The scaffold protein Homer1b/c links metabotropic glutamate receptor 5 to extracellular signal-regulated protein kinase cascades in neurons.

Group I metabotropic glutamate receptors (mGluRs) increase cellular levels of inositol-1,4,5-triphosphate (IP3) and thereby trigger intracellular Ca2+ release. Also, group I mGluRs are organized with members of Homer scaffold proteins into multiprotein complexes involved in postreceptor signaling. In this study, we investigated the relative importance of the IP3/Ca2+ signaling and novel Homer proteins in group I mGluR-mediated activation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in cultured rat striatal neurons. We found that selective activation of mGluR5, but not mGluR1, increased ERK1/2 phosphorylation. Whereas the IP3/Ca2+ cascade transmits a small portion of signals from mGluR5 to ERK1/2, the member of Homer family Homer1b/c forms a central signaling pathway linking mGluR5 to ERK1/2 in a Ca2+-independent manner. This was demonstrated by the findings that the mGluR5-mediated ERK1/2 phosphorylation was mostly reduced by a cell-permeable Tat-fusion peptide that selectively disrupted the interaction of mGluR5 with the Homer1b/c and by small interfering RNAs that selectively knocked down cellular levels of Homer1b/c proteins. Furthermore, ERK1/2, when only coactivated by both IP3/Ca2+- and Homer1b/c-dependent pathways, showed the ability to phosphorylate two transcription factors, Elk-1 and cAMP response element-binding protein, and thereby facilitated c-Fos expression. Together, we have identified two coordinated signaling pathways (a conventional IP3/Ca2+ vs a novel Homer pathway) that differentially mediate the mGluR5-ERK coupling in neurons. Both the Ca2+-dependent and -independent pathways are corequired to activate ERK1/2 to a level sufficient to achieve the mGluR5-dependent synapse-to-nucleus communication imperative for the transcriptional regulation.

A novel Ca2+-independent signaling pathway to extracellular signal-regulated protein kinase by coactivation of NMDA receptors and metabotropic glutamate receptor 5 in neurons.

The specification and organization of glutamatergic synaptic transmission require the coordinated interaction among glutamate receptors and their synaptic adaptor proteins closely assembled in the postsynaptic density (PSD). Here we investigated the interaction between NMDA receptors and metabotropic glutamate receptor 5 (mGluR5) in the integral regulation of extracellular signal-regulated protein kinase (ERK) and gene expression in cultured rat striatal neurons. We found that coapplication of NMDA and the mGluR5 agonist (S)-3,5-dihydroxyphenylglycine synergistically increased ERK phosphorylation. Interestingly, the synergistic increase in ERK phosphorylation was dependent on the cross talk between NMDA receptor-associated synaptic adaptor protein PSD-95 and the mGluR5-linked adaptor protein Homer1b/c but not on the conventional Ca2+ signaling derived from NMDA receptors (Ca2+ influx) and mGluR5 (intracellular Ca2+ release). This was demonstrated by the findings that the synergistic phosphorylation of ERK induced by coactivation of NMDA receptors and mGluR5 was blocked by either a Tat peptide that disrupts NMDA receptor/PSD-95 binding or small interfering RNAs that selectively reduce cellular levels of Homer1b/c. Furthermore, ERK activated through this PSD-95/Homer1b/c-dependent and Ca2+-independent pathway was able to phosphorylate the two key transcription factors Elk-1 and cAMP response element-binding protein, which further leads to facilitation of c-Fos expression. Together, we have identified a novel Ca2+-independent signaling pathway to ERK by the synergistic interaction of NMDA receptors and mGluR5 via their adaptor proteins in the PSD of neurons, which underlies a synapse-to-nucleus communication important for the transcriptional regulation.

Distinct expression of phosphorylated N-methyl-D-aspartate receptor NR1 subunits by projection neurons and interneurons in the striatum of normal and amphetamine-treated rats.

N-methyl-D-aspartate (NMDA) receptors are heteromeric assemblies of subunits (NR1 and NR2A-D), and are enriched in the striatum. Receptor phosphorylation has recently been demonstrated on the NR1 subunit at three serine residues, 897, 896, and 890, which appear to correspond to the level of receptor activity. In this study, expression of phospho-specific NR1 subunits at serine 897 (pNR1S897), serine 896 (pNR1S896), or serine 890 (pNR1S890) in neurochemically identified neurons of the adult rat striatum was detected by using double-immunofluorescent labeling or combined in situ hybridization and immunohistochemistry. In both the dorsal and ventral striatum, pNR1S897 was expressed at high levels in projection neurons containing >55% dynorphin (striatonigral) and >90% enkephalin (striatopallidal) and in interneurons that were 100% positive for choline, >90% positive for parvalbumin, and >45% positive for somatostatin (co-containing neuropeptide Y and neuronal nitric oxide synthase). Low levels of pNR1S896 were present in a small portion of projection neurons (<15% for both populations of projection neurons) and were almost lacking in the three types of interneurons. Interestingly, pNR1S890 was exclusively expressed in most parvalbumin-containing interneurons (70-80%). Acute administration of a psychostimulant, amphetamine, increased the number of dynorphin-containing projection neurons and parvalbumin interneurons showing detectable levels of pNR1S896 and pNR1S890, respectively. These results demonstrate the distinct expression of phospho-NR1 subunits in different populations of striatal projection neurons and interneurons at variable levels in normal rats; they also demonstrate that phosphorylation of NR1, at least on serine 896 and 890 sites, is sensitive to drug exposure.

Glutamate signaling to Ras-MAPK in striatal neurons: mechanisms for inducible gene expression and plasticity.

Extracellular signals can regulate mitogen-activated protein kinase (MAPK) cascades through a receptor-mediated mechanism in postmitotic neurons of adult mammalian brain. Both ionotropic and metabotropic glutamate receptors (mGluRs) are found to possess such an ability in striatal neurons. NMDA and AMPA receptor signals seem to share a largely common route to MAPK phosphorylation which involves first activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) via Ca2+ influx, followed by subsequent induction of phosphoinositide 3-kinase (PI3-kinase). Through its lipid and protein kinase activity, active PI3-kinase may transduce signals to Ras-MAPK cascades via at least two distinct pathways. A novel, Ca(2+)-independent pathway is believed to mediate mGluR signals to Ras-MAPK activation. As an information superhighway between the surface membrane and the nucleus, Ras-MAPK cascades, through activating their specific nuclear transcription factor targets, are actively involved in the regulation of gene expression. Emerging evidence shows that MAPK-mediated genomic responses in striatal neurons to drug exposure contribute to the development of neuroplasticity related to addictive properties of drugs of abuse.

Regulation of MAPK/ERK phosphorylation via ionotropic glutamate receptors in cultured rat striatal neurons.

Extracellular signals may regulate mitogen-activated protein kinase (MAPK) cascades through a receptor-mediated mechanism. As a signaling superhighway to the nucleus, active Ras-MAPK cascades phosphorylate transcription factors and facilitate gene expression. In cultured rat striatal neurons, the present work systemically examined the linkage between glutamate receptors and the extracellular signal-regulated kinase 1/2 (ERK1/2) subclass of MAPK. We found that glutamate induced a rapid and transient phosphorylation of ERK1/2. Similar responses of ERK1/2 phosphorylation were also induced by the ligands selective for each of three subtypes of ionotropic receptors (NMDA, AMPA and kainate), although not by the subgroup-selective agonists for three subgroups of metabotropic glutamate receptors after 8-9 days in culture. The ERK1/2 phosphorylation induced by all ionotropic receptor agents was dose-, time- and Ca(2+) influx-dependent and occurred in neurons, but not glia. The NMDA-, AMPA- and kainate-induced ERK1/2 phosphorylation was blocked only by the antagonists selective for respective subtypes. The ERK1/2 phosphorylation induced by these agents was also sensitive to the MAPK kinase 1 (MEK1) inhibitor PD98059 and the MEK1/2 inhibitor U0126. In a further attempt to evaluate the role of active ERK1/2 in activating a downstream transcription factor cAMP response element-binding protein (CREB), NMDA, AMPA, and kainate were found to increase CREB phosphorylation. The NMDA- and AMPA/kainate-induced CREB phosphorylation was completely and partially blocked by U0126, respectively. These results revealed a positive linkage between ionotropic glutamate receptors and MEK-sensitive ERK1/2 phosphorylation in striatal neurons. The active ERK1/2 cascade activates the downstream transcription factor CREB to participate in the regulation of gene expression.