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Biochemical Society Transactions (200 I ) Volume 29, Part 3 A89 g5 Use of GST pull-down assays to identify new interactions 96 GABAB receptor binds a novel scaffolding protein that between the PICKl and GRIP proteins and the glutamate receptors. 0.Perestenko; H. Hirbec; K.Dev; A. Nishimune; G . Meyer; J.C. Francis; J.M. Henley M R C Centre f o r Synaptic Plasticity, University of Bristol, UK. forms multiple signal transduction protein complex. A.Nishirnune, S. Kantamneni, N. Vinh, G . Meyer, and J. M. Henley M R C Centre for Synaptic Plasticity, University of Bristol, University Walk,Bristol,BS8 1TD,UK. PDZ domain containing proteins are believed to play a key role in the targeting, expression and regulation of the proteins involved in synaptic transmission and plasticity in the CNS. Yeast two hybrid (Y2H) screens have allowed the identification of two of these proteins, PICKl and GRIP, as direct partners of the AMPA receptor subunits GluR2/3 and the metabotropic glutamate receptor mGluR7a. However several reports have suggested that Y2H assays can fail to detect certain protein:protein interactions. We have therefore undertaken a series of GST pull-down experiments to identify additional interactions between PICKl / GRIP and the glutamate receptor (GluR) subunits. GST-fusions were made for the cytoplasmic domain of GluRs and used in pull-downs assays. Using this approach we detected interactions that are not seen with the Y2H system. Furthermore, these data suggest that PICKl and GRIP do not distinguish between type I and type I1 PDZ binding motifs since both proteins interact with a wide range of GluRs subtypes. We are currently using these approaches to investigate whether PICKl and GRIP may play a more general role than originally believed in the localisation of GluRs. Using the intracellular C-terminal domain of the GABA-B-R1 subunit, we isolated a partial clone homologous to the human A kinase anchoring protein 450 (AKAP450). We isolated multiple cDNA clones containing a novel open reading frame (ORF) from hippocampal library. These cDNAs all contain an O R F that encodes the same 130-kDa protein that corresponds to the Cterminal domain of the AKAP450, but lacks PKA anchoring sites. We tried to identify proteins contained in the protein complex. We found several other signal transduction components binds to this protein and we named our novel protein as a GABA-B-R anchoring scaffold protein (GASP). When GASP was fused to GFP and expressed in COS cells, it was retained in the ER. In the two-hybrid assay, the binding of GASP was observed specifically to GABA-B-Rl subunit. Since it has been demonstrated that GABA-B-R1 subunit has specific ER retention signal, the interaction is likely to occur in the ER in vivo. We are now analysing the complex as a possible GABA-B receptor sorting machinery to synaptic plasma me 0 200 I Biochemical Society