Protein Kinase C Phosphorylation of the Metabotropic Glutamate ReceptormGluR5 on Serine 839 Regulates Ca2+Oscillations

Abstract

The activation of Group 1 metabotropic glutamate receptors, mGluR5 and mGluR1␣, triggers intracellular calcium release; however, mGluR5 activation is unique in that it elicits Ca 2؉ oscillations. A short region of the mGluR5 C terminus is the critical determinant and differs from the analogous region of mGluR1␣ by a single amino acid residue, Thr-840, which is an aspartic acid (Asp-854) in mGluR1␣. Previous studies show that mGluR5-elicited Ca 2؉ oscillations require protein kinase C (PKC)-dependent phosphorylation and identify Thr-840 as the phosphorylation site. However, direct phosphorylation of mGluR5 has not been studied in detail. We have used biochemical analyses to directly investigate the phosphorylation of the mGluR5 C terminus. We showed that Ser-839 on mGluR5 is directly phosphorylated by PKC, whereas Thr-840 plays a permissive role. Although Ser-839 is conserved in mGluR1␣ (Ser-853), it is not phosphorylated, as the adjacent residue (Asp-854) is not permissive; however, mutagenesis of Asp-854 to a permissive alanine residue allows phosphorylation of Ser-853 on mGluR1␣. We investigated the physiological consequences of mGluR5 Ser-839 phosphorylation using Ca 2؉ imaging. Mutations that eliminate Ser-839 phosphorylation prevent the characteristic mGluR5-dependent Ca 2؉ oscillations. However, mutation of Thr-840 to alanine, which prevents potential Thr-840 phosphorylation but is still permissive for Ser-839 phosphorylation, has no effect on Ca 2؉ oscillations. Thus, we showed that it is phosphorylation of Ser-839, not Thr-840, that is absolutely required for the unique Ca 2؉ oscillations produced by mGluR5 activation. The Thr-840 residue is important only in that it is permissive for the PKC-dependent phosphorylation of Ser-839. Metabotropic glutamate receptors (mGluRs)1 play important roles throughout the nervous system, including the activation of ion channels and the regulation of synaptic plasticity (1). In addition, they have been implicated in a variety of neurological diseases (2-5). There are eight different mGluRs, and these are subdivided into three groups based on sequence identity and pharmacological properties. Group 1 mGluRs (mGluR1 and mGluR5) are linked to phospholipase C, whereas Group 2 mGluRs (mGluR2 and mGluR3) and Group 3 mGluRs (mGluR4, mGluR6, mGluR7, and mGluR8) are negatively linked to adenylate cyclase. Activation of Group 1 mGluRs triggers phospholipase C, resulting in increases in IP 3 and diacylglycerol production and the concomitant release of intracellular Ca 2ϩ and activation of protein kinase C (PKC) (6). Group 1 mGluR-elicited Ca 2ϩ release stimulates PKC translocation to the plasma membrane and oscillations of both PKC activation and IP 3 production (7, 8).Although both mGluR1 and mGluR5 stimulate intracellular Ca 2ϩ release, they differ in that mGluR5 activation results in Ca 2ϩ oscillations, whereas mGluR1 activation results in a single Ca 2ϩ transient with or without subsequent low frequency oscillations (9, 10). A previous study (10) demonstrated that a short stret...

Calmodulin dynamically regulates the trafficking of the metabotropic glutamate receptor mGluR5

“…Early studies have demonstrated that mGluR5 activity elicits high-frequency Ca 2ϩ oscillations that are PKC-dependent. These oscillations are regulated by the direct phosphorylation of the mGluR5 C terminus, with phosphorylation of S839 being required (8,10). We now show that phosphorylation of a second site, S901, is a key mechanism in the regulation of mGluR5-mediated high-frequency Ca 2ϩ oscillations.…”

“…Group I mGluRs are G protein-coupled receptors (GPCRs), which are coupled to phospholipase C, and receptor activation triggers phosphoinositide turnover, release of intracellular Ca 2ϩ , and activation of Protein Kinase C (PKC) (7). Although PKC activity regulates mGluR5-mediated Ca 2ϩ signaling and receptor function (8)(9)(10)(11), there are no studies linking PKC phosphorylation of mGluR5 to receptor surface expression, endocytosis, or intracellular trafficking.…”

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

“…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.…”

“…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).…”

“…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. CaM binds to two sites in the C-terminal tail of the mGlu5 receptor, which blocks PKC phosphorylation; reciprocally, PKC phosphorylation blocks CaM binding (Minakami et al, 1997;Lee et al, 2008).…”