SUMO E3 ligases are expressed in the retina and regulate SUMOylation of the metabotropic glutamate receptor 8b

Abstract

The central nervous system regulates neuronal excitability by macromolecular signalling complexes that consist of functionally related proteins, including neurotransmitter receptors, enzymes and scaffolds. The composition of these signal complexes is regulated by post-translational modifications, such as phosphorylation and SUMOylation (SUMO is small ubiquitin-related modifier). In the present study, we searched for proteins interacting with the intracellular C-termini of the metabotropic glutamate receptors mGluR8a and mGluR8b and identified proteins of the SUMOylation and NEDDylation machinery. The SUMO E3 ligases Pias1 [Pias is protein inhibitor of activated STAT (signal transducer and activator of transcription)] and Pias3L interacted strongly with mGluR8b, and were co-localized with the E2-conjugating Ubc9, SUMO1 and mGluR8b in cell bodies present in the ganglion cell layer of the mammalian retina. SUMO1 conjugation of Lys882, present in a bona fide consensus sequence for SUMOylation (VKSE) in the mGluR8b C-terminus, was enhanced by addition of Pias1, consistent with an interaction between both proteins. Mutation of Lys882 to arginine reduced, but did not abolish, mGluR8b SUMOylation. Co-mutating a second lysine residue (Lys903) located in the mGluR8b isoform-specific C-terminus largely prevented SUMO1 conjugation by Ubc9. Modelling studies suggested that Lys903 contacts Ubc9 and thus is part of the non-canonical SUMOylation site VKSG. In summary, the results of the present study show in vivo SUMOylation of the complete mGluR8b and co-localize proteins of the SUMOylation machinery in the retina.

Structure of metabotropic glutamate receptor C-terminal domains in contact with interacting proteins

“…Although originally described for nuclear proteins, the covalent attachment of SUMO to targets regulates a variety of physiological processes, including transport of proteins, synaptic excitability and protein-protein interactions (Wilkinson et al, 2010). Thus, as protein phosphorylation, also SUMOylation regulates neuronal function and indeed, enzymes of the SUMOylation machinery physically interact with neurotransmitter receptors and an association between SUMOylation and neurodegeneration is discussed (Tang et al, 2005; Martin et al, 2007; Wilkinson et al, 2010; Dütting et al, 2011). …”

“…However, SUMOylation of the full-length mGluR7a was undetectable (Wilkinson and Henley, 2011). In contrast, two intracellular lysine residues located in the C-terminus of the complete mGluR8b receptor protein were SUMOylated in mammalian cells (Dütting et al, 2011). The mGluR8b C-terminus contains one bona-fide SUMOylation site (VKSE) that is located in the proximal region of the mGluR8b C-terminus, which is identical between the mGluR8a and mGluR8b isoforms (Figure 1B).…”

“…In addition, mGluR8b—but not mGluR8a—carries a second non-canonical SUMOylation motif (VKSG) located in the isoform specific, distal part of the C-terminus. The lysine residues present at position +2 in both motifs were covalently linked to SUMO proteins and this process was enhanced in the presence of the E3-ligase PIAS1 (Dütting et al, 2011). …”

“…Structure-based computational techniques predicted an extended backbone conformation for the two mGluR8b motifs VKSE and VKSG after complex formation (Dütting et al, 2011). In both sequences, Val+1 interacts with a hydrophobic pocket on the Ubc9 surface, while the side chain of Ser+3 points away from the mGluR8b/Ubc9 interface.…”

Regulation of Neuronal Protein Trafficking and Translocation by SUMOylation

“…In yeast two-hybrid assays the c-termini of mGluR8a and 8b interact with Ubc9 and SUMO1, in addition to the SUMO E3 ligases PIAS1, PIASγ, PIASxβ. Subsequently, it was shown that PIAS1 interacts with the c-termini of all group III mGluRs [21] and that the mGluR8 c-terminus can also interact with the E3 ligases Pc2 and PIAS3L [20]. In addition, full-length mGluR8b can be SUMOylated in HEK293 cells co-transfected with SUMO1 [20].…”