G protein-coupled receptors (GPCRs) are best known for their role as plasma membrane-localized receptors that respond to extracellular ligands. However, evidence is building that there may be complete GPCR signaling systems in the nucleus as well. Kumar et al. show that a particular type of metabotropic glutamate receptor, mGluR5, can couple to Gq/11 and stimulate phosphatidylinositol-phospholipase C (PI-PLC) to generate inositol trisphosphate (IP3) and oscillatory calcium signals in nuclei from transfected human embryonic kidney (HEK) 293 cells or cultured dissociated striatal neurons. Hemagglutinin-tagged mGluR5 expressed in HEK293 cells exhibited both plasma membrane and nuclear membrane localization. To analyze the nuclear mGluR5 signaling, nuclei were isolated from cells either expressing the hemagglutinin-tagged wild-type mGluR5 or a point mutant that could not couple to G proteins (F767S mutant). Bath application of glutamate to the isolated nuclei showed that only those from cells expressing wild-type mGluR5 produced oscillations in calcium concentration (measured with a fluorescent calcium indicator). Glutamate-induced calcium oscillations required PLC activity and were inhibited by the pan-PLC inhibitor U73122 and the PI-PLC-specific inhibitor ET-18-OCH3. Although mGluR5 frequently exhibits constitutive activity in heterologous systems, a modest glutamate-induced increase in IP3 concentration was detected in nuclei from cells expressing mGluR5. Transfection of a biosensor that binds to membranes through an interaction with phosphatidylinositol 4,5-bisphosphate (PIP2) and then is released from membranes when IP3 is produced and PIP2 concentrations decrease revealed that the isolated nuclei from the mGluR5-expressing cells had glutamate-stimulated oscillatory changes in the PIP2 to IP3 ratio, leading to repeated release from and recruitment to the nuclear membrane of the biosensor. Pharmacological inhibitors of IP3 receptors and ryanodine receptors inhibited glutamate-induced calcium oscillations in nuclei isolated from cells expressing mGluR5. Several types of glutamate receptors are present in striatal neurons. To isolate the mGluR5 response, the authors added the agonist quisqualate in the presence of inhibitors of both AMPA-type and mGluR1 receptors and found that the neurons exhibited both cytosolic and nuclear calcium oscillations. The quisqualate-stimulated calcium signals were blocked if the neurons were transfected with a dominant-negative Gαq or if PLCβ1 was knocked down with siRNA. Application of pharmacological inhibitors to isolated striatal neuron nuclei suggested that these cells also exhibited PI-PLC-dependent calcium signaling in response to quisqualate. Thus, there appears to be a complete GPCR signaling system in the nucleus. How these receptors are targeted to the nucleus and how they are activated when their ligand-binding domains are in the nuclear lumen remain to be investigated.
V. Kumar, Y.-J. I. Jong, K. L. O’Malley, Activated nuclear metabotropic glutamate receptor mGlu5 couples to nuclear Gq/11 proteins to generate inositol 1,4,5-trisphosphate-mediated nuclear Ca2+ release. J. Biol. Chem. 283, 14072-14083 (2008). [Abstract] [Full Text]