PKC Phosphorylation Regulates mGluR5 Trafficking by Enhancing Binding of Siah-1A

Abstract

Glutamate is the major excitatory neurotransmitter in the mammalian CNS and acts on both ionotropic and metabotropic glutamate receptors (mGluRs). The mGluRs are widely distributed in the CNS and modulate a variety of neuronal processes including neurotransmitter release and ion channel function. In hippocampus and cortex, mGluR5 is highly expressed and plays an important role in the regulation of synaptic plasticity. CaM binding dynamically regulates mGluR5 surface expression; however, the mechanisms linking CaM to mGluR5 trafficking are not clear. Recent studies showed that CaM binding to mGluR7 regulates its trafficking in a phosphorylation-dependent manner by disrupting the binding of PICK1. The E3 ligase seven in absentia homolog (Siah)-1A binds to mGluR5 and competes with CaM binding making it an intriguing molecule to regulate phosphorylation-dependent trafficking of mGluR5. In the present study, we find that CaM competes with Siah-1A for mGluR5 binding in a phosphorylation-dependent manner in rat hippocampal neurons. Specifically, phosphorylation of mGluR5 S901 favors Siah-1A binding by displacing CaM. We identified critical residues regulating Siah-1A binding to mGluR5 and showed that binding is essential for the Siah-1A effects on mGluR5 trafficking. Siah-1A binding decreases mGluR5 surface expression and increases endosomal trafficking and lysosomal degradation of mGluR5. Thus CaM-regulated Siah-1A binding to mGluR5 dynamically regulates mGluR5 trafficking. These findings support a conserved role for CaM in regulating mGluR trafficking by PKC-dependent regulation of receptor binding proteins.

Location-Dependent Signaling of the Group 1 Metabotropic Glutamate Receptor mGlu5

“…As briefly described above and thoroughly reviewed elsewhere (Dhami and Ferguson, 2006;Esseltine and Ferguson, 2013), there are reports indicating that mGlu5 receptors undergo agonist-dependent and -independent endocytosis that may or may not involve b-arrestin (Fourgeaud et al, 2003;Dhami and Ferguson, 2006;Ko et al, 2012). These data suggest that there are multiple complex mechanisms associated with trafficking and regulating mGlu5 receptor location.…”

“…As scaffolding proteins that interact with proline-rich sequences, Homers bring together mGlu1/mGlu5 receptors with IP 3 and ryanodine receptors, Shank, phosphoinositide 3 kinase enhancer-long, and Dynamin III (Bockaert et al, 2010). So-called long Homers have Regulates receptor signaling and internalization Emery et al (2010Emery et al ( , 2012, Rajagopal et al (2010), Kenakin (2011Kenakin ( , 2014, Reiter et al (2012) Regulates receptor signaling and localization Minakami et al (1997), Ishikawa et al (1999), Lee et al (2008), and Ko et al (2012) CaMKIIa Regulates receptor signaling and desensitization Mao et al (2008) and Jin et al (2013) Regulates protein-protein interactions, and mediates receptor signaling and desensitization Catania et al (1991), Aronica et al (1993), Gereau and Heinemann (1998), Kammermeier and Ikeda (2002), Kim et al (2005Kim et al ( , 2008 Decreases receptor surface expression and increases receptor endocytosis Ishikawa et al (1999) and Ko et al (2012) Tamalin Promotes receptor intracellular trafficking and cell surface expression Kitano et al (2002) CAIN, calcineurin inhibitor protein; CAL, cystic fibrosis transmembrane conductance regulator-associated ligand; CaN, calcineurin; NECAB2, neuronal Ca 2+ -binding protein 2; NHERF-2, Na + /H + exchanger regulatory factor 2; PP1g1/2, protein phosphatase 1g1/2; PP2A, protein phosphatase 2A; PP2B, protein phosphatase 2B; Pyk2, prolinerich tyrosine kinase 2. a C-terminal coiled coil domain that allows them to form multiprotein complexes (Hayashi et al, 2006). Short Homers (e.g., Homer 1a) lack the coiled coil domain and thus act as dominant negative proteins (Xiao et al, 2000;Fagni et al, 2002).…”

“…Other key mGlu5 receptor interacting proteins, such as PKC, CaM, Siah-1A, and Norbin, also play central roles in integrating synaptic signals to direct mGlu5 receptor signaling and trafficking (Kim et al, 2005;Ko et al, 2012). For example, mGlu1/mGlu5-activated PKC feeds back to phosphorylate and desensitize these receptors (Catania et al, 1991;Aronica et al, 1993;Gereau and Heinemann, 1998;Kammermeier and Ikeda, 2002) and PKC-mediated phosphorylation of serine 839 is involved in the regulation of mGlu5 receptor-mediated Ca 21 oscillations (Kim et al, 2005.…”

“…Besides PKC and CaM, Siah-1A also interacts with mGlu5 receptors competing with CaM for mGlu5 receptor binding (Ishikawa et al, 1999;Ko et al, 2012). In hippocampal neurons, activation of mGlu5 receptors leads to PKC phosphorylation, which decreases CaM binding.…”

“…In hippocampal neurons, activation of mGlu5 receptors leads to PKC phosphorylation, which decreases CaM binding. Subsequently, Siah-1A binds and ubiquitinates mGlu5 receptors, resulting in endocytosis and downregulation (Kim et al, 2005;Ko et al, 2012). Finally, Norbin also interacts with mGlu5 receptors via binding sites overlapping with those of CaM and Siah-1A ).…”