Pias1 Interaction and Sumoylation of Metabotropic Glutamate Receptor 8

Zhongshu Tang,Oussama El Far,Astrid Scheschonka,Heinrich Betz

doi:10.1074/jbc.m508168200

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, 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.

Highlights

(mGluR4, -6, -7, and -8) are activated by L(ϩ)-2-amino-4phosphonobutyric acid (L-AP4), negatively coupled to adenylate cyclase and, apart from mGluR6, exclusively localized presynaptically
Yeast Two-hybrid Screen and Yeast Mating—To identify mGluR8-Cinteracting proteins, we performed a yeast two-hybrid screen of an adult rat brain cDNA library, using the entire C-terminal domain (CTD) coding regions of the mGluR8a and mGluR8b cDNAs as baits
All clones were in-frame with the B42 activation domain, and interactions were confirmed in yeast mating assays using the respective CTDs of mGluR8 (TABLE ONE)

Summary

EXPERIMENTAL PROCEDURES

Yeast Two-hybrid Screen and Yeast Mating—Yeast two-hybrid screening was carried out using the DupLEX-A system Expression Constructs—Glutathione S-transferase (GST) fusion proteins of the CTDs of mGluR4, -6, -7a, -7b, -8a, and -8b, and the mGluR7a truncations GST-7a-N38 and GST-7a-C27 have been described previously [28]. Flanking EcoRI and SalI sites were used to shuttle inserts between pGEX-5X-1 and pEGFP-C2 to generate bacterial or mammalian fusion proteins. MBP-ube2a and MBP-Pias␥ were generated by transferring in-frame inserts between EcoRI and XhoI sites from the identified clones to pMAL-c2 (New England Biolabs, Frankfurt, Germany). BamHI and HindIII flanked full-length cDNAs of ube2a and sumo were generated by PCR and subcloned into the BglII and Hind III sites of pEYFP-C1 (Clontech) and pECFP-C1, respectively. Protein expression in the bacterial lysates and cell homogenates was confirmed by Western blotting with anti-GST, anti-MBP, and anti-GFP antibodies. Western blotting was performed after 8% SDS-PAGE using guinea pig anti-mGluR8a-C [11] or monoclonal mouse anti-sumo (anti-Sentrin, Zytomed, Berlin, Germany)

RESULTS

Fragments identified

DISCUSSION