Abstract
Recently we have shown that the metabotropic glutamate 5 (mGlu5) receptor can be expressed on nuclear membranes of heterologous cells or endogenously on striatal neurons where it can mediate nuclear Ca2+ changes. Here, pharmacological, optical, and genetic techniques were used to show that upon activation, nuclear mGlu5 receptors generate nuclear inositol 1,4,5-trisphosphate (IP3) in situ. Specifically, expression of an mGlu5 F767S mutant in HEK293 cells that blocks Gq/11 coupling or introduction of a dominant negative Galphaq construct in striatal neurons prevented nuclear Ca2+ changes following receptor activation. These data indicate that nuclear mGlu5 receptors couple to Gq/11 to mobilize nuclear Ca2+. Nuclear mGlu5-mediated Ca2+ responses could also be blocked by the phospholipase C (PLC) inhibitor, U73122, the phosphatidylinositol (PI) PLC inhibitor 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine (ET-18-OCH3), or by using small interfering RNA targeted against PLCbeta1 demonstrating that PI-PLC is involved. Direct assessment of inositol phosphate production using a PIP2/IP3 "biosensor" revealed for the first time that IP3 can be generated in the nucleus following activation of nuclear mGlu5 receptors. Finally, both IP3 and ryanodine receptor blockers prevented nuclear mGlu5-mediated increases in intranuclear Ca2+. Collectively, this study shows that like plasma membrane receptors, activated nuclear mGlu5 receptors couple to Gq/11 and PLC to generate IP3-mediated release of Ca2+ from Ca2+-release channels in the nucleus. Thus the nucleus can function as an autonomous organelle independent of signals originating in the cytoplasm, and nuclear mGlu5 receptors play a dynamic role in mobilizing Ca2+ in a specific, localized fashion.
Highlights
Such a model of nuclear Ca2ϩ release is bolstered by data documenting the presence and activation of the inositol 1,4,5-trisphosphate receptors (IP3Rs)2 and ryanodine receptors (RyRs) on inner nuclear membranes and the nucleoplasmic reticulum (3, 4, 6 –9)
Given the importance of nuclear Ca2ϩ signaling and the unlikelihood, at least in neurons, that IP3 could diffuse long distances from its presumed site of synthesis in neuronal processes, it seems reasonable to propose that IP3 is generated in situ via the actions of G protein-coupled receptors (GPCRs) located on the inner nuclear membrane
MGlu5 Receptors Are Localized at Both the Plasma and Nuclear Membranes—We have previously shown that metabotropic glutamate 5 (mGlu5) is expressed on plasma membranes as well as on many intracellular membranes including nuclear membranes [18, 19]
Summary
Like plasma membrane receptors, activated nuclear mGlu5 receptors couple to Gq/11 and phosphatidylinositol (PI)-PLC in mGlu5-expressing HEK cells as well as striatal nuclei to generate IP3-mediated release of Ca2ϩ via Ca2ϩ release channels in the nucleus. To test whether F767S could mediate Ca2ϩ changes, HEK cells stably expressing wild type or against mGlu5, the inner nuclear marker, lamin
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