Pias1 interaction and sumoylation of metabotropic glutamate receptor 8.
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, Piasgamma, Piasxbeta) 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.
Tang
Zhongshu
Z
Department of Neurochemistry, Max-Planck-Institute for Brain Research, 60528 Frankfurt, Germany.
El Far
Oussama
O
Betz
Heinrich
H
Scheschonka
Astrid
A
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007473:Ion Channels / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D011485:Protein Binding; D050828:Protein Inhibitors of Activated STAT / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000378:metabolism; D018094:Receptors, Metabotropic Glutamate / Q000737:chemistry* / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012685:Sepharose / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D025841:Small Ubiquitin-Related Modifier Proteins / Q000502:physiology*; D014162:Transfection; D020798:Two-Hybrid System Techniques; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
D018076:DNA, Complementary; D007473:Ion Channels; C106529:PIAS1 protein, human; C414349:Piasy protein, mouse; D050828:Protein Inhibitors of Activated STAT; D043562:Receptors, G-Protein-Coupled; D018094:Receptors, Metabotropic Glutamate; D011993:Recombinant Fusion Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; C111435:metabotropic glutamate receptor 8; D049452:Green Fluorescent Proteins; D012685:Sepharose; D001120:Arginine; C512786:Ube2a protein, mouse; C465394:Ube2b protein, mouse; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D005982:Glutathione Transferase; C484534:Pias2 protein, rat; D005978:Glutathione; D008239:Lysine