G-protein-coupled receptors are well known for converting an extracellular signal into an intracellular response. Here we showed that the metabotropic glutamate receptor 5 (mGlu5) plays a dynamic intracellular role in signal transduction. Activation of endogenously expressed mGlu5 on striatal nuclear membranes leads to rapid, sustained calcium (Ca 2؉ ) responses within the nucleoplasm that can be blocked by receptor-specific antagonists. Extracellular ligands such as glutamate and quisqualate reach nuclear receptors via both sodiumdependent transporters and cystine glutamate exchangers. Inhibition of either transport system blocks radiolabeled agonist uptake as well as agonist-induced nuclear Ca 2؉ changes. can enter the nucleoplasm via the nuclear pore complex or from the nuclear lumen, the presence of nuclear mGlu5 receptors appeared to amplify the latter process generating a faster nuclear response in heterologous cells. In isolated striatal nuclei, nuclear receptor activation results in the de novo appearance of phosphorylated CREB protein. Thus, activation of nuclear mGlu5 receptors initiates a signaling cascade that is known to alter gene transcription and regulate many paradigms of synaptic plasticity. These studies demonstrated that mGlu5 receptors play a dynamic role in signaling both on and off the plasma membrane.The structure and function of G-protein-coupled receptors have received intense scrutiny over the past decades. These studies point to a dynamic environment in which receptors are not static but rather move on and off the plasma membrane according to environmental stimuli, specific targeting information, protein-protein interactions, etc. In this model, intracellular receptors are considered transitional, i.e. receptors that are either ready to be inserted into the plasma membrane or that have just been sequestered from such a site. Emerging data, however, suggest that some intracellular receptors may have intracellular functions as well. For example, a number of G-protein-coupled receptors such as the apelin, angiotensin AT1 and ATII, bradykinin B2, and lysophosphatidate LP1 receptors have been localized within the nucleoplasm itself (1-3). In contrast, prostaglandin E 2 receptors have been found on the nuclear envelope together with their ligand-generating enzymes (4, 5). Similarly, endothelin receptors A and B are also localized to the perinuclear region of cardiac ventricular myocytes where they mediate nuclear Ca 2ϩ levels and activate nuclear protein kinases (6). Finally, we have shown that the metabotropic glutamate receptor, mGlu5, 1 can be expressed on nuclear membranes where it can couple with endogenous signaling components to induce changes in nuclear Ca 2ϩ (7). Because most studies investigating the properties of nuclear receptors have been performed in heterologous cell types with overexpressed receptors, the question arises as to whether such phenomena are physiologically relevant and, in the case of mGlu5, how a ligand such as glutamate has access to this receptor.Widely expre...

G-protein-coupled receptors are thought to transmit extracellular signals to the cytoplasm from their position on the cell surface. Some receptors, including the metabotropic glutamate receptor 5 (mGluR5), are also highly expressed on intracellular membranes where they serve unknown functions. Here, we show that activation of cell surface versus intracellular mGluR5 results in unique Ca 2؉ signatures leading to unique cellular responses. Specifically, activation of either cell surface or intracellular mGluR5 leads to JNK, Ca 2؉ /calmodulin-dependent protein kinase (CaMK), and cyclic adenosine 3 ,5 -monophosphate-responsive element-binding protein phosphorylation, whereas activation of only intracellular mGluR5 leads to ERK1/2 and Elk-1 phosphorylation. Using pharmacological and genetic approaches, the present findings support a role for CaMK kinase in mediating mGluR5-dependent cyclic adenosine 3 ,5 -monophosphate-responsive element-binding protein phosphorylation, whereas CaMKII is upstream of intracellular mGluR5-mediated Elk-1 phosphorylation. Consistent with models showing Elk-1 regulating cascades of gene expression, the known Elk-1 targets c-fos and egr1 were up-regulated following intracellular mGluR5 activation, whereas a representative non-Elk-1 target, c-jun, was not. These findings emphasize that glutamate not only serves as a neurotransmitter for cell surface receptors but, when transported into the cell, can also activate intracellular receptors such as mGluR5. Glutamate activation of intracellular mGluR5 serves an important role in the regulation of nuclear Ca 2؉ , transcriptional activation, and gene expression necessary for physiological processes such as synaptic plasticity.G-protein-coupled receptors are known for converting extracellular signals into intracellular responses. Some receptors, however, are also localized on intracellular membranes where they perform unknown functions. For instance, 50 -90% of the metabotropic glutamate receptor, mGluR5, 2 which plays an active role in neuronal excitability, synaptic transmission, as well as in various neurological (anxiety, seizures, and addiction) and neurodevelopmental (Fragile X syndrome and autism) disorders, is found on endoplasmic reticulum and nuclear membranes (1-3).In the striatum, we have shown that mGluR5 is expressed on nuclear membranes where it can couple to G q/11 leading to phosphoinositide signaling and release of Ca 2ϩ from nuclear stores. Moreover, results demonstrate that both sodium-dependent and -independent transporters are involved in moving agonist across extra-and intracellular membranes; inhibition of either transport system blocks agonist-induced nuclear Ca 2ϩ changes (4, 5). Thus, mechanisms exist whereby intracellular receptors like mGluR5 may be activated and in turn trigger intracellular signal transduction processes.What are the functional consequences of activating intracellular mGluR5? In the nucleus, Ca 2ϩ is generated by diffusion of cytosolic Ca 2ϩ waves through nuclear pore complexes (6) or by release from t...

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 Ca 2؉ independent of cytosolic Ca 2؉ 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. Changes in nuclear Ca2ϩ play an integral role in cellular functions such as protein import, apoptosis, and gene transcription (1, 2). Nuclear Ca 2ϩ may be generated from a number of sources including diffusion of cytosolic Ca 2ϩ waves through nuclear pore complexes (2). Because the outer nuclear envelope is continuous with the endoplasmic reticulum, which serves as an internal store of Ca 2ϩ , rises in nuclear Ca 2ϩ may also be attributable to a luminal source (3). Recent studies using high speed imaging of intracellular Ca 2ϩ have shown that waves of Ca 2ϩ 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 (IP 3 ) 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 IP 3 can stimulate IP 3 receptors located on the inner nuclear membrane and cADP ribose has been shown to activate nuclear ryanodine receptors (7,8). Luminal Ca 2ϩ is refilled at least in part by the nuclear Ca 2ϩ -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 Ca 2ϩ transporters on the nuclear envelope argues for a nuclear Ca 2ϩ regulatory system that may be independent of cytosolic Ca 2ϩ 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 Ca 2ϩ influx into the nucleus (22,23). Taken togeth...

Metabotropic glutamate receptor 5 (mGluR5) is widely expressed throughout the CNS and participates in regulating neuronal function and synaptic transmission. Recent work in the striatum led to the groundbreaking discovery that intracellular mGluR5 activation drives unique signaling pathways, including upregulation of ERK1/2, Elk-1 (Jong et al., 2009) and Arc (Kumar et al., 2012). To determine whether mGluR5 signals from intracellular membranes of other cell types, such as excitatory pyramidal neurons in the hippocampus, we used dissociated rat CA1 hippocampal cultures and slice preparations to localize and characterize endogenous receptors. As in the striatum, CA1 neurons exhibited an abundance of mGluR5 both on the cell surface and intracellular membranes, including the endoplasmic reticulum and the nucleus where it colocalized with the sodium-dependent excitatory amino acid transporter, EAAT3. Inhibition of EAAT3 or sodium-free buffer conditions prevented accumulations of radiolabeled agonist. Using a pharmacological approach to isolate different pools of mGluR5, both intracellular and cell surface receptors induced oscillatory Ca 2ϩ responses in dissociated CA1 neurons; however, only intracellular mGluR5 activation triggered sustained high amplitude Ca 2ϩ rises in dendrites. Consistent with the notion that mGluR5 can signal from intracellular membranes, uncaging glutamate on a CA1 dendrite led to a local Ca 2ϩ rise, even in the presence of ionotropic and cell surface metabotropic receptor inhibitors. Finally, activation of intracellular mGluR5 alone mediated both electrically induced and chemically induced long-term depression, but not long-term potentiation, in acute hippocampal slices. These data suggest a physiologically relevant and important role for intracellular mGluR5 in hippocampal synaptic plasticity.

Oxidative stress is a key player in a variety of neurodegenerative disorders including Parkinson's disease. Widely used as a parkinsonian mimetic, 6-hydroxydopamine (6-OHDA) generates reactive oxygen species (ROS) as well as coordinated changes in gene transcription associated with the unfolded protein response (UPR) and apoptosis. Whether 6-OHDA-induced UPR activation is dependent on ROS has not yet been determined. The present study used molecular indicators of oxidative stress to place 6-OHDA-generated ROS upstream of the appearance of UPR markers such as activating transcription factor 3 (ATF3) and phosphorylated stress-activated protein kinase (SAPK/JNK) signaling molecules. Antioxidants completely blocked 6-OHDA-mediated UPR activation and rescued cells from toxicity. Moreover, cytochrome c release from mitochondria was observed after the appearance of early UPR markers, suggesting that cellular stress pathways are responsible for its release. Mechanistically, the 6-OHDA-induced UPR was independent of intracellular calcium changes. Rather, evidence of protein oxidation was observed before the expression of UPR markers, suggesting that the rapid accumulation of damaged proteins triggered cell stress/UPR. Taken together, 6-OHDAmediated cell death in dopaminergic cells proceeds via ROSdependent UPR up-regulation which leads to an interaction with the intrinsic mitochondrial pathway and downstream caspase activation.