Karen L. O'Malley’s research while affiliated with Washington University in St. Louis and other places

Metabotropic glutamate receptor 5 (mGlu5) plays a fundamental role in synaptic plasticity, potentially serving as a therapeutic target for various neurodevelopmental and psychiatric disorders. Previously, we have shown that mGlu5 can also signal from intracellular membranes in the cortex, hippocampus, and striatum. Using cytoplasmic Ca2+ indicators, we showed that activated cell surface mGlu5 induced a transient Ca2+ increase, whereas the activation of intracellular mGlu5 mediated a sustained Ca2+ elevation in striatal neurons. Here, we used the newly designed ER-targeted Ca2+ sensor, ER-GCaMP6-150, as a robust, specific approach to directly monitor mGlu5-mediated changes in ER Ca2+ itself. Using this sensor, we found that the activation of cell surface mGlu5 led to small declines in ER Ca2+, whereas the activation of ER-localized mGlu5 resulted in rapid, more pronounced changes. The latter could be blocked by the Gq inhibitor FR9000359, the PLC inhibitor U73122, as well as IP3 and ryanodine receptor blockers. These data demonstrate that like cell surface and nuclear mGlu5, ER-localized receptors play a pivotal role in generating and shaping intracellular Ca2+ signals.

Metabotropic glutamate receptor 5 (mGlu5) is widely expressed throughout the central nervous system (CNS) and is involved in neuronal function, synaptic transmission, and a number of neuropsychiatric disorders such as depression, anxiety, and autism. Recent work from this lab showed that mGlu5 is one of a growing number of G protein coupled receptors (GPCRs) that can signal from intracellular membranes where it drives unique signaling pathways, including upregulation of extracellular signal-regulated kinase (ERK1/2), ETS transcription factor Elk-1 and activity regulated cytoskeleton associated protein (Arc). To determine the roles of cell surface mGlu5 as well as the intracellular receptor in a well-known mGlu5 synaptic plasticity model such as long-term depression (LTD), we used pharmacological isolation and genetic and physiological approaches to analyze spatially restricted pools of mGlu5 in striatal cultures and slice preparations. Here we show that both intracellular and cell surface receptors activate the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway whereas only intracellular mGlu5 activates protein phosphatase 2 (PP2A) and leads to fragile X mental retardation protein (FMRP) degradation and de novo protein synthesis followed by a protein-synthesis-dependent increase in Arc and post-synaptic density protein 95 (PSD-95). However, both cell surface and intracellular mGlu5 activation lead to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluA2 internalization and chemically induced LTD albeit via different signaling mechanisms. These data underscore the importance of intracellular mGlu5 in the cascade of events associated with sustained synaptic transmission in the striatum.

Studies over the last two decades have firmly cemented the notion that G protein‐coupled receptors (GPCRs) can signal from a variety of intracellular membranes, not just the cell surface. In as much as GPCRs maintain and regulate cellular responses to a wide range of external stimuli, it makes sense that these master regulators also play critical roles within cells coordinating the complex spatial and temporal interactions required for maintenance and function. Current data indicate >120 GPCRs can signal from intracellular membranes including the nucleus, various endosomal vesicles as well as the trans ‐Golgi network (TGN), the Golgi, endoplasmic reticulum (ER), and even mitochondria and lysosomes. Although many GPCRs can be activated at the cell surface and subsequently endocytosed and transported to a unique intracellular site, other intracellular GPCRs can be activated in situ via de novo ligand synthesis, diffusion of permeable ligands, or active transport of nonpermeable ligands. The wealth of data demonstrating intracellular GPCRs playing a dynamic role in a host of biological functions including learning and memory, contractility, pain, angiogenesis, and cancer opens the door for new pharmacological opportunities and the development of more effective therapeutic tools. Further in‐depth study of compartmentalized signaling and associated drug discovery studies will provide valuable insights and new location‐specific drug targets.

Emerging data indicate that G-protein coupled receptor (GPCR) signaling is not only determined by the agonist and a given receptor but also a variety of cell type-specific factors that can influence a receptor’s response. For example, the metabotropic glutamate receptor, mGlu5, which is implicated in a number of neuropsychiatric disorders such as depression, anxiety, and autism, also signals from inside the cell which leads to sustained Ca2+ mobilization versus rapid transient responses. Because mGlu5 is an important drug target, many negative allosteric modulators (NAMs) have been generated to modulate its activity. Here we show that NAMs such as AFQ056, AZD2066 and RG7090 elicit very different endpoints when tested in postnatal neuronal cultures expressing endogenous mGlu5 receptors. For example, AFQ056 fails to block mGlu5-mediated Ca2+ increases whereas RG7090 is very effective. These differences are not due to differential receptor levels since about the same number of mGlu5 receptors are present on neurons from the cortex, hippocampus and striatum based on pharmacological, biochemical and molecular data. Moreover, biotinylation studies reveal that more than 90% of the receptor is intracellular in these neurons. Taken together, these data indicate that the tested NAMs exhibit both location-dependent and cell type specific bias for mGlu5-mediated Ca2+ mobilization which may affect clinical outcomes.

The trillions of synaptic connections within the human brain are shaped by experience and neuronal activity both of which underlie synaptic plasticity and ultimately learning and memory. G protein-coupled receptors (GPCRs) play key roles in synaptic plasticity by strengthening or weakening synapses and/or shaping dendritic spines. While most studies of synaptic plasticity have focused on cell surface receptors and their downstream signaling partners, emerging data point to a critical new role for the very same receptors to signal from inside the cell. Intracellular receptors have been localized to nuclear, endoplasmic reticulum, lysosomes and mitochondria. From these intracellular positions, such receptors may couple to different signaling systems, display unique desensitization patterns and/or exhibit distinct patterns of subcellular distribution. Intracellular GPCRs can be activated at the cell surface, endocytosed and transported to an intracellular site or simply activated in situ by de novo ligand synthesis, diffusion of permeable ligands or active transport of nonpermeable ligands. Current findings reinforce the notion that intracellular GPCRs play a dynamic role in synaptic plasticity and learning and memory. As new intracellular GPCR roles are defined, the need to selectively tailor agonists and/or antagonists to both intracellular and cell surface receptors may lead to the development of more effective therapeutic tools.

Traditionally, signal transduction from GPCRs is thought to emanate from the cell surface where receptor interactions with external stimuli can be transformed into a broad range of cellular responses. However, emergent data show that numerous GPCRs are also associated with various intracellular membranes where they may couple to different signalling systems, display unique desensitization patterns and/or exhibit distinct patterns of subcellular distribution. Although many GPCRs can be activated at the cell surface and subsequently endocytosed and transported to a unique intracellular site, other intracellular GPCRs can be activated in situ either via de novo ligand synthesis, diffusion of permeable ligands or active transport of nonpermeable ligands. Current findings reinforce the notion that intracellular GPCRs play a dynamic role in various biological functions including learning and memory, contractility and angiogenesis. As new intracellular GPCR roles are defined, the need to selectively tailor agonists and/or antagonists to both intracellular and cell surface receptors may lead to the development of more effective therapeutic tools. Linked articles: This article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.21/issuetoc.

Traditionally G-protein coupled receptors (GPCR) are thought to be located on the cell surface where they transmit extracellular signals to the cytoplasm. However recent studies indicate that some GPCRs are also localized to various subcellular compartments such as the nucleus where they appear required for various biological functions. For example, the metabotropic glutamate receptor 5, mGluR5, is concentrated at the inner nuclear membrane (INM) where it mediates Ca2+ changes in the nucleoplasm by coupling with Gq/11. Here we identified a region within the C-terminal domain (amino acids 852-876) which is necessary and sufficient for INM localization of the receptor. Because these sequences do not correspond to known NLS motifs they represent a new motif for INM trafficking. mGluR5 is also trafficked to the PM where it undergoes re-cycling/degradation in a separate receptor pool, one that does not interact with the nuclear mGluR5 pool. Finally, our data suggest that once at the INM, mGluR5 is stably retained via interactions with molecular structures such as chromatin and nuclear Lamins. Thus mGluR5 is perfectly positioned to regulate nucleoplasmic Ca2+ in situ.