The Scaffold Protein Homer1b/c Links Metabotropic Glutamate Receptor 5 to Extracellular Signal-Regulated Protein Kinase Cascades in Neurons

Abstract

Group I metabotropic glutamate receptors (mGluRs) increase cellular levels of inositol-1,4,5-triphosphate (IP 3 ) and thereby trigger intracellular Ca 2ϩ 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 IP 3 /Ca 2ϩ 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 IP 3 /Ca 2ϩ 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 Ca 2ϩ -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 IP 3 /Ca 2ϩ -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 IP 3 /Ca 2ϩ vs a novel Homer pathway) that differentially mediate the mGluR5-ERK coupling in neurons. Both the Ca 2ϩ -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.

Glutamate Delta-1 Receptor Regulates Metabotropic Glutamate Receptor 5 Signaling in the Hippocampus

“…Homer proteins are known to link mGluRs to other postsynaptic density proteins (Shiraishi-Yamaguchi and Furuichi, 2007;Niswender and Conn, 2010) and play a vital role in coupling the receptors with downstream effectors (Mao et al, 2005;Jung et al, 2007;Won et al, 2009). Disrupted interaction between mGlu5 and the long isoform of Homer leads to deficits in mTOR signaling, protein translation, as well as synaptic plasticity in the FMR1 knockout mouse model (Ronesi and Huber, 2008;Ronesi et al, 2012).…”

A Signaling Mechanism from Gαq-Protein-Coupled Metabotropic Glutamate Receptors to Gene Expression: Role of the c-Jun N-Terminal Kinase Pathway

“…To monitor the transcription mediated by activator protein-1 (AP-1), we used a pAP1-Luc reporter plasmid (Clontech, Palo Alto, CA) containing four tandem copies of the AP-1 core consensus sequence fused to the sequence coding the firefly luciferase gene. Transfections of the plasmid (1 g/well) into cultured striatal neurons were performed using the cationic lipid Lipofectamine 2000 (Invitrogen, Carsbad, CA) by following an optimized procedure described previously Mao et al, 2005a). Eighteen hours after the transfection, cells were treated with the indicated agents, harvested with 1ϫ reporter lysis buffer (Promega, Madison, WI) into 1.5 ml tubes on ice, and assayed for luciferase activity by adding the substrate luciferin of luciferase from a luciferase reporter assay kit (Promega).…”

“…Single or double immunofluorescent labeling on eight-chamber coverslip slides was performed as described previously Mao et al, 2005a). Briefly, cultures were fixed in cold 4% paraformaldehyde (10 min), followed by incubation in 4% normal donkey serum and 1% bovine serum album (20 min) to block nonspecific staining.…”

“…Agonist ligands for ionotropic glutamate receptors (NMDA or AMPA) or for group I mGluRs increased striatal ERK1/2 phosphorylation via distinct signaling mechanisms involving Ca 2ϩ -sensitive or Ca 2ϩ -insensitive kinases (Sgambato et al, 1998;Perkinton et al, 1999Perkinton et al, , 2002; Vanhoutte et al, 1999;Thandi et al, 2002;Mao et al, 2004Mao et al, , 2005aYang et al, 2004). Similarly, glutamate increased JNK phosphorylation on their Thr 183 and Tyr 185 sites in striatal neurons (Schwarzschild et al, 1997;Mukherjee et al, 1999;Vanhoutte et al, 1999;Crossthwaite et al, 2004).…”

Loss of Metabotropic Glutamate Receptor-Dependent Long-Term Depression via Downregulation of mGluR5 after Status Epilepticus

“…This isoform is not constitutively expressed, but is powerfully induced by activity. Long Homer isoforms are known to link mGluR5 activation to intracellular signal transduction, such as the extracellular signal-regulated protein kinase cascade (Yang et al, 2004;Mao et al, 2005), or inositol triphosphate receptor mediated signaling (Sala et al, 2005). We first examined whether the abundance of long Homer isoforms might be altered in protein extracts from the CA1 region of control versus pilocarpine-treated rats.…”