Pias1 Interaction and Sumoylation of Metabotropic Glutamate Receptor 8

Abstract

Group III presynaptic metabotropic glutamate receptors (mGluRs) play a central role in regulating presynaptic activity through G-protein effects on ion channels and signal transducing enzymes. Like all Class C G-protein-coupled receptors, mGluR8 has an extended intracellular C-terminal domain (CTD) presumed to allow for modulation of downstream signaling. In a yeast two-hybrid screen of an adult rat brain cDNA library with the CTDs of mGluR8a and 8b (mGluR8-C) as baits, we identified sumo1 and four different components of the sumoylation cascade (ube2a, Pias1, Pias␥, Piasx␤) as interacting proteins. Binding assays using recombinant GST fusion proteins confirmed that Pias1 interacts not only with mGluR8-C but also with all group III mGluR CTDs. Pias1 binding to mGluR8-C required a region N-terminal to a consensus sumoylation motif and was not affected by arginine substitution of the conserved lysine 882 within this motif. Co-transfection of fluorescently tagged mGluR8a-C, sumo1, and enzymes of the sumoylation cascade into HEK293 cells showed that mGluR8a-C can be sumoylated in vivo. Arginine substitution of lysine 882 within the consensus sumoylation motif, but not other conserved lysines within the CTD, abolished in vivo sumoylation. Our results are consistent with post-translational sumoylation providing a novel mechanism of group III mGluR regulation.G-protein-coupled metabotropic glutamate receptors (mGluRs) 4 have been implicated in the regulation of transmitter release, short and long term modulation of synaptic transmission, neuronal development, and synaptic plasticity (1-5). mGluRs are structurally distinct from family A and B G-protein-coupled receptors (GPCRs), as they possess a large extracellular ligand binding domain and an extended intracellular C terminus (4, 6). At least eight different mGluR isoforms have been identified and classified into three subgroups based on sequence homology, downstream effectors, and agonist specificity. Group III mGluRs (mGluR4, are specifically activated by L(ϩ)-2-amino-4-phosphonobutyric acid (L-AP4), negatively coupled to adenylate cyclase and, apart from mGluR6, exclusively localized presynaptically. Aside from mGluR6, which is only found in the retina, group III mGluRs are expressed throughout the brain.Compared with other mGluRs, mGluR8 expression is regionally restricted, with highest mRNA levels detected in olfactory bulb, pontine gray, thalamus, and mamillary body (7,8). Alternative splicing and outof-frame insertion generates two splice variants of mGluR8, named mGluR8a and 8b, which differ in the last 16 amino acids of the cytoplasmic C-terminal domain (CTD) (9). Both splice variants share similar pharmacological profiles and expression levels throughout the brain, except for the lateral reticular and ambiguous nuclei where only mGluR8a mRNA is found (9). Pronounced mGluR8 expression in the dentate gyrus and CA3 region of the hippocampus (10, 11) and performance deficits of mice lacking mGluR8 in learning tasks (12) suggest that this receptor plays a role ...

SUMO modification regulates inactivation of the voltage-gated potassium channel Kv1.5

“…SUMOylation may thus be a widespread mechanism of channel regulation. The recent description of SUMOylation of the glucose transporters GLUT1 and GLUT4 (30,40), the two-pore potassium leak channel K2P1 (16), and the G protein-coupled receptor mGluR8 (31) further supports the view that SUMOylation is an important regulatory mechanism for integral membrane proteins. In this report we have demonstrated that disruption of Kv1.5 SUMO modification has a significant effect on the biophysical properties of the channel.…”

“…To date, SUMOylation has been most extensively studied in the context of nuclear proteins, and, in the case of sequence-specific transcription factors, SUMOylation exerts a direct and context-dependent inhibitory role (27,28). SUMOylation, however, is not restricted to this compartment because the SUMO conjugation machinery is found throughout the cell and multiple proteins not associated with the nuclear compartment, such as the plasma membrane K ϩ channel K2P1 (16), are targets for SUMO modification (30)(31)(32).…”

Knock-In Mice Lacking the PDZ-Ligand Motif of mGluR7a Show Impaired PKC-Dependent Autoinhibition of Glutamate Release, Spatial Working Memory Deficits, and Increased Susceptibility to Pentylenetetrazol

“…We attribute this to interaction with mGluR7a residues located N-terminally to the -LVI motif that contribute to PICK1 binding, as described for other PDZ-binding motifs (Hung and Sheng, 2002). Alternatively, PICK1 might bind indirectly to mGluR7a after sumoylation (Tang et al, 2005); this protein modification may occur N-terminally to the -LVI site and mediate interactions with proteins harboring Sumo-binding motifs, as present in PICK1 (Scheschonka et al, 2007).…”

A Caspase-3-cleaved Fragment of the Glial Glutamate Transporter EAAT2 Is Sumoylated and Targeted to Promyelocytic Leukemia Nuclear Bodies in Mutant SOD1-linked Amyotrophic Lateral Sclerosis

“…There are two other reports of sumoylation of membrane-bound proteins. Tang et al (50) reported that metabotropic glutamate receptor 8a (mGluR8a) can be sumoylated, although no functional consequence of sumoylation was assigned, whereas Giorgino et al (51) reported that the glucose transporters GLUT1 and GLUT4 are sumoylated in insulin-sensitive cells, resulting in differential regulation of protein levels. However, these observations were not reported in a disease context.…”