Mg(C2H3O2)2, CaCl2, CaM, and ATP for either 90 seconds at 4°C or 10 minutes at 30°C, and samples were immunoblotted for total or phospho-Thr286 (p-Thr286) CaMKII. Although quantitative analysis (right) indicated that there was a similarly robust Thr286 autophosphorylation using these two conditions, the 10-minute/30°C incubation resulted in a substantial reduction in electrophoretic mobility due to phosphorylation at additional unidentified sites. Data are plotted as the mean ± S.E.M. (n = 3) and analyzed using a one-way ANOVA (P = 0.0167, F = 8.746, R2 = 0.7446) with Sidak’s post hoc test for multiplicity adjusted P values: control (Ctrl) vs. 4°C, P = 0.031; control vs. 30°C, P = 0.035; 4°C vs. 30°C, P = 1.00. (B) The GST-mGlu5a-CTD binds CaMKIIα after selective autophosphorylation at Thr286. GST-mGlu5a-CTD was incubated with purified CaMKIIα that had been preincubated as in (A), and complexes were isolated using glutathione agarose. Immunoblot analyses revealed that CaMKIIα binding to the CTD was strongly enhanced by selective Thr286 autophosphorylation at 4°C, but that the autophosphorylation of additional sites on CaMKII at 30°C substantially reduced binding. Data are plotted as the mean ± S.E.M. (n = 3) and were analyzed using a one-way ANOVA (P = 0.005, F = 2.477, R2 = 0.829) with Sidak’s post hoc test for multiplicity-adjusted P values: control vs. 4°C, P = 0.009; 4°C vs. 30°C, P = 0.011; control vs. 30°C, P = 1.00. (C) Binding of activated CaMKIIα to GST-mGlu5a-CTD is disrupted by Ca2+/CaM. Purified CaMKIIα was autophosphorylated for 90 seconds at 4°C [see (A)] and then incubated with GST-mGlu5a-CTD in the absence or presence of excess Ca2+/CaM (see Materials and Methods). Complexes were isolated using glutathione-agarose and then immunoblotted as indicated. Data are plotted as the mean ± S.E.M.; excess Ca2+/CaM significantly reduced CaMKIIα binding (P < 0.0001 relative to a theoretical value of 1.00, one-sample t test; n = 4). In all panels, the symbols *, ** and *** indicate P< 0.05, 0.01 and 0.001, respectively, with n.s. indicating non-significant (P>0.05).

We then performed glutathione-agarose cosedimentation experiments to test the interaction of GST-mGlu5-CTD with CaMKIIα in these different activation states (after terminating the autophosphorylation reactions by chelating metal ions with excess EGTA and EDTA). The selective Thr286-autophosphorylation (4°C) protocol resulted in a robust enhancement of CaMKIIα binding to GST-mGlu5-CTD relative to the nonphosphorylated kinase, but this interaction was substantially reduced after more extensive in vitro phosphorylation at 30°C (Fig. 2B). The short exposure times used for the development of these immunoblots failed to detect weak binding of inactive CaMKIIα to GST-mGlu5-CTD. In combination, these data show that although activation and Thr286 autophosphorylation of CaMKIIα strongly enhance binding to the mGlu5a-CTD, the interaction can be reduced by autophosphorylation at additional non-Thr286 sites.

Binding of Activated CaMKIIα to the GST-mGlu5a-CTD Is Disrupted by Ca2+/CaM.

Ca2+/CaM binds to residues 889–917 within the CTD of mGlu5a with important functional consequences (Minakami et al., 1997; Lee et al., 2008; Choi et al., 2011). Moreover, it was previously reported that excess Ca2+/CaM disrupts the binding of inactive CaMKIIα to the mGlu5a-CTD (Jin et al., 2013b). Therefore, we tested whether excess Ca2+/CaM also disrupts the binding of activated CaMKIIα to GST-mGlu5a-CTD. Thr286-autophosphorylated CaMKIIα (4°C protocol) robustly binds to GST-mGlu5a-CTD, as noted above, but this interaction was essentially eliminated by the inclusion of excess Ca2+/CaM in the binding assay (Fig. 2C). Thus, binding of activated CaMKIIα to the mGlu5a CTD is also blocked by Ca2+/CaM, suggesting that multiple Ca2+-sensitive proteins are involved in the regulation of mGlu5 signaling.

Identification of a CaMKIIα-Binding Determinant in the mGlu5 CTD.

As an initial approach to identify key CaMKIIα-binding determinants in the mGlu5a CTD, we compared residues 827–964 of mGlu5 with CaMKIIα-binding domains that have been previously identified in other proteins. Our laboratory recently showed that activated CaMKIIα binds to the N-terminal domains of CaV1.2 and CaV1.3 L-type voltage-gated Ca2+ channels, and that this interaction is disrupted by the mutation of three basic residues (Arg83-Lys-Arg85) to alanine (Wang et al., 2017). Similar tribasic residue motifs are also present within CaMKIIα-binding domains that have been previously identified in the intracellular loops of the D2 dopamine receptor (D2R) and D3 dopamine receptor (D3R) (Liu et al., 2009; Zhang et al., 2014) and the mGlu1-CTD (Jin et al., 2013a). Notably, the CaMKIIα-binding fragment of the mGlu5a-CTD also contains a tribasic residue motif (residues Lys866-Arg867-Arg868) (Fig. 3A). We found that substituting alanines for Lys866-Arg867-Arg868 in the mGlu5a-CTD essentially abolished the binding of activated CaMKIIα to GST-mGlu5-CTD in vitro (Fig. 3B). These data identify a key determinant for CaMKII binding to the CTD of mGlu5a.

Fig. 3.
Fig. 3.

Identification of CaMKII-binding determinants in the mGlu5a-CTD. (A) Alignment of part of the mGlu5a-CTD with amino acid sequences surrounding known CaMKII-binding domains. Tribasic residue motifs (highlighted with blue asterisks above) were identified within CaMKII-binding domains from other proteins, as well as within the CaMKII binding fragment in the CTD of mGlu5a. Mutation of R83K84R85 to AAA in the CaV1.3 N-terminal domain disrupts the binding of CaMKII (Wang et al., 2017). The red and blue fonts indicate residues in each domain that are identical and homologous, respectively, with residues in the mGlu5a sequence. Underlined residues in the mGlu1-CTD and the D2R and D3R (IL3, third intracellular loop) demark the sequences of synthetic peptides that were shown to compete for CaMKII binding (Jin et al., 2013a; Zhang et al., 2014). (B) Mutation of the tribasic residue motif in the mGlu5a-CTD disrupts CaMKII binding. Thr286 autophosphorylated CaMKIIα (90 seconds/4°C protocol) was incubated with GST-mGlu5a-CTD (WT or with a K866R867R868 to AAA mutation), and complexes were analyzed as in Fig. 1. The K866R867R868/AAA mutation essentially abolishes CaMKII binding. Data are plotted as the mean ± S.E.M. (P = 0.003, one-sample t test; n = 4). In panel B, the symbol ** indicates P<0.01.

CaMKIIα Activation Increases CaMKIIα-mGlu5a Association in Heterologous Cells.

To better understand the interaction of CaMKIIα with full-length mGlu5a we conducted coimmunoprecipitation experiments from lysates of transfected HEK293A cells. We first tested the hypothesis that CaMKIIα activation would increase the association with full-length mGlu5a, as with in vitro binding of CaMKIIα to GST-mGlu5a-CTD. We expressed mApple-tagged WT CaMKIIα in the absence or presence of mGlu5a with an N-terminal HA-epitope tag in HEK293A cells. Prior to HA immunoprecipitation, the cell lysates were split into two aliquots and preincubated with either excess EGTA and EDTA or with Ca2+/CaM, Mg2+, ATP, and phosphatase inhibitors to stimulate CaMKIIα autophosphorylation. CaMKII activation in the lysates resulted in a robust increase in autophosphorylation at Thr286, without the large shift in electrophoretic mobility that was observed after autophosphorylation of purified CaMKIIα at 30°C (Fig. 4A). HA-immunoprecipitation from the two preincubated lysates yielded similar amounts of the monomeric and dimeric species of HA-mGlu5a, but CaMKIIα activation resulted in a statistically significant ∼3-fold increase in the amount of coimmunoprecipitated CaMKIIα (Fig. 4A). These data show that full-length mGlu5a preferentially interacts with activated WT CaMKIIα.

Fig. 4.
Fig. 4.

Role of the CTD in full-length mGlu5a binding to activated CaMKIIα. (A) CaMKII activation enhances interaction with full length mGlu5. Solubilized fractions of HEK293A cells expressing HA-tagged mGlu5a and/or mApple-tagged WT CaMKIIα (as indicated above lanes) were pre-incubated with Ca2+/CaM, MgAc2, and ATP in the presence or absence of excess EDTA (± activation, respectively) and then immunoprecipitated (IP) using antibodies to the HA epitope. Lysates and immune complexes were analyzed by immunoblotting, as indicated. CaMKIIα activation results in robust Thr-286 autophosphorylation, which increases CaMKIIα association with HA-mGlu5a. Data are plotted as the mean ± S.E.M. (P = 0.043, one-sample t test; n = 4). Open and closed arrowheads indicate dimeric and monomeric species of mGlu5a. (B) CaMKII association with full-length mGlu5a is disrupted by mutation of the CTD tribasic residue motif. Solubilized fractions of HEK293A cells expressing HA-mGlu5 (WT or with the K866R867R868/AAA mutation) and mApple-tagged CA-CaMKIIα were immunoprecipitated using antibodies to the HA epitope. Lysates and the immune complexes were analyzed by immunoblotting, as indicated. The K866R867R868/AAA mutation reduced the association of CA-CaMKIIα with HA-mGlu5. Data are plotted as the mean ± S.E.M. (P = 0.028, one sample t test; n = 4). p-Thr286, phospho-Thr286. In the bar graphs for both panels, * indicates P<0.05.

Association of Activated CaMKIIα with Full-Length mGlu5a Requires Arg83-Lys-Arg85.

We next investigated whether the association of activated CaMKIIα with full-length mGlu5a involves the CTD. To avoid complications that might arise from preincubating cell lysates to activate WT-CaMKIIα, we used an mApple-tagged CA-CaMKIIα (mApple-CA-CaMKIIα); the phosphomimetic T286D mutation results in constitutive CaMKIIα activity, and the phospho-null T305A/T306A mutations prevent CaMKIIα phosphorylation at these sites, which interferes with the binding of Ca2+/CaM and α-actinin (Jalan-Sakrikar et al., 2012). The mApple-CA-CaMKIIα was expressed alone, or coexpressed with either HA-mGlu5a or HA-mGlu5a-AAA (with Lys866-Arg867-Arg868 mutated to alanines). HA-immunoprecipitation from cell lysates confirmed a robust association of mApple-CA-CaMKIIα with WT mGlu5a that was partially (∼50%) reduced by the triple alanine mutation in the CTD (Fig. 4B). These data demonstrate that the Lys866-Arg867-Arg868 residues in the mGlu5a-CTD play an important role in the association of activated CaMKIIα with the full-length mGlu5 receptor.

CaMKIIα Increases Basal mGlu5a Surface Expression.

Since the CTD is known to modulate mGlu5 cell-surface expression and consequently mGlu5 signaling, we investigated the effect of CaMKIIα on the cell-surface expression of full-length mGlu5a. Intact HEK293A cells expressing mGlu5 with or without mApple-CA-CaMKIIα were incubated with sulfo-NHS-SS-biotin to biotinylate all surface-expressed proteins. Streptavidin-conjugated magnetic beads were then used to isolate cell-surface proteins from cell lysates. Immunoblotting of total cell lysates and isolated cell-surface proteins revealed that the coexpression of mApple-CA-CaMKIIα increased the proportion of mGlu5a expressed on the cell surface by 3.0 ± 0.7-fold (S.E.M.) under basal conditions (P = 0.036; one-sample t test vs. hypothetical value of 1) (Fig. 5). To determine whether CaMKIIα interaction with the mGlu5a-CTD is important for this effect, we examined the cell-surface expression of mGlu5a-AAA. In the absence of coexpressed CaMKII, the surface expression of mGlu5a-AAA was not significantly different from those of WT mGlu5a [1.6 ± 0.6-fold (S.E.M.); n = 5; P = 0.35; one-sample t test vs. hypothetical value of 1]. Moreover, the coexpression of mApple-CA-CaMKIIα had no effect on cell-surface expression of mGlu5a-AAA. These data demonstrate that interaction with the mGlu5a-CTD is necessary for CaMKIIα-mediated increases in mGlu5a cell-surface expression.

Fig. 5.
Fig. 5.

CaMKII enhances the cell-surface expression of mGlu5a via interaction with the CTD. Cell-surface biotinylation analyses of HEK293A cells expressing mGlu5a (WT or with K866R867R868/AAA mutation in the CTD) with either mApple or mApple-tagged CA-CaMKIIα. The coexpression of CA-CaMKIIα increased steady-state surface expression levels of WT mGlu5a (P = 0.036, one-sample t test; n = 6), but not of the K866R867R868/AAA mutant. Data are plotted as the mean ± S.E.M. (P = 0.569, one-sample t test; n = 5). *P<0.05; n.s., non-significant.

CaMKIIα Reduces mGlu5a-Stimulated Peak Ca2+ Mobilization.

To investigate the effect of CaMKIIα on mGlu5a signaling, we measured glutamate-induced Ca2+ mobilization in populations of 293A-5aLOW cells that stably express mGlu5a and were transiently transfected to coexpress mApple or mApple-tagged CaMKIIα (either WT or CA). A similar fraction of the total cells expressed detectable levels of mApple-tagged WT- or CA-CaMKIIα in each transfection (typically ∼60%). After loading glutamate-starved cells with Fluo-4-AM, a fluorescent Ca2+ indicator, we measured fluorescence responses of total cell populations to increasing glutamate concentrations (0.01–100 µM) (Fig. 6A). An overlay of raw traces from cells expressing mApple, mApple-WT-CaMKIIα, or mApple-CA-CaMKIIα in a representative experiment is shown in Fig. 6B. Peak Ca2+ responses (increased fluorescence) at each glutamate concentration were expressed as a ratio to the maximum response to a saturating concentration of glutamate (100 µM) in mApple-expressing control cells for each individual experiment, and then data were averaged across five independent experiments. Glutamate increased the peak fluorescence in a concentration-dependent manner, with an apparent EC50 value of 0.38 ± 0.03 µM in control cells, similar to previous analyses (Schoepp et al., 1999; Hammond et al., 2010). The glutamate response was unaffected by the coexpression of mApple-WT-CaMKIIα, but the coexpression of mApple-CA-CaMKIIα reduced peak Ca2+ responses at the highest concentrations of glutamate by approximately 20%, without affecting the apparent EC50 value (Fig. 6C). As an alternative measure of Ca2+ responses, we determined the area under the curve of the initial Ca2+ peak at the highest glutamate concentration. There was no difference in the area under the curve between cells expressing mApple or mApple-CaMKIIα-WT, but the coexpression of mApple-CA-CaMKIIα significantly reduced the area under the curve (control, 109.2 ± 2.7; WT, 101.3 ± 7.4; CA, 82.6 ± 4.1; one-way analysis of variance (ANOVA), P = 0.011, F = 6.280; Sidak’s post hoc test for multiplicity–adjusted P values: WT vs. control, P = 0.51; CA vs. control, P = 0.0073) (data not shown). Since mApple-CA-CaMKIIα is expressed in only a fraction of the cell population in each well, the measured reductions in maximal Ca2+ responses presumably underestimate the actual impact of expressing CA-CaMKIIα in each cell. However, these data cannot differentiate whether this effect reflects decreased Ca2+ mobilization within each cell or a decrease in the fraction of responsive cells. Nevertheless, the data indicate that the coexpression of CA-CaMKIIα, but not WT-CaMKIIα, can reduce mGlu5a-stimulated peak Ca2+ mobilization.

Fig. 6.
Fig. 6.

CaMKIIα regulates mGlu5a-stimulated Ca2+ mobilization in 293A-5aLOW cells. Time courses of intracellular Ca2+ responses to glutamate were measured by changes in Fluo-4 fluorescence in stable 293A-5aLOW cells in 96-well plates. (A) Time courses of Ca2+ responses. Example of calcium responses to increasing glutamate concentrations was collected in a row of eight wells. (B) Overlay of individual Ca2+ responses to increasing concentrations of glutamate [labeled by colors in (A)] from 293A-5aLOW cells transiently transfected to express mApple control, mApple-CaMKIIα-WT, or mApple-CA-CaMKIIα from a representative experiment. (C) Concentration-response curves. Initial peak Ca2+ responses (ΔF/F0) at each concentration were normalized to the maximal glutamate-stimulated response in control (mApple-transfected) cells within each experiment. Normalized Ca2+ responses are plotted as the mean ± S.E.M. (n = 5 experiments) as a function of glutamate concentration. The expression of CaMKIIα-WT had no impact on the Ca2+ response curve, but the expression of CA-CaMKIIα reduced peak Ca2+ responses (multiple-comparisons two-way ANOVA: sources of variation: CaMKII, P < 0.0001; interaction, P = 0.029. Tukey’s post hoc test for multiplicity–adjusted P values: mApple vs. WT, P = 0.926; mApple vs. CA-CaMKIIα, P < 0.0001; WT vs. CA-CaMKIIα, P = 0.0002). The inset table shows the maximum response (Max), EC50 (µM), and Hill coefficient (±S.E.M.) obtained by fitting the data in GraphPad Prism. In panel C, *** and **** indicate P<0.001 and 0.0001, respectively.

CaMKIIα Prolongs mGlu5a-Mediated Ca2+ Signaling.

To address caveats associated with studies investigating the effects of mApple-CA-CaMKIIα on Ca2+ mobilization in 293A-5aLOW cells, we also examined Ca2+ mobilization in single HEK293A cells transfected to express full-length mGlu5a with either mApple alone (control) or mApple-CA-CaMKIIα. After loading all cells with Fura-2/AM, a ratiometric Ca2+ indicator, single cells were selected for analysis based on the presence of mApple as a marker of transfection. Application of 100 µM glutamate to cells coexpressing soluble mApple or mApple-CA-CaMKIIα with WT mGlu5a produced an initial peak of Fura-2 fluorescence followed by highly variable changes of fluorescence over the next 10 minutes (Fig. 7A). In a majority of cells in each group (53%–68%) Ca2+ signals waned over time, sometimes with a secondary shoulder, but subpopulations of the cells displayed clear Ca2+ oscillations that either returned to baseline between oscillations (10%–21%) or were superimposed on a more sustained Ca2+ elevation (18%–25%) (Supplemental Fig. 1, A–C). However, the percentage of WT mGlu5a cells exhibiting Ca2+ oscillations was unaffected by the coexpression of mApple-CA-CaMKIIα (Supplemental Fig. 1D). Since it is unclear whether oscillating and nonoscillating cell responses cause different physiologic effects, we developed an approach to analyze the responses of all cells (both oscillating and nonoscillating) across five independent experiments, revealing that the initial peak fluorescence was significantly reduced (P = 0.009) in cells expressing mApple-CA-CaMKIIα versus cells expressing mApple alone (Fig. 7B), consistent with data from stably transfected cell populations (Fig. 6). Moreover, the Ca2+ signal was relatively prolonged in cells expressing mApple-CA-CaMKIIα versus cells expressing mApple alone, as reflected by a statistically significant increase in the half-life of the fluorescence signal (Fig. 7A, inset; Fig. 7C). We also analyzed responses in subsets of the cells within each population that exhibited at least three baseline Ca2+ oscillations (Supplemental Fig. 2A). There was no statistically significant difference in the total number of mGlu5a-mediated Ca2+ oscillations between transfection conditions (Supplemental Fig. 2B). However, coexpression of mApple-CA-CaMKIIα reduced the relative rate of decay of peak Ca2+ signals in successive oscillations (Supplemental Fig. 2C). Coexpression of mApple-CA-CaMKIIα also increased the frequency of Ca2+ oscillations, as reflected by a reduction of the interevent intervals (Supplemental Fig. 2D). In combination, these data confirm that CaMKIIα can reduce the amplitude of initial mGlu5a-dependent Ca2+ mobilization and indicate that the relative duration of Ca2+ signals is extended, with increases of the frequency of oscillations when they are present.

Fig. 7.
Fig. 7.

CaMKIIα binding to the CTD is required for the modulation mGlu5a-stimulated Ca2+ mobilization. HEK293A cells were transiently transfected to express mGlu5a (WT or K866RR868/AAA) with either mApple or mApple-CA-CaMKIIα for single-cell Fura-2 Ca2+ imaging (see Materials and Methods). Representative data from a single experiment. Averaged normalized changes in fluorescence from 58 to 114 cells (ΔF/F0, mean ± S.E.M.) expressing mGlu5a-WT (A) or mGlu5-K866R867R868/AAA (D) in the presence (blue lines) or absence (red lines) of mApple-CA-CaMKIIα. The inset graphs show line fits for time courses of the decline of Ca2+ signals from the peak ΔF/F0 under each condition. (B, C, E, and F) Summary data. The bar graphs depict mean ± S.E.M. values for peak Ca2+ signals (ΔF/F0) (B and E) and half-lives for the decline in Ca2+ signals (C and F) with superimposed data points from each experiment (n = 5). The expression of constitutively active mApple-CA-CaMKII decreases the peak Ca2+ signal but increases the half-life of the Ca2+ signal with mGlu5a-WT [(B) P = 0.009; (C) P = 0.001], but has no significant effect on the mGlu5a-K866R867R868/AAA mutant that disrupts CaMKII binding to the CTD [(E), P = 0.155; (F), P = 0.415]. Paired Student’s t tests were used for statistical comparisons in each panel. Max, maximal. In all panels, ** indicates P<0.01 and n.s. indicates non-significant.

To test the hypothesis that CaMKIIα binding to the mGlu5a-CTD is necessary for the modulation of Ca2+ mobilization, we examined the effect of coexpressing mApple-CA-CaMKIIα with mGlu5a-AAA (Fig. 7D), The replacement of Lys866-Arg867-Arg868 in the CTD with alanines had little effect on glutamate-stimulated Ca2+ mobilization in cells expressing mApple. Moreover, the coexpression of mApple-CA-CaMKIIα with mGlu5a-AAA had no statistically significant effect on either the initial peak (Fig. 7E) or the duration (Fig. 7F) of the glutamate-stimulated Ca2+ signal relative to control cells expressing mApple alone. Furthermore, the CTD mutation had no statistically significant effect on the responses of cells displaying baseline Ca2+ oscillations (Supplementary Fig. 1D) but abrogated the CaMKII-dependent modulation, as reflected by a lack of effect on the peak height decay of successive Ca2+ oscillations (Supplemental Fig. 2C) and the Ca2+ oscillation frequency (Supplemental Fig. 2D). These data indicate that binding to the mGlu5a-CTD is important for both the increase of initial peak Ca2+ signals and for the prolonged Ca2+ signaling induced by coexpression of CA-CaMKIIα.

Discussion

In a previous report (Jin et al., 2013b), the membrane proximal region of the mGlu5a-CTD was shown to bind inactive CaMKII. Here we extend these findings by further characterizing the physical and functional relationship between these key regulators of synaptic transmission. We confirmed that CaMKIIα and mGlu5 specifically interact in mouse brain. However, our data show that mGlu5a-CTD residues 827–964 bind more strongly to CaMKIIα in an active, Thr286-autophosphorylated conformation, but that this interaction is disrupted by excess Ca2+/CaM or by robust CaMKII autophosphorylation at additional undefined sites. Furthermore, our data indicate that CaMKII binding to the CTD exerts complex effects on mGlu5a surface expression and downstream Ca2+ mobilization.

There is a growing appreciation that specific physiologic actions of CaMKII are modulated in part through dynamically regulated interactions with CaMKII-associated proteins (CaMKAPs). Several CaMKAPs preferentially interact with activated conformations of CaMKII; these CaMKAPs can be subclassified based on differences between the amino acid sequences of their CaMKII-binding domains. CaMKII-binding domains in the N-methyl-d-aspartate receptor GluN2B subunits and calcium channel β1 and β2 subunits resemble the CaMKII regulatory domain (Strack et al., 2000; Grueter et al., 2008). In contrast, the amino acid sequence of a CaMKII-binding domain in densin has similarity with a naturally occurring CaMKII inhibitor protein (Jiao et al., 2011). Here, we show here that the binding domain for activated CaMKII in the mGlu5a-CTD does not resemble these CaMKAPs. Rather, this novel interaction requires three basic residues (Lys866-Arg867-Arg868), similar to the recently identified interaction of activated CaMKII with the N-terminal domains of L-type voltage-gated Ca2+ channels (Wang et al., 2017). Interestingly, triple basic residue motifs can also be identified in CaMKII-binding domains of other G protein–coupled receptors, including intracellular loops of the Gαi-coupled D2R and D3R (Liu et al., 2009; Zhang et al., 2014) and the CTD of the mGlu1 receptor (Jin et al., 2013a,b), which also couples to Gαq/11 (Fig. 3). Thus, it will be interesting to investigate the role of these triple basic residue motifs in CaMKII binding to additional G protein–coupled receptors.

One unusual aspect of CaMKII binding to the mGlu5a-CTD is that, whereas the in vitro interaction requires CaMKIIα activation and Thr286 autophosphorylation, additional autophosphorylation at non-Thr286 sites after incubation at 30°C reduces the binding. Our recent proteomics analyses of purified CaMKIIα autophosphorylated in vitro using a similar 30°C protocol detected 17 autophosphorylation sites, in addition to Thr-286 (Baucum et al., 2015). Presumably, the autophosphorylation at one or more of these non-Thr286 sites interferes with in vitro CaMKIIα binding to mGlu5a. Although this is a potentially interesting finding, parallel proteomics analyses of CaMKII isolated from mouse brain failed to detect phosphorylation at many of these in vitro sites (Baucum et al., 2015). However, it is possible that this observation explains why Jin et al. (2013b) found that autophosphorylated CaMKII did not bind to mGlu5a in vitro because their autophosphorylation reactions were incubated at 30°C.

Our data show that CaMKIIα activation enhances the association with full-length mGlu5a, and that this interaction involves the Lys866-Arg867-Arg868 motif in the CTD (Fig. 3). However, triple alanine substitution of CTD residues 866–868 reduced the interaction by only ∼50%, suggesting that CaMKII may interact with additional regions in mGlu5a or bind to the receptor through an indirect interaction. Indeed, a CaMKII interaction with the second intracellular loop of mGlu5 has been reported previously (Raka et al., 2015), although we have been unable to detect direct binding of purified CaMKII to a GST fusion protein containing the mGlu5 second intracellular loop (data not shown). Although our data cannot preclude a role for a secondary or indirect interaction, our analyses in heterologous cells indicate that CaMKIIα interaction with the CTD is critical for several novel functional effects of CaMKII on mGlu5a signaling. First, we show here that CaMKIIα can increase cell-surface expression of mGlu5a. Second, we found that CaMKIIα has complex effects on mGlu5a-dependent Ca2+ mobilization. As noted previously, mGlu5 activation can induce temporally diverse intracellular Ca2+ responses in heterologous cells and in neurons (Flint et al., 1999; Mao and Wang, 2003; Kim et al., 2005; Uematsu et al., 2015; Jong and O’Malley, 2017). The coexpression of CaMKII had little effect on the proportion of cells exhibiting different oscillatory or nonoscillatory response patterns (Supplementary Fig. 1). However, we found that the coexpression of CA-CaMKIIα reduces the amplitude of the initial peak Ca2+ signals (Fig. 6C; Fig. 7B) but prolongs the duration of the Ca2+ signals (Fig. 7C; Supplementary Fig. 2C) in either the total responding cell population or only in cells that exhibit baseline Ca2+ oscillations. The coexpression of CA-CaMKIIα also increases the frequency of baseline Ca2+ oscillations (Supplementary Fig. 2D). All of these effects are prevented by the triple alanine substitution for Lys866-Arg867-Arg868 in the CTD (Fig. 7, D–F; Supplementary Fig. 2, C and D). Presumably, the effect of CaMKIIα to increase basal cell-surface expression contributes to the prolongation of Ca2+ signaling, but the mechanisms underlying the reduced initial peak Ca2+ signal, observed in both stable 293A-5aLOW cell populations and in single transiently transfected cells, remains unclear. Taken together, our data show that binding of CaMKIIα can play an important role in modulating cellular responses to mGlu5a activation. Further examination into the contribution of these mechanisms in synaptic plasticity and neuronal Ca2+ signaling are warranted in future studies.

Interestingly, cell-surface expression of mGlu5a is also modulated by direct binding of Ca2+/CaM to the CTD, similar to the effects of CaMKIIα binding to the CTD reported herein, and Ca2+/CaM also prolongs mGlu5-mediated Ca2+ signaling (Lee et al., 2008). The Ca2+/CaM binding domain involved in mediating these effects is located 30–40 residues C-terminal to the tribasic residue motif that is critical for CaMKII binding. Nevertheless, we found that Ca2+/CaM competes for the binding of activated CaMKII to the mGlu5a-CTD in vitro (Fig. 2C). Taken together, our data suggest an intriguing model in which the binding of CaM might confer a relatively transient Ca2+-dependent modulation of mGlu5a surface expression and signaling, but that increased CaMKIIα autophosphorylation at Thr286 would result in sustained binding to the CTD and longer-term modulation of mGlu5a surface expression and Ca2+ mobilization. Since Thr286 autophosphorylation of CaMKII is sensitive to changes in the source, duration, or frequency of Ca2+ signals originating from multiple channels (Pasek et al., 2015), such as those occurring during synaptic plasticity, as well as to the regulated activities of protein phosphatases, this may provide a mechanism for cross talk with other signaling pathways.

As noted above, CaMKII has also been shown to interact with a membrane-proximal region in the CTDs of mGlu1 (Jin et al., 2013a), and the CaMKII-binding domain in mGlu1 contains a tri-basic residue motif, similar to the motif we have identified here as being critical for CaMKII binding to the mGlu5a-CTD. However, CaMKII was shown to desensitize mGlu1 signaling, whereas we found that CaMKII prolongs mGlu5 signaling. This apparently differential modulation of mGlu1 and mGlu5 by CaMKII may contribute to their distinct neuronal roles (Mannaioni et al., 2001; Valenti et al., 2002; Volk et al., 2006). Interestingly, the effects of CaMKII on mGlu1 signaling are mediated in part by phosphorylation at Thr871, which lies within the CaMKII-binding domain. Therefore, it will be interesting to investigate whether phosphorylation is required for the effects of CaMKII on mGlu5 signaling, as well as the physical interaction demonstrated here.

The effects of CaMKIIα on mGlu5a must also interface with the known modulation of mGlu5 signaling by other mechanisms. Prior studies have shown that several protein kinases modulate mGlu5 via the CTD. For example, PKC phosphorylates Ser901 in the mGlu5a-CTD to inhibit Ca2+/CaM binding and antagonize the aforementioned modulation by Ca2+/CaM (Lee et al., 2008). In addition, PKA phosphorylates Ser870 in mGlu5a, prolonging Ca2+ mobilization, similar to the effects of CaMKII reported here, and enhancing ERK activation (Uematsu et al., 2015). However, it was previously reported that CaMKII reduces mGlu5-stimulated ERK1/2 activation and increases agonist-induced mGlu5 internalization (Raka et al., 2015). It is possible that the enhanced agonist-induced internalization in part results from the increased basal surface expression reported herein (Fig. 5). Although the mechanistic relationships between these different modes of mGlu5 regulation remain to be more clearly established, the convergence of Ca2+/CaM, CaMKII, PKA, and PKC actions within an ∼60–amino acid region in the long CTD (345 amino acids) suggests that the actions of mGlu5 are tightly controlled across different time frames, presumably fine-tuning neuronal responses such as different forms of synaptic plasticity.

Acknowledgments

We thank Hyekyung Plumley Cho for help with Flex Station experiments, and Dr. David A. Jacobson and Prasanna Dadi for providing access to equipment for the single-cell Ca2+ imaging and for helpful discussions. We also thank Drs. David Piston and Winship Herr for generously providing various plasmids, as detailed in the text.

Authorship Contributions

Participated in research design: Marks, Shonesy, Wang, Niswender, Colbran.

Conducted experiments: Marks.

Contributed new reagents or analytic tools: Marks, Wang, Stephenson, Niswender.

Performed data analysis: Marks, Shonesy.

Wrote or contributed to the writing of the manuscript: Marks, Shonesy, Colbran.

Footnotes

    • Received May 29, 2018.
    • Accepted September 19, 2018.

  • This work was supported by grants from the National Institutes of Health [R01-MH-063232] and [R01-NS-078291] to R.J.C., [F31-MH-109196] to C.R.M., [T32-DK-07563] to C.R.M.; and the American Heart Association [14PRE18420020] to X.W. and [15PRE25110020] to C.R.M. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or other funding agencies. CRISPR/Cas9 pronuclear injections were performed by the Vanderbilt University School of Medicine Transgenic Mouse/ES Cell Shared Resource, which was supported through Cancer Center Support Grant CA68485, the Vanderbilt Diabetes Research and Training Center [DK020593], and the Center for Stem Cell Biology.

  • https://doi.org/10.1124/mol.118.113142.

  • ↵Embedded ImageThis article has supplemental material available at molpharm.aspetjournals.org.

Abbreviations

AM
acetoxymethyl ester
ANOVA
analysis of variance
bp
base pair
CA
constitutively active
CA-CaMKII
constitutively active Ca2+/calmodulin-dependent protein kinase II
CaM
calmodulin
CaMKII
Ca2+/calmodulin-dependent protein kinase II
CaMKAP
Ca2+/calmodulin-dependent protein kinase II–associated protein
CTD
C-terminal domain
D2R
D2 dopamine receptor
D3R
D3 dopamine receptor
DMEM
Dulbecco’s modified Eagle’s medium
DTT
dithiothreitol
FBS
fetal bovine serum
F/F0
fluorescence intensity ratio
GST
glutathione S-transferase
HA
hemagglutinin
HEK293A
human embryonic kidney 293A
HRP
horseradish peroxidase
IR
infrared
KO
knockout
mApp
mApple control vector
mGlu
metabotropic glutamate receptor
PBS
phosphate-buffered saline
PKA
protein kinase A
PKC
protein kinase C
PMSF
phenylmethane sulfonyl fluoride
sulfo-NHS-SS-biotin
sulfosuccinimidyl 2-(biotinamido)-ethyl-1,3-dithiopropionate
TTBS
Tween Tris-buffered saline
WT
wild type

References

  1. ↵
    1. Ade KK,
    2. Wan Y,
    3. Hamann HC,
    4. O’Hare JK,
    5. Guo W,
    6. Quian A,
    7. Kumar S,
    8. Bhagat S,
    9. Rodriguiz RM,
    10. Wetsel WC, et al.
    (2016) Increased metabotropic glutamate receptor 5 signaling underlies obsessive-compulsive disorder-like behavioral and striatal circuit abnormalities in mice. Biol Psychiatry 80:522–533.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Baucum AJ II.,
    2. Shonesy BC,
    3. Rose KL, and
    4. Colbran RJ
    (2015) Quantitative proteomics analysis of CaMKII phosphorylation and the CaMKII interactome in the mouse forebrain. ACS Chem Neurosci 6:615–631.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Cho HP,
    2. Garcia-Barrantes PM,
    3. Brogan JT,
    4. Hopkins CR,
    5. Niswender CM,
    6. Rodriguez AL,
    7. Venable DF,
    8. Morrison RD,
    9. Bubser M,
    10. Daniels JS, et al.
    (2014) Chemical modulation of mutant mGlu1 receptors derived from deleterious GRM1 mutations found in schizophrenics. ACS Chem Biol 9:2334–2346.
    OpenUrl
  4. ↵
    1. Choi KY,
    2. Chung S, and
    3. Roche KW
    (2011) Differential binding of calmodulin to group I metabotropic glutamate receptors regulates receptor trafficking and signaling. J Neurosci 31:5921–5930.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. Coultrap SJ,
    2. Freund RK,
    3. O’Leary H,
    4. Sanderson JL,
    5. Roche KW,
    6. Dell’Acqua ML, and
    7. Bayer KU
    (2014) Autonomous CaMKII mediates both LTP and LTD using a mechanism for differential substrate site selection. Cell Rep 6:431–437.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Enz R
    (2012) Structure of metabotropic glutamate receptor C-terminal domains in contact with interacting proteins. Front Mol Neurosci 5:52.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Flint AC,
    2. Dammerman RS, and
    3. Kriegstein AR
    (1999) Endogenous activation of metabotropic glutamate receptors in neocortical development causes neuronal calcium oscillations. Proc Natl Acad Sci USA 96:12144–12149.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Foster DJ and
    2. Conn PJ
    (2017) Allosteric modulation of GPCRs: new insights and potential utility for treatment of schizophrenia and other CNS disorders. Neuron 94:431–446.
    OpenUrl
  9. ↵
    1. Giese KP,
    2. Fedorov NB,
    3. Filipkowski RK, and
    4. Silva AJ
    (1998) Autophosphorylation at Thr286 of the alpha calcium-calmodulin kinase II in LTP and learning. Science 279:870–873.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Gregory KJ,
    2. Noetzel MJ,
    3. Rook JM,
    4. Vinson PN,
    5. Stauffer SR,
    6. Rodriguez AL,
    7. Emmitte KA,
    8. Zhou Y,
    9. Chun AC,
    10. Felts AS, et al.
    (2012) Investigating metabotropic glutamate receptor 5 allosteric modulator cooperativity, affinity, and agonism: enriching structure-function studies and structure-activity relationships. Mol Pharmacol 82:860–875.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Grueter BA,
    2. McElligott ZA,
    3. Robison AJ,
    4. Mathews GC, and
    5. Winder DG
    (2008) In vivo metabotropic glutamate receptor 5 (mGluR5) antagonism prevents cocaine-induced disruption of postsynaptically maintained mGluR5-dependent long-term depression. J Neurosci 28:9261–9270.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Hammond AS,
    2. Rodriguez AL,
    3. Townsend SD,
    4. Niswender CM,
    5. Gregory KJ,
    6. Lindsley CW, and
    7. Conn PJ
    (2010) Discovery of a novel chemical class of mGlu(5) allosteric ligands with distinct modes of pharmacology. ACS Chem Neurosci 1:702–716.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Hu NW,
    2. Nicoll AJ,
    3. Zhang D,
    4. Mably AJ,
    5. O’Malley T,
    6. Purro SA,
    7. Terry C,
    8. Collinge J,
    9. Walsh DM, and
    10. Rowan MJ
    (2014) mGlu5 receptors and cellular prion protein mediate amyloid-β-facilitated synaptic long-term depression in vivo. Nat Commun 5:3374.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Huber KM,
    2. Roder JC, and
    3. Bear MF
    (2001) Chemical induction of mGluR5- and protein synthesis--dependent long-term depression in hippocampal area CA1. J Neurophysiol 86:321–325.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Jalan-Sakrikar N,
    2. Bartlett RK,
    3. Baucum AJ II., and
    4. Colbran RJ
    (2012) Substrate-selective and calcium-independent activation of CaMKII by α-actinin. J Biol Chem 287:15275–15283.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. Jia Z,
    2. Lu Y,
    3. Henderson J,
    4. Taverna F,
    5. Romano C,
    6. Abramow-Newerly W,
    7. Wojtowicz JM, and
    8. Roder J
    (1998) Selective abolition of the NMDA component of long-term potentiation in mice lacking mGluR5. Learn Mem 5:331–343.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Jiao Y,
    2. Robison AJ,
    3. Bass MA, and
    4. Colbran RJ
    (2008) Developmentally regulated alternative splicing of densin modulates protein-protein interaction and subcellular localization. J Neurochem 105:1746–1760.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Jiao Y,
    2. Jalan-Sakrikar N,
    3. Robison AJ,
    4. Baucum AJ,
    5. 2nd Bass MA, and
    6. Colbran RJ
    (2011) Characterization of a central Ca2+/calmodulin-dependent protein kinase IIα/β binding domain in densin that selectively modulates glutamate receptor subunit phosphorylation. J Biol Chem 286:24806–24818.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Jin D-Z,
    2. Guo M-L,
    3. Xue B,
    4. Fibuch EE,
    5. Choe ES,
    6. Mao L-M, and
    7. Wang JQ
    (2013a) Phosphorylation and feedback regulation of metabotropic glutamate receptor 1 by calcium/calmodulin-dependent protein kinase II. J Neurosci 33:3402–3412.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Jin D-Z,
    2. Guo M-L,
    3. Xue B,
    4. Mao L-M, and
    5. Wang JQ
    (2013b) Differential regulation of CaMKIIα interactions with mGluR5 and NMDA receptors by Ca(2+) in neurons. J Neurochem 127:620–631.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Jin DZ,
    2. Xue B,
    3. Mao LM, and
    4. Wang JQ
    (2015) Metabotropic glutamate receptor 5 upregulates surface NMDA receptor expression in striatal neurons via CaMKII. Brain Res 1624:414–423.
    OpenUrl
  22. ↵
    1. Joly C,
    2. Gomeza J,
    3. Brabet I,
    4. Curry K,
    5. Bockaert J, and
    6. Pin JP
    (1995) Molecular, functional, and pharmacological characterization of the metabotropic glutamate receptor type 5 splice variants: comparison with mGluR1. J Neurosci 15:3970–3981.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Jong YI and
    2. O’Malley KL
    (2017) Mechanisms associated with activation of intracellular metabotropic glutamate receptor, mGluR5. Neurochem Res 42:166–172.
    OpenUrl
  24. ↵
    1. Kim CH,
    2. Braud S,
    3. Isaac JT, and
    4. Roche KW
    (2005) Protein kinase C phosphorylation of the metabotropic glutamate receptor mGluR5 on Serine 839 regulates Ca2+ oscillations. J Biol Chem 280:25409–25415.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Lee JH,
    2. Lee J,
    3. Choi KY,
    4. Hepp R,
    5. Lee JY,
    6. Lim MK,
    7. Chatani-Hinze M,
    8. Roche PA,
    9. Kim DG,
    10. Ahn YS, et al.
    (2008) Calmodulin dynamically regulates the trafficking of the metabotropic glutamate receptor mGluR5. Proc Natl Acad Sci USA 105:12575–12580.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Li MZ and
    2. Elledge SJ
    (2012) SLIC: a method for sequence- and ligation-independent cloning. Methods Mol Biol 852:51–59.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Liu XY,
    2. Mao LM,
    3. Zhang GC,
    4. Papasian CJ,
    5. Fibuch EE,
    6. Lan HX,
    7. Zhou HF,
    8. Xu M, and
    9. Wang JQ
    (2009) Activity-dependent modulation of limbic dopamine D3 receptors by CaMKII. Neuron 61:425–438.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Lüscher C and
    2. Huber KM
    (2010) Group 1 mGluR-dependent synaptic long-term depression: mechanisms and implications for circuitry and disease. Neuron 65:445–459.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Mannaioni G,
    2. Marino MJ,
    3. Valenti O,
    4. Traynelis SF, and
    5. Conn PJ
    (2001) Metabotropic glutamate receptors 1 and 5 differentially regulate CA1 pyramidal cell function. J Neurosci 21:5925–5934.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Mao L and
    2. Wang JQ
    (2003) Metabotropic glutamate receptor 5-regulated Elk-1 phosphorylation and immediate early gene expression in striatal neurons. J Neurochem 85:1006–1017.
    OpenUrlPubMed
  31. ↵
    1. Mao LM,
    2. Liu XY,
    3. Zhang GC,
    4. Chu XP,
    5. Fibuch EE,
    6. Wang LS,
    7. Liu Z, and
    8. Wang JQ
    (2008) Phosphorylation of group I metabotropic glutamate receptors (mGluR1/5) in vitro and in vivo. Neuropharmacology 55:403–408.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Mao LM and
    2. Wang Q
    (2016) Phosphorylation of group I metabotropic glutamate receptors in drug addiction and translational research. J Transl Neurosci (Beijing) 1:17–23.
    OpenUrl
  33. ↵
    1. McNeill RB and
    2. Colbran RJ
    (1995) Interaction of autophosphorylated Ca2+/calmodulin-dependent protein kinase II with neuronal cytoskeletal proteins. Characterization of binding to a 190-kDa postsynaptic density protein. J Biol Chem 270:10043–10049.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Michalon A,
    2. Sidorov M,
    3. Ballard TM,
    4. Ozmen L,
    5. Spooren W,
    6. Wettstein JG,
    7. Jaeschke G,
    8. Bear MF, and
    9. Lindemann L
    (2012) Chronic pharmacological mGlu5 inhibition corrects fragile X in adult mice. Neuron 74:49–56.
    OpenUrlCrossRefPubMed
  35. ↵
    1. Miller SG,
    2. Patton BL, and
    3. Kennedy MB
    (1988) Sequences of autophosphorylation sites in neuronal type II CaM kinase that control Ca2(+)-independent activity. Neuron 1:593–604.
    OpenUrlCrossRefPubMed
  36. ↵
    1. Minakami R,
    2. Iida K,
    3. Hirakawa N, and
    4. Sugiyama H
    (1995) The expression of two splice variants of metabotropic glutamate receptor subtype 5 in the rat brain and neuronal cells during development. J Neurochem 65:1536–1542.
    OpenUrlPubMed
  37. ↵
    1. Minakami R,
    2. Jinnai N, and
    3. Sugiyama H
    (1997) Phosphorylation and calmodulin binding of the metabotropic glutamate receptor subtype 5 (mGluR5) are antagonistic in vitro. J Biol Chem 272:20291–20298.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    1. Mockett BG,
    2. Guévremont D,
    3. Wutte M,
    4. Hulme SR,
    5. Williams JM, and
    6. Abraham WC
    (2011) Calcium/calmodulin-dependent protein kinase II mediates group I metabotropic glutamate receptor-dependent protein synthesis and long-term depression in rat hippocampus. J Neurosci 31:7380–7391.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    1. Mukherji S,
    2. Brickey DA, and
    3. Soderling TR
    (1994) Mutational analysis of secondary structure in the autoinhibitory and autophosphorylation domains of calmodulin kinase II. J Biol Chem 269:20733–20738.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    1. Niswender CM and
    2. Conn PJ
    (2010) Metabotropic glutamate receptors: physiology, pharmacology, and disease. Annu Rev Pharmacol Toxicol 50:295–322.
    OpenUrlCrossRefPubMed
  41. ↵
    1. Noetzel MJ,
    2. Rook JM,
    3. Vinson PN,
    4. Cho HP,
    5. Days E,
    6. Zhou Y,
    7. Rodriguez AL,
    8. Lavreysen H,
    9. Stauffer SR,
    10. Niswender CM, et al.
    (2012) Functional impact of allosteric agonist activity of selective positive allosteric modulators of metabotropic glutamate receptor subtype 5 in regulating central nervous system function. Mol Pharmacol 81:120–133.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    1. Oliet SH,
    2. Malenka RC, and
    3. Nicoll RA
    (1997) Two distinct forms of long-term depression coexist in CA1 hippocampal pyramidal cells. Neuron 18:969–982.
    OpenUrlCrossRefPubMed
  43. ↵
    1. Palmer MJ,
    2. Irving AJ,
    3. Seabrook GR,
    4. Jane DE, and
    5. Collingridge GL
    (1997) The group I mGlu receptor agonist DHPG induces a novel form of LTD in the CA1 region of the hippocampus. Neuropharmacology 36:1517–1532.
    OpenUrlCrossRefPubMed
  44. ↵
    1. Pasek JG,
    2. Wang X, and
    3. Colbran RJ
    (2015) Differential CaMKII regulation by voltage-gated calcium channels in the striatum. Mol Cell Neurosci 68:234–243.
    OpenUrl
  45. ↵
    1. Raka F,
    2. Di Sebastiano AR,
    3. Kulhawy SC,
    4. Ribeiro FM,
    5. Godin CM,
    6. Caetano FA,
    7. Angers S, and
    8. Ferguson SS
    (2015) Ca(2+)/calmodulin-dependent protein kinase II interacts with group I metabotropic glutamate and facilitates receptor endocytosis and ERK1/2 signaling: role of β-amyloid. Mol Brain 8:21.
    OpenUrlCrossRefPubMed
  46. ↵
    1. Rich RC and
    2. Schulman H
    (1998) Substrate-directed function of calmodulin in autophosphorylation of Ca2+/calmodulin-dependent protein kinase II. J Biol Chem 273:28424–28429.
    OpenUrlAbstract/FREE Full Text
  47. ↵
    1. Roche KW,
    2. Tu JC,
    3. Petralia RS,
    4. Xiao B,
    5. Wenthold RJ, and
    6. Worley PF
    (1999) Homer 1b regulates the trafficking of group I metabotropic glutamate receptors. J Biol Chem 274:25953–25957.
    OpenUrlAbstract/FREE Full Text
  48. ↵
    1. Romano C,
    2. van den Pol AN, and
    3. O’Malley KL
    (1996) Enhanced early developmental expression of the metabotropic glutamate receptor mGluR5 in rat brain: protein, mRNA splice variants, and regional distribution. J Comp Neurol 367:403–412.
    OpenUrlCrossRefPubMed
  49. ↵
    1. Ronesi JA,
    2. Collins KA,
    3. Hays SA,
    4. Tsai NP,
    5. Guo W,
    6. Birnbaum SG,
    7. Hu JH,
    8. Worley PF,
    9. Gibson JR, and
    10. Huber KM
    (2012) Disrupted Homer scaffolds mediate abnormal mGluR5 function in a mouse model of fragile X syndrome. Nat Neurosci 15:431–440, S1.
    OpenUrlCrossRefPubMed
  50. ↵
    1. Saito H,
    2. Kimura M,
    3. Inanobe A,
    4. Ohe T, and
    5. Kurachi Y
    (2002) An N-terminal sequence specific for a novel Homer1 isoform controls trafficking of group I metabotropic glutamate receptor in mammalian cells. Biochem Biophys Res Commun 296:523–529.
    OpenUrlCrossRefPubMed
  51. ↵
    1. Schoepp DD,
    2. Jane DE, and
    3. Monn JA
    (1999) Pharmacological agents acting at subtypes of metabotropic glutamate receptors. Neuropharmacology 38:1431–1476.
    OpenUrlCrossRefPubMed
  52. ↵
    1. Shonesy BC,
    2. Jalan-Sakrikar N,
    3. Cavener VS, and
    4. Colbran RJ
    (2014) CaMKII: a molecular substrate for synaptic plasticity and memory. Prog Mol Biol Transl Sci 122:61–87.
    OpenUrlCrossRefPubMed
  53. ↵
    1. Silva AJ,
    2. Paylor R,
    3. Wehner JM, and
    4. Tonegawa S
    (1992a) Impaired spatial learning in alpha-calcium-calmodulin kinase II mutant mice. Science 257:206–211.
    OpenUrlAbstract/FREE Full Text
  54. ↵
    1. Silva AJ,
    2. Stevens CF,
    3. Tonegawa S, and
    4. Wang Y
    (1992b) Deficient hippocampal long-term potentiation in alpha-calcium-calmodulin kinase II mutant mice. Science 257:201–206.
    OpenUrlAbstract/FREE Full Text
  55. ↵
    1. Simonyi A,
    2. Schachtman TR, and
    3. Christoffersen GR
    (2005) The role of metabotropic glutamate receptor 5 in learning and memory processes. Drug News Perspect 18:353–361.
    OpenUrlCrossRefPubMed
  56. ↵
    1. Stephenson JR,
    2. Wang X,
    3. Perfitt TL,
    4. Parrish WP,
    5. Shonesy BC,
    6. Marks CR,
    7. Mortlock DP,
    8. Nakagawa T,
    9. Sutcliffe JS, and
    10. Colbran RJ
    (2017) A novel human CAMK2A mutation disrupts dendritic morphology and synaptic transmission, and causes ASD-related behaviors. J Neurosci 37:2216–2233.
    OpenUrlAbstract/FREE Full Text
  57. ↵
    1. Strack S,
    2. McNeill RB, and
    3. Colbran RJ
    (2000) Mechanism and regulation of calcium/calmodulin-dependent protein kinase II targeting to the NR2B subunit of the N-methyl-D-aspartate receptor. J Biol Chem 275:23798–23806.
    OpenUrlAbstract/FREE Full Text
  58. ↵
    1. Uematsu K,
    2. Heiman M,
    3. Zelenina M,
    4. Padovan J,
    5. Chait BT,
    6. Aperia A,
    7. Nishi A, and
    8. Greengard P
    (2015) Protein kinase A directly phosphorylates metabotropic glutamate receptor 5 to modulate its function. J Neurochem 132:677–686.
    OpenUrlCrossRefPubMed
  59. ↵
    1. Valenti O,
    2. Conn PJ, and
    3. Marino MJ
    (2002) Distinct physiological roles of the Gq-coupled metabotropic glutamate receptors Co-expressed in the same neuronal populations. J Cell Physiol 191:125–137.
    OpenUrlCrossRefPubMed
  60. ↵
    1. Volk LJ,
    2. Daly CA, and
    3. Huber KM
    (2006) Differential roles for group 1 mGluR subtypes in induction and expression of chemically induced hippocampal long-term depression. J Neurophysiol 95:2427–2438.
    OpenUrlCrossRefPubMed
  61. ↵
    1. Wang X,
    2. Marks CR,
    3. Perfitt TL,
    4. Nakagawa T,
    5. Lee A,
    6. Jacobson DA, and
    7. Colbran RJ
    (2017) A novel mechanism for Ca2+/calmodulin-dependent protein kinase II targeting to L-type Ca2+ channels that initiates long-range signaling to the nucleus. J Biol Chem 292:17324–17336.
    OpenUrlAbstract/FREE Full Text
  62. ↵
    1. Yang E and
    2. Schulman H
    (1999) Structural examination of autoregulation of multifunctional calcium/calmodulin-dependent protein kinase II. J Biol Chem 274:26199–26208.
    OpenUrlAbstract/FREE Full Text
  63. ↵
    1. Zhang S,
    2. Xie C,
    3. Wang Q, and
    4. Liu Z
    (2014) Interactions of CaMKII with dopamine D2 receptors: roles in levodopa-induced dyskinesia in 6-hydroxydopamine lesioned Parkinson’s rats. Sci Rep 4:6811.
    OpenUrl
  64. ↵
    1. Zhou Y,
    2. Takahashi E,
    3. Li W,
    4. Halt A,
    5. Wiltgen B,
    6. Ehninger D,
    7. Li GD,
    8. Hell JW,
    9. Kennedy MB, and
    10. Silva AJ
    (2007) Interactions between the NR2B receptor and CaMKII modulate synaptic plasticity and spatial learning. J Neurosci 27:13843–13853.
    OpenUrlAbstract/FREE Full Text
Back to top

In this issue

Download PDF
Article Alerts
Email Article
Citation Tools

Research ArticleArticle

CaMKII Modulates mGlu5 by Interaction with the CTD

Christian R. Marks, Brian C. Shonesy, Xiaohan Wang, Jason R. Stephenson, Colleen M. Niswender and Roger J. Colbran
Molecular Pharmacology December 1, 2018, 94 (6) 1352-1362; DOI: https://doi.org/10.1124/mol.118.113142
Reddit logo Twitter logo Facebook logo Mendeley logo

Jump to section

Advertisement