Activation of Metabotropic Glutamate Receptor mGlu5 on Nuclear Membranes Mediates Intranuclear Ca2+ Changes in Heterologous Cell Types and Neurons*

Karen L. O'Malley {ddagger} §, Yuh-Jiin I. Jong {ddagger}, Yuri Gonchar {ddagger}, Andreas Burkhalter {ddagger} and Carmelo Romano ¶

From the Departments of {ddagger}Anatomy and Neurobiology and Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, January 23, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Nuclear Ca2+ plays a critical role in many cellular functions although its mode (s) of regulation is unclear. This study shows that the metabotropic glutamate receptor, mGlu5, mobilizes nuclear Ca2+ independent of cytosolic Ca2+ regulation. Immunocytochemical, ultrastructural, and subcellular fractionation techniques revealed that the metabotropic glutamate receptor, mGlu5, can be localized to nuclear membranes in heterologous cells as well as midbrain and cortical neurons. Nuclear mGlu5 receptors derived from HEK cells or cortical cell types bound [3H]quisqualate. When loaded with Oregon Green BAPTA, nuclei isolated from mGlu5-expressing HEK cells responded to the addition of glutamate with rapid, oscillatory [Ca2+] elevations that were blocked by antagonist or EGTA. In contrast, carbachol-activation of endogenous muscarinic receptors led to cytoplasmic but not nuclear Ca2+ responses. Similarly, activation of mGlu5 receptors expressed on neuronal nuclei led to sustained Ca2+ oscillatory responses. These results suggest mGlu5 may mediate intranuclear signaling pathways.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Changes in nuclear Ca2+ play an integral role in cellular functions such as protein import, apoptosis, and gene transcription (1, 2). Nuclear Ca2+ may be generated from a number of sources including diffusion of cytosolic Ca2+ waves through nuclear pore complexes (2). Because the outer nuclear envelope is continuous with the endoplasmic reticulum, which serves as an internal store of Ca2+, rises in nuclear Ca2+ may also be attributable to a luminal source (3). Recent studies using high speed imaging of intracellular Ca2+ have shown that waves of Ca2+ can invade the nucleus by emptying intracellular stores (4). Calcium release from internal stores is controlled by various channels including the inositol 1,4,5-trisphosphate (IP3)1 receptor and ryanodine receptor families (5, 6) both of which are present on nuclear membranes (7, 8). Calcium itself is an activator of these channels (1) although nuclear IP3 can stimulate IP3 receptors located on the inner nuclear membrane and cADP ribose has been shown to activate nuclear ryanodine receptors (7, 8). Luminal Ca2+ is refilled at least in part by the nuclear Ca2+-ATPase (9, 10) located on the outer nuclear membrane. Thus, although signals originating at the plasma membrane may be transmitted to the nucleus (4), the presence of specific Ca2+ transporters on the nuclear envelope argues for a nuclear Ca2+ regulatory system that may be independent of cytosolic Ca2+ regulation.

Many components of G protein signaling pathways are also found in the nucleus or associated with nuclear membranes. These include phospholipase C isozymes (11, 12), nuclear inositol phosphates (12, 13), DAG (13), PKC isozymes (14), adenylate cyclase (15), regulators of G protein signaling (RGS proteins; Refs. 16 and 17) as well as heterotrimeric G proteins themselves (18). These observations raise the possibility that plasma membrane-based signaling components may also serve a similar function at nuclear membranes. Indeed, several recent reports are consistent with the notion that nuclear G protein-coupled receptors directly modulate nuclear signal transduction pathways. For example, angiotensin II receptors were found on hepatocyte nuclear membranes (19), opioid binding sites were described on ventricular myocardial nuclei (20) and endothelin-1 receptors were reported on vascular smooth muscle nuclear membranes (21). Direct evidence of nuclear receptor G protein signaling has also been demonstrated for prostaglandin receptors which, when stimulated, cause rapid Ca2+ influx into the nucleus (22, 23). Taken together, these data suggest that nuclear signaling may represent a versatile, independent system by which nuclear function is regulated.

Glutamate, the major excitatory amino acid in the brain, activates a wide range of both ion channels and G protein-coupled receptors. The latter, so-called metabotropic glutamate receptors, are widely expressed throughout the central nervous system where they are thought to be involved with many critical processes including neuronal development, learning and memory, and neurodegeneration (24). Structural and functional criteria have further subdivided the metabotropic receptors into 3 groups; Group I include mGlu1 and mGlu5 receptors, which stimulate PLC activity and release Ca2+ from cytoplasmic stores (24, 25). Calcium can also modulate the glutamate sensitivity of Group I receptors (Refs. 2628; but see Ref. 29).

Although mGlu1 and mGlu5 utilize similar signal transduction pathways, activation of mGlu1 receptors elicits a single Ca2+ peak (30) whereas mGlu5 activation leads to a marked oscillatory response in heterologous cells (3032) as well as neocortical, hippocampal (33), and spinal cord neurons (34). It has been estimated that 90% of the Ca2+ oscillations that occur spontaneously in immature cortical neurons can be ascribed to mGlu5 receptors (33). Inasmuch as Ca2+ oscillations can differentially affect gene expression (35), they play an important role in the control of developmental events.

Electron microscopic studies have shown that Group l mGlu receptors can be localized at the edge of asymmetric and in symmetric synapses (3638) as well as extrasynaptically (38, 39) in a variety of brain regions. In addition, mGlu5 and mGlu1 have also been localized intracellularly where they are predominately associated with the endoplasmic reticulum (ER) or nuclear membranes (3638). Because these receptors are activated by contact with their endogenous ligands at the cell surface, intracellular accumulation is presumed to represent either receptor sequestration and/or "extra" receptors poised to be inserted into the membrane if needed. Here, we show that activation of nuclear mGlu5 receptors can trigger Ca2+ oscillations in both the cytoplasm as well as isolated nuclei. These data suggest that nuclear receptors can independently activate rises in nuclear Ca2+ and that intracellular receptors can have functions other than serving as spare receptors.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Plasmids—HEK cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Invitrogen) as described (32). Midbrain cultures were prepared and maintained exactly as described (40). The mGlu5 stable cell line was generated using standard transfection techniques followed by repetitive rounds of limiting dilution (32). The N-terminal HA-tagged mGlu5 clone was generated previously (32). An expression plasmid containing the full-length clone of GABAB2 was obtained from Dr. Bernhard Bettler (Novartis, AG, Basel, Switzerland). A GFP-tagged Gq plasmid was obtained from Dr. Thomas Hughes (Yale, New Haven, CT).

The Semliki Forest virus vector used to deliver mGlu5 into postmitotic neurons was obtained from Dr. Kenneth Lundstrom (Hoffman-La Roche, Basel, Switzerland). This cloning vector, pSFV(PD) contains two point mutations inhibiting cytotoxicity (41). The construct, pSFV(PD)-mGlu5, was created using standard cloning methodologies. SFV stocks were produced as described (42). Briefly, in vitro transcribed RNA molecules from pSFV(PD)-mGlu5 were cotransfected via electroporation into BHK cells with the pSFV-Helper2 RNA. Viral stocks were harvested 18 h later and filter-sterilized. Viral stocks were subsequently activated with {alpha}-chymotrypsin (Sigma) and the reaction terminated with aprotinin (Sigma) as described (42). Approximate viral titers were determined by transduction of HEK cells with serial dilutions of SFV(PD)-mGlu5 followed by immunocytochemistry 1 day later.

Subcellular Fractionation—The mGlu5 receptor stable cell line was grown to near confluency, washed twice with phosphate-buffered saline and harvested by scraping followed by centrifugation. Postnatal day 3 mouse forebrains were processed by mincing tissue on ice with a razor blade into 1 mm cubes. Tissue cubes or cellular pellets were resuspended in 10 volumes of Buffer "A" medium containing 2.0 mM MgCl2, 25 mM KCl, 10 mM HEPES (pH 7.5), and protease inhibitors (Complete Tablets; Roche Applied Science, Indianapolis, IN). After swelling for 10 min on ice, cells were homogenized in a Wheaton glass homogenizer using 15 strokes with a "B" pestle. The homogenate was filtered through 3 layers of sterile gauze and centrifuged at 1000 x g for 10 min. The nuclear pellet was resuspended in 3 ml of Buffer N containing 0.25 M sucrose in buffer A. Resuspended nuclei were layered over 2 ml of medium containing 1.1 M sucrose in buffer A, and then recentrifuged at 1000 x g. This step was repeated twice. The final nuclear pellet was resuspended in SDS sample buffer as described (32). The supernatant from the first pellet was concurrently further fractionated by centrifugation at 35,000 x g for 40 min. This second supernatant represented soluble, cytoplasmic proteins whereas the high speed pellet contained plasma membrane proteins. Aliquots from each fraction were used for gel electrophoresis as well as membrane binding. Protein concentrations were determined using the Bradford assay (Bio-Rad, Richmond, CA).

Immunocytochemical and Confocal Microscopic Studies—Cells for immunocytochemical and confocal microscopic studies were grown on poly-D-lysine-treated glass coverslips, 8-well chamber slides, and/or dishes with 35-mm glass grids (Mat-Tek, Ashland, MA). Fixation, blocking, and antibody incubation were as described (32) with the exception of experiments permeabilizing membranes with digitonin. In the latter cases, permeabilization, fixation, staining, etc. were performed exactly as described by Watkins et al. (43). Primary antibodies included affinity-purified anti-C-terminal mGlu5 (1:300; 44) and anti-mGlu1a (1:300; 45), polyclonal anti-HA (1:250; Babco, Berkeley, CA), monoclonal anti-lamin B2 (1:100; Zymed Laboratories, San Francisco, CA), monoclonal anti-ryanodine receptor (1:200; MA3–925, Affinity Bioreagents, Inc., Golden, CO), and polyclonal anti-GFP (1:500; Chemicon, Temecula, CA). GABAB2 was detected with rabbit antibody AbC22 (1:1000; 46). Secondary antibodies included goat anti-rabbit and/or anti-mouse Cy3 (both at 1:300; Jackson ImmunoResearch, West Grove, PA) and goat anti-mouse Alexa 488 (1:300; Molecular Probes, Eugene, OR) or goat anti-rabbit Alexa 488 (1:2000; Molecular Probes). Confocal microscopy was performed using an Olympus Fluoview microscope and associated software.

Electron Microscopy—Adult Long-Evans rats were perfused, fixed, and sectioned exactly as described (47). Thin sections were mounted on nickel grids and staining was performed on drops of rabbit anti-mGlu5 antibody followed by a goat anti-rabbit secondary conjugated to 15-nm gold particles (1:25; Amersham Biosciences). Sections were stained with uranyl acetate and lead citrate, and ultrastructural analysis was performed in a Jeol-100 electron microscope.

[3H]Quisqualate Binding Assay—Nuclear and plasma membrane fractions were prepared as described. Both pellets were subsequently washed three times in 2 mM EDTA, 2 mM HEPES, pH 7.5, with protease inhibitors followed by centrifugation at 17,000 x g for 35 min. The final pellets were resuspended in buffer containing 40 mM HEPES, pH 7.5, 2.5 mM Ca2+, and protease inhibitors. Binding was performed as described (32).

Fluorescent Measurements of Intracellular Ca2+For whole cell measurements, mGlu5 expressing HEK cells were grown on glass coverslips in 35-mm Petri dishes overnight, washed with serum-free medium (SFM), and incubated with 10 µM Oregon Green 488 BAPTA-1 AM/0.00l% pluronic acid (Molecular Probes) in SFM for 1 h at 37 °C. Cells were washed three times with SFM and then imaged in real time using an Olympus Fluoview laser scanning confocal microscope. Drugs at 100x concentrations were added to the side of the dish and allowed to diffuse over the cells at room temperature. Glutamate was added at a final concentration of 1 mM, the mGlu5-specific antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP; Tocris) at 1 µM, the muscarinic agonist, carbachol, at 1 mM, and the glutamate transport blockers, DL-threo-{beta}-benzoxylaspartate (TBOA) at 1, 10, and 100 µM and threo-{beta}-OH-aspartic acid (TEA; Sigma) at 100 µM final concentration. In some experiments after dye loading, SFM was replaced with a standard ringers solution in which choline was substituted for sodium (138 mM choline, 4 mM KCl, 10 mM HEPES, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, pH 7.25).

For whole cell measurements of SFV-transduced midbrain cultures, cells plated on glass ovals with grids were maintained in Neurobasal media until DIV 7 when they were transduced with SFV-mGlu5 or SFV-Pael-R viral particles at an MOI of 1 for 60 min. SFV-Pael-R served as a non-mGlu5-related control. Cells were subsequently allowed to recover and express foreign gene product for 5 h prior to being loaded with Oregon Green BAPTA and imaged as described above.

To measure Ca2+ changes in individual nuclei, cells were plated at low density on 35-mM glass bottom, dishes with grids, and grown overnight. Cells were loaded with Oregon Green BAPTA as above, washed with SFM and then phosphate-buffered saline. Dishes were placed on ice and covered with medium containing 0.125 M sucrose, 2.0 mM MgCl2, 25 mM KCl, 10 mM HEPES (pH 7.5), and protease inhibitors for 10 min. These conditions were empirically found to gently lyse plasma cell membranes while leaving nuclear membranes intact and nuclei attached to the dish. Subsequently, the lysing medium was removed, and cells were washed three times with phosphate-buffered saline and then SFM. Nuclei were imaged immediately thereafter. Drug treatments were identical to whole cells. Following image collection, cells/nuclei were fixed, stained, with anti-mGlu5 and/or lamin B2, and field relocated to verify nuclear designations.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
mGlu1 Receptors Are Localized at Both the Nuclear and Plasma Membranes—Electron microscopic analyses have revealed Group l mGlu receptors on nuclear and ER membranes (3638). To establish a system in which nuclear receptor function could be addressed, cDNAs for each receptor were transiently transfected into a variety of cell types including human embryonic kidney (HEK) cells. Antibodies against the C-terminal portion of the mGlu5 protein stained the plasma membrane as well as ER and nuclear membranes in transiently transfected (Fig. 1) and stable HEK cell lines (Fig. 2B). Nuclear localization was determined by co-staining with the nuclear membrane marker lamin B2 (Fig. 1). In contrast, GABAB2 was predominately localized at the plasma membrane (Fig. 1B). Ryanodine receptor family members are expressed in the ER as well as nuclear membranes (48). Anti-ryanodine receptor-2 co-localized with mGlu5 around the nuclear membrane (Fig. 1C). Treatment with 1 mM glutamate or baclofen did not produce an obvious re-distribution of receptor subtypes in these cultures (not shown). Naive HEK cells or ones expressing un-related receptors never exhibited mGlu5 immunoreactivity (Ref. 44 and not shown)



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FIG. 1.
Co-localization of mGlu5 receptors with lamin B2. A–C, HEK-293 cells were transiently transfected with the cDNA for the indicated receptors. After 24 h the cells were washed, fixed, permeabilized, with Triton-X-100 and processed for immunocytochemistry using receptor-specific antibodies as well as anti-lamin B2 (A, B) or anti-ryanodine receptor (C). Cells were analyzed by confocal microscopy to detect receptor localization (green) and lamin B2 or ryanodine receptor distribution (red). Photographs represent single optical sections of 0.4 µm merged such that yellow indicates co-localization of the specific antigens. D and E, mGlu5 but not GABAB2 receptor localizes to the nuclear membrane. HEK cells were processed for immunocytochemistry using either the Digitonin-Fix-Triton protocol (D) or Digitonin-Fix (E) and the indicated antibodies. Permeabilization of cells prior to fixing markedly reduced the detection of GABAB2 (D and E). The lack of anti-lamin B2 staining under Digitonin-Fix conditions shows the lack of antibody accessibility to the nucleus in the absence of Triton-X-100 (E).

 


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FIG. 2.
Localization of mGlu5 N terminus to nuclear membranes. A, N-terminal-tagged mGlu5 receptors are present on nuclear membranes. HEK cells were transiently transfected with the cDNA for HA-tagged mGlu5 and then processed using digitonin permeabilization techniques as indicated. The lack of anti-HA staining under Digitonin-Fix conditions shows the lack of antibody accessibility to the nuclear lumen in the absence of Triton-X-100. B, anti-mGlu5 labels isolated nuclei from mGlu5 stable cell line. Aliquots of nuclei used for subcellular fractionation were allowed to adhere to a poly-D-lysine-treated coverslip prior to fixation, permeabilization, and incubation with antibodies directed against mGlu5 receptors (green), lamin B2 (red), or ryanodine receptors (red). Nuclei were analyzed by confocal microscopy. Merged images of single 0.4-µm optical sections confirm mGlu5/lamin B2 co-localization in yellow. C, GFP-tagged Gq is widely expressed in transfected HEK cells including the nuclear envelop. HEK cells transiently transfected with GFP-tagged Gq were fixed and permeabilized with Triton-X-100 and processed for immunocytochemistry using receptor-specific antibodies as well as anti-GFP.

 

To confirm the presence of mGlu5 on the nuclear membrane, conditions were used in which the plasma membrane was briefly permeabilized with digitonin (43). Cells were then fixed and stained (Dig-Fix) or fixed, treated with Triton X-100, and stained (Dig-Fix-Triton). Without Triton X-100 the nuclear envelope and nuclear pore complexes remained intact, restricting antibody access. Thus, inner nuclear proteins such as lamin B2 were not observed using Dig-Fix conditions versus buffers containing Triton X-100 (Fig. 1D versus Fig. 1E). This strategy allowed mGlu5 receptors to be clearly visualized at the nuclear membrane in close proximity to lamin B2 (Fig. 1D). Because anti-mGlu5 antibodies labeled these receptors in the absence of Triton X-100 (Fig. 1E), many of these receptors must be on the outer nuclear membrane with their C-terminal domains cytoplasmically accessible. In contrast, the GABAB2 receptor was never seen on the nuclear membrane using either standard fixation or digitonin permeabilization techniques (Fig. 1, B, D, and E). The latter process essentially removed all detectable GABAB2 receptor from the plasma membrane (Fig. 1, D and E). Identical intracellular localization patterns were observed for mGlu5 and GABAB2 receptors in CHO, PC12, and MN9D cells (not shown). These data suggest that a significant fraction of mGlu5 receptors but not GABAB2 can be expressed on nuclear membranes in heterologous cell types.

Since the antibody used to determine the subcellular localization of mGlu5 was directed against its C terminus, HEK cells were transiently transfected with an N-terminal HA-tagged mGlu5 receptor (32) to determine whether the N-terminal portion of the receptor was also co-localized with nuclear membranes. Anti-HA antibodies also showed co-localization of this marker with lamin B2 (Fig. 2A). These data suggest that the entire mGlu5 receptor is present on the nuclear membrane, not just the C terminus. Using digitonin permeabilization techniques, N-terminally tagged mGlu5 receptors were only observed on nuclear membranes in the presence of Triton-X-100 (Fig. 2A). This finding suggests that the N terminus of mGlu5 is within the nuclear lumen and thus inaccessible to antibody.

To confirm and extend the nuclear localization of mGlu5, aliquots of HEK nuclei prepared as described below were allowed to adhere to a poly-D-lysine-coated glass disc, fixed and processed for immunohistochemistry. The mGlu5 receptor staining co-localized with lamin B2 as well as the ryanodine receptor (Fig. 2B). Staining was confined essentially to the periphery of the nucleus or in regions of the endoplasmic reticulum adjacent to it. These results further corroborate the whole cell staining showing the presence of mGlu5 receptors on intracellular membranes.

Group l mGlu receptors couple preferentially to Gq/11 proteins leading to the activation of PLC and subsequent Ca2+ mobilization. Hughes et al. (49) have reported Gq to be present on nuclear membranes. The presence of GFP-tagged Gq on nuclear membranes was confirmed in these studies (Fig. 2C). Collectively, both mGlu5 receptors as well as their effector proteins can be localized to nuclear membranes.

Nuclear and Plasma Membrane mGlu5 Receptors Exhibit Similar Binding Characteristics—To confirm the co-localization of mGlu5 with nuclear membranes and to extend this observation in vivo, subcellular fractionation experiments were performed using an mGlu5/HEK stable cell line (45) as well as postnatal day 3 (P3) cortical tissues. In either case, the mGlu5 receptor was clearly expressed in both nuclear and plasma membrane fractions as indicated by membrane-specific markers, lamin B2, and Na+,K+-ATPase, respectively (Fig. 3).



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FIG. 3.
Co-fractionation of mGlu5 with nuclear and plasma membranes in HEK and brain cells. Subcellular fractionation of mGlu5-expressing HEK cells and P3 forebrain (WC, whole cell extracts) shows that mGlu5 receptor can be detected in fractions containing both the nuclear (N) and plasma membranes (PM). Thirty micrograms of protein from each fraction were separated on reducing SDS gels and transferred to nylon membranes. The same blot was sequentially probed with antibodies against mGlu5, the inner nuclear marker, lamin B2, and the plasma membrane marker, Na+,K+-ATPase.

 

To determine whether nuclear receptors exhibited similar binding characteristics as did plasma membrane receptors, binding studies using [3H]quisqualate as a radioligand were performed on the same cortical fractions used for Western blotting (Fig. 3). The ability of quisqualate or the endogenous ligand, glutamate, to inhibit [3H]quisqualate binding was similar for nuclear and co-fractionated plasma membrane receptors. Specifically, P3 mouse forebrain plasma membrane receptors exhibited an IC50 value for quisqualate of 1.74 ± 0.39 µM versus 10.33 ± 0.38 µM for glutamate. P3 mouse forebrain nuclear membranes exhibited IC50 values of 0.94 ± 0.23 µM and 3.26 ± 0.97 µM, respectively. Inasmuch as nuclear receptors can bind agonist and display similar rank order potencies for glutamate and quisqualate, it would appear that they are correctly folded and inserted into the nuclear envelope.

Embryonic Mesencephalic and Adult Cortical Neurons Exhibit Nuclear mGlu5 Localization—Although associated with synaptic membranes in a variety of brain regions, Hubert et al., (36) described a predominately intracellular pattern of expression for mGlu5 in neurons from rat and monkey substantia nigra. To test whether mGlu5 expression was apparent in primary cultures of mouse mesencephalon, dissociated neurons derived from embryonic day 14 mouse pups were grown in vitro for 10 days and then stained for mGlu5. At this developmental time point no obvious mGlu5 staining was observed (not shown). Sibling cultures were then transduced at an MOI of 1 for 1 h with a SFV vector driving mGlu5 expression. Viral particles were removed and cells allowed to recover overnight. Cultures were subsequently fixed and stained for mGlu5 and lamin B2. mGlu5 expression was observed in soma and dendrites as well as at nuclear membranes (Fig. 4A).



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FIG. 4.
Embryonic mesencephalic and adult cortical neurons exhibit nuclear mGlu5 localization. A, primary cultures of mesencephalic neurons were transduced with SFV(PD)-mGlu5 viral particles at an MOI of 1. Virus was removed 1 h later and cultures were allowed to recover for 16 h before being fixed and stained for mGlu5 (green) or lamin B2 (red). Merged images reveal areas of co-localization (yellow). B and C, electron micrographs of mGlu5 receptor immunogold labeling in rat visual cortex. Immunogold particles (black dots) are associated with postsynaptic density (PSD) of asymmetric synapse (B) as well as inner (IN) and outer nuclear (ON) membranes and rough ER (C) from the same preparation. AT, axon terminal; D, dendrite.

 

In other studies immunogold staining with antibodies directed against the C-terminal portion of mGlu5 was used to assess the subcellular location of this receptor in rat visual cortex. Immunogold particles were associated with postsynaptic densities of asymmetrical (excitatory) synapses (Fig. 4B) as well as rough ER and nuclear membranes (Fig. 4C). These data confirm and extend the observations of Hubert et al. (36) indicating that mGlu5 can be expressed on nuclear membranes in embryonic and adult neurons as well as heterologous cell types.

Glutamate Treatment Triggers Cytosolic and Nuclear Ca2+ Oscillations—Previously we have utilized fura-2 epifluorescence microscopy to analyze glutamate-induced mobilization of Ca2+ in transiently transfected HEK cells (32). Although those experiments measured cytosolic Ca2+, we reasoned that if nuclear mGlu5 receptors coupled to nuclear G proteins nucleoplasmic Ca2+ changes should also be apparent. To test this hypothesis, mGlu5 stably transfected HEK cells (32) were grown on glass slides, loaded with the Ca2+ indicator, Oregon Green BAPTA whose spatio-temporal distribution was analyzed in the confocal microscope. Oregon Green BAPTA exhibited a relatively homogeneous distribution throughout both the cytoplasm and nucleoplasm (Fig. 5A). When glutamate was bath-applied, Ca2+ oscillations were observed in both intracellular compartments (Fig. 5, A–C). Both sets of fluctuations were blocked with the addition of the antagonist, MPEP (1 µM). Fig. 5D represents compiled data from multiple experiments. Addition of carbachol led to a cytosolic rise in Ca2+ but not to increased nuclear Ca2+ (Figs. 5E and 6D). Moreover, activation of the endogenous muscarinic receptor never led to intracellular Ca2+ oscillations (Fig. 5E and Ref. 32). Bath application of glutamate to non-transfected HEK cells had no effect (Fig. 5G). These data are consistent with nuclear mGlu5 receptors coupling to G proteins localized on nuclear membranes but they do not rule out the possibility that the observed Ca2+ fluctuations are due to other, indirect causes.



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FIG. 5.
Single cell imaging of intracellular [Ca2+] in mGlu5/HEK or control cells. A, confocal images of Oregon Green BAPTA-loaded mGlu5/HEK cells treated at indicated times (sec) with 1 mM glutamate (Glu) or 1 µM MPEP. Bar at right of last panel represents {Delta}F/Fo as a pseudocolor scale with red being the highest. Times correspond to those for traces in B, nucleoplasmic [Ca2+] (white circle) and C, cytoplasmic [Ca2+] (blue circle) where oscillations are represented as the fractional change in fluorescence relative to the basal value. D, compiled data from maximum response ({Delta}F/Fo,%) from >10 cells from three independent experiments. *, p < 106. E, calcium rise in cytoplasm following 1 mM carbachol treatment of mGlu5/HEK cells. F, Glu-induced [Ca2+] changes in nucleus of mGlu5/HEK cells incubated in choline-substituted ringer solution as described in text. G, effect of Glu on non-mGlu5-expressing HEK cells.

 


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FIG. 6.
Glutamate-mediated Ca2+ changes in isolated nuclei. A, imaging of nucleus derived from mGlu5/HEK cells loaded with Oregon Green BAPTA and treated at indicated times with 1 mM Glu or 1 µM MPEP. First panel, transmitted light image of selected nucleus; second panel, lamin B2 staining of selected nucleus following drug treatment and posthoc field relocation. Third panel, white circle corresponds to area measured for representative trace shown in B where Glu-mediated oscillations are represented as the fractional change in fluorescence relative to the basal level. Bar at right of last panel represents {Delta}F/Fo as a pseudocolor scale with red being the highest. C, compiled data from peak {Delta}F/Fo (%) from n > 5 cells in three independent experiments. *, p < 104. D, effect of 1 mM carbachol on a representative mGlu5/HEK nucleus. E, effect of Glu on a representative HEK nucleus.

 

Direct Activation of Nuclear mGlu5 Receptors Triggers Nuclear Ca2+ Oscillations—To determine whether mGlu5 receptors present on the nuclear membrane could directly induce nuclear Ca2+ oscillations, cells were grown on dishes with grids, loaded with Oregon Green BAPTA, and then hypotonically shocked in situ. Isolated nuclei were initially identified by morphology and subsequently confirmed by lamin B2 staining and field relocation (Fig. 6A). Bath-application of 1 mM glutamate elicited an oscillatory Ca2+ response that could be blocked by the addition of 1 µM MPEP (Fig. 6, A–C) or 0.2 mM EGTA (not shown). The mGlu5-mediated Ca2+ oscillations lasted for at least 5 min although of diminishing intensity. Carbachol did not elicit a nuclear Ca2+ response (Fig. 6D). Glutamate treatment of non-mGlu5 receptor expressing HEK cells did not elicit a response (Fig. 6E). These data demonstrate that nuclear mGlu5 receptors couple to functional nuclear G proteins to increase nuclear Ca2+ levels.

Several studies have suggested that bivalent cations like Ca2+ affect Group I mGlu receptors (2628). Conceivably, increased cytosolic Ca2+ levels due to activation of plasma membrane mGlu5 receptors activate nuclear mGlu5 receptors in turn perhaps via entry through nuclear Ca2+-ATPase carriers and/or IP4 receptors. However, as shown above, no nuclear oscillations were observed in stably transfected mGlu5/HEK cells treated with carbachol despite increased cytosolic Ca2+ (Fig. 5E and 6D). Similarly, no nuclear oscillations were seen in mGlu5-expressing HEK cells loaded with caged Ca2+ after uncaging (not shown). Collectively, these experiments indicate that increased cytosolic Ca2+ per se does not activate nuclear mGlu5 receptors.

To test the idea that glutamate entering via endogenous glutamate transporters (EAAT3 and Ref. 50) could activate nuclear receptors, sodium-containing ringer solutions were replaced with choline-substituted buffers. Thus, sodium-dependent glutamate transporters such as EAAT3 would be non-functional in its absence (51). Despite the lack of Na+ both cytosolic (not shown) and nuclear compartments exhibited glutamate-induced Ca2+ fluctuations (Fig. 5F). Similarly transport inhibitors such as TBOA or TEA did not block glutamate-induced oscillations either (not shown). Although sodium-independent transporters and/or other carriers remain a possibility, these data suggest that sodium-dependent plasma membrane glutamate transporter activity is not responsible for nuclear mGlu5 receptor activation.

Besides Gq/11, Group l mGlu receptors have also been shown to couple to Gs or Gi/o proteins in various model systems. Preliminary experiments treating mGlu5-expressing HEK cells with pertussis toxin overnight did not block glutamate-induced cytoplasmic or nuclear oscillations (n = 4, not shown) thus it seems unlikely that Gi/o proteins play a major role in the observed response.

Activation of mGlu5-expressing Neurons Also Triggers Cytosolic and Nuclear Ca2+ Oscillations—To extend these studies to neurons, primary mesencephalic cultures grown on glass coverslips with grids were transduced with the SFV vector driving mGlu5 expression. SFV is known to exhibit a marked preference for neurons in vitro and in vivo (90–95%; 41). SFV-transduced midbrain cultures similarly expressed viral particles only in cell types that co-stained with the neuronal marker, NeuN, not with the astrocytic marker, GFAP (not shown). Cultures loaded with Oregon Green BAPTA were analyzed as before. Cells were subsequently fixed, stained for mGlu5 and field-relocated to assess specificity of response. As in HEK cells, mGlu5-transduced neurons exhibited Ca2+ oscillations in both nuclear and cytoplasmic compartments in response to bath-applied glutamate (Fig. 7). In contrast, bath application of glutamate to non-transduced cultures led to single, transient intracellular Ca2+ rises in many neurons but not to the oscillatory patterns observed in mGlu5 transduced cells (not shown). Application of glutamate had no effect on cultures transduced with an un-related receptor (Pael-R; Fig. 7E)) or on non-transduced controls (not shown). Thus, these data confirm and extend the results from heterologous cell types indicating that neuronal mGlu5 receptors expressed on nuclear membranes can trigger large, sustained oscillatory Ca2+ responses.



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FIG. 7.
Glutamate-mediated Ca2+ changes in primary midbrain neurons. A, confocal images of Oregon Green BAPTA loaded SFV-mGlu5/midbrain neurons treated at indicated times (sec) with 1 mM glutamate (Glu) or 1 µM MPEP. Bar at right of last panel represents {Delta}F/Fo as a pseudocolor scale with red being the highest. Times correspond to those for traces in B, nucleoplasmic [Ca2+] (white circle) and C, cytoplasmic [Ca2+] (red circle) where oscillations are represented as the fractional change in fluorescence relative to the basal value. D, compiled data from maximum response ({Delta}F/Fo,%) from >10 cells from three independent experiments. *, p < 106. E, effect of Glu on SFV-Pael-R-transduced midbrain cultures.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Although nuclear Ca2+ plays a critical role in many cellular functions such as apoptosis and gene transcription, its regulation remains unclear. The present study shows that the metabotropic receptor mGlu5 can be expressed on nuclear membranes where it can couple with endogenous signaling components to induce changes in nuclear Ca2+. Glutamate-induced Ca2+ oscillations can be blocked by the mGlu5 specific antagonist, MPEP, as well as by Ca2+ chelation. Thus, besides being regulated by external signals and intracellular stores, nuclear Ca2+ can also be regulated in situ by G-protein-coupled receptors.

The functionality of nuclear mGlu5 receptors is supported by a number of observations. First, purified nuclear membranes from HEK or cortical cell types bound radiolabeled quisqualate, the Group I-specific agonist. Second, glutamate-treated intact cells exhibited Ca2+ oscillatory patterns in the cytoplasm and the nucleus that could be blocked by the mGlu5 antagonist, MPEP (Fig. 5). Third, direct stimulation of isolated nuclei from mGlu5 expressing cells but not non-expressing cells induced nuclear Ca2+ oscillations that could also be blocked by MPEP (Fig. 6). Fourth, nuclear Ca2+ oscillations were specific since carbachol stimulation of endogenous muscarinic receptors did not induce this pattern of Ca2+ release. Fifth, glutamate-induced nuclear Ca2+ oscillations also occurred in mGlu5-transduced neurons demonstrating a physiological role for this phenomenon (Fig. 7). Taken together, these findings suggest a major role for nuclear mGlu5 receptors in the regulation of nuclear Ca2+.

Activation of Nuclear Receptors—Because the luminal side of the ER corresponds to the extracellular side of the plasma membrane, the mGlu5 receptor topology is predicted to be such that ligand binding domains are within the lumen of the nuclear envelop (Fig. 8). This interpretation is supported by data obtained by differential permeabilization indicating that while some receptors co-localize with lamin B2 (Fig. 1D) others are cytoplasmically accessible (Fig. 1E). Moreover, the in accessibility of the N-terminal-tagged mGlu5 receptor to antibodies in the absence of Triton-X-100 further supports this model (Fig. 2A). This notion is also consistent with electron microscopic images from neurons showing that immunogold labeling directed against the mGlu5 C-terminal cytoplasmic tail is present not only at synapses (Fig. 4B) but also on the outside of ER/nuclear membranes and the nucleoplasmic face of the inner nuclear membrane (Fig. 4C). Given the many studies documenting the occurrence and directionality of the IP3 and ryanodine receptors on the inner nuclear membrane (8, 48) as well as the existence of a nuclear phosphoinositol cycle (13, 12), mGlu5 receptors oriented in the latter fashion would be ideally situated to activate nuclear PLC/IP3/Ca2+ cascades.



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FIG. 8.
Proposed model of nuclear and plasma membrane mGlu5 receptors. Proposed topology of nuclear mGlu5 receptors together with relevant nuclear proteins. Calcium within the nuclear envelope is continuous with the ER pool. Alternatively nuclear Ca2+ stores can be filled via nuclear Ca2+-ATPases and/or IP4 receptors (IP4R). For simplicity, homer proteins have not been included. IP3R, IP3 receptor; RyR, ryanodine receptor.

 

The question arises then as to what ligand activates these receptors and how does it do so. The Group I mGlu receptors are homologous to the Ca2+-sensing receptors, which are activated by bivalent cations (24). Indeed a number of studies (2628) although not all (29) have shown that mGlu receptor-mediated functions can be affected by alterations in extracellular Ca2+ concentrations. However, the proposed luminal binding sites would already be exposed to several hundred micromolar Ca2+ due to the continuous nature of the ER/nuclear lumen (3). Moreover, activation of mGlu5 cell surface receptors leads to the loss of luminal stores, not increased ER Ca2+. Finally, neither uncaging Ca2+ nor increasing cytoplasmic levels via activation of muscarinic receptors led to nuclear oscillations (Fig. 5E). Thus, it seems unlikely that Ca2+ per se activates nuclear receptors.

As the natural ligand, glutamate itself may activate intracellular receptors. Glutamate uptake is mediated by at least five sodium-dependent transporter proteins that are present on the plasma membrane of glial and neuronal cells as well as many peripheral tissues including pancreas and kidney (51). Many other glutamate carriers exist, however, including sodium-independent plasma membrane transporters (52) as well as chloride-dependent uptake systems (53), and various heteroexchangers (54). Intracellular glutamate carriers also abound including mitochondrial (55) and vesicular transporters (56) as well as intracellular pools of plasma membrane transporters (57). In theory, some combination of plasma membrane and intracellular transporter could transfer glutamate into the ER lumen.

Previous studies have shown that HEK cells endogenously express the EAAT3 glutamate transporter subtype (50). Thus, one potential glutamate source for nuclear mGlu 5 receptors is extracellular glutamate uptake via the endogenous transporter. Current data, however, does not support this hypothesis since transporter inhibitors such as TBOA or AP-4 had no effect on agonist-mediated nuclear Ca2+ oscillations. Moreover, replacement of sodium, which is required for glutamate transporter activity, did not block nuclear Ca2+ oscillations either (Fig. 5F). Hence it seems unlikely in these cells that extracellular glutamate activates nuclear mGlu5 receptors via high affinity, sodium-dependent glutamate transporters.

Glutamate is involved in intermediary metabolism as well as neurotransmission. In neurons, for example, intracellular glutamate levels have been estimated at about 10 mM rising to 100–200 mM within glutamatergic vesicles (58, 51). Moreover, glutamate formation from its precursor glutamine plays a pivotal regulatory role in many cellular processes (59). Recent data have also implicated glutamate as an intracellular messenger in the regulation of insulin secretion (EC50, 2–6 mM; Refs. 60 and 61) as well as in the regulation of glutamate transporter cell surface expression (62). Conceivably, activation of plasma membrane mGlu5 receptors could trigger adaptive responses that lead to increased intracellular glutamate formation and/or mobilization, which activate nuclear mGlu5 receptors in turn. Pharmacological manipulation of key enzymes involved in glutamine conversion to glutamate and/or other glutamate carriers may help define nuclear mGlu5 ligand formation.

Many studies have shown that the in vivo trafficking of Group 1 mGlu receptors is dynamically modulated by Homer proteins (63, 64). For example, some Homer subtypes can prevent cell surface expression of Group 1 receptors whereas others can promote trafficking to specific membrane destinations (63, 64). Homer interactions can even lead to agonist-independent receptor activation (65). Thus potentially Homer family members could not only influence the cellular localization of mGlu 1/5 receptors but might also serve as perfectly positioned (cytoplasmic) activators of that function. Studies are currently under way to address these hypotheses.

Nuclear GPCRs—In the last few years the roster of nuclear signaling components has become extensive including heterotrimeric G proteins themselves as well as many of their effector molecules (18). Despite the presence of signal transduction machinery, few GPCRs have been described on nuclear membranes, most with unknown physiological relevance. Recently, however, PGE2 receptors have been found on the nuclear envelope (22, 23) together with their ligand-generating enzymes. Nuclear effects of ligand stimulation of these receptors included induction of immediate-early genes such as c-fos as well as the regulation of endothelial nitric-oxide synthase (eNOS) expression in endothelial cells (22, 23). The physiological relevance of nuclear PGE2 receptors was also strengthened by experiments demonstrating the requirement of plasma membrane prostaglandin transporters for receptor activation (22). These data support the notion that uptake via cell surface transporters contributes to signaling by activation of intracellular receptors.

Glutamate transporters are located on astrocytes where they participate in the glutamate/glutamine cycle, and are located postsynaptically (51). For example, the glutamate transporter, EAAT4, is highly enriched on Purkinje cell spines (66) where it is thought to modulate postsynaptic excitation (67, 68). Similarly, EAAC1 is associated with dendritic spines and shafts in the CA1 region of the hippocampus (57). Because Group 1 mGlu receptors exhibit overlapping distributions with these transporters in the cerebellum (mGlu1) and hippocampus (mGlu5; Ref. 63) it is conceivable that glutamate uptake serves many purposes including signal termination, modulation of firing, as well as activation of extra and intracellular mGlu receptors. It is worth noting that both EAAC1 (57) and EAAT4 (66) are associated with intracellular membranes as well. Thus, transport systems are already in place in vivo that are poised to deliver ligand to intracellular receptors.

In conclusion, contrary to the idea that intracellular mGlu5 receptors are non-functional and merely constitute an internal reserve of receptors waiting to go to the cell surface, the present data argue strongly for a dynamic intracellular role in signal transduction. Given that the ER, nuclear lumen and mitochondria all serve as unique intracellular stores of Ca2+, intracellular receptors such as mGlu5 may play a pivotal role in generating and shaping intracellular Ca2+ signals.


    FOOTNOTES
 
* This work was supported by National Institutes of Health Grant MH57817. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Dept. of Anatomy and Neurobiology, Washington University School of Medicine, 660 S. Euclid, St. Louis, MO 63110. Tel.: 314-362-7087; Fax: 314-362-3446; E-mail: omalleyk{at}pcg.wustl.edu.

1 The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; PLC, phospholipase C; ER, endoplasmic reticulum; HEK, human embryonic kidney; GFP, green fluorescent protein; GABA, {gamma}-aminobutyric acid; GPCR, G protein-coupled receptor; HA, hemagglutinin; TBOA, DL-threo-{beta}-benzoxylaspartate; TEA, threo-{beta}-OH-aspartic acid; MPEP, 2-methyl-6-(phenylethynyl)-pyridine. Back


    ACKNOWLEDGMENTS
 
We thank Seta Dikranian, Steve Harmon, Karen Myhr, Daphne Hasbani, and Dennis Oakley for technical assistance and insightful comments. We also thank Drs. B. Bettler and T. Hughes for the kind gift of plasmids and Dr. Rachel Wong for critical reading of the manuscript.



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