Gγ13 Interacts with PDZ Domain-containing Proteins

Abstract

The G protein ␥13 subunit (G␥13) is expressed in taste and retinal and neuronal tissues and plays a key role in taste transduction. We identified PSD95, Veli-2, and other PDZ domain-containing proteins as binding partners for G␥13 by yeast two-hybrid and pulldown assays. In two-hybrid assays, G␥13 interacted specifically with the third PDZ domain of PSD95, the sole PDZ domain of Veli-2, and the third PDZ domain of SAP97, a PSD95-related protein. G␥13 did not interact with the other PDZ domains of PSD95. Coexpression of G␥13 with its G␤1 partner did not interfere with these two-hybrid interactions. The physical interaction of G␥13 with PSD95 in the cellular milieu was confirmed in pull-down assays following heterologous expression in HEK293 cells. The interaction of G␥13 with the PDZ domain of PSD95 was via the C-terminal CAAX tail of G␥13 (where AA indicates the aliphatic amino acid); alanine substitution of the CTAL sequence at the C terminus of G␥13 abolished its interactions with PSD95 in twohybrid and pull-down assays. Veli-2 and SAP97 were identified in taste tissue and in G␥13-expressing taste cells. Coimmunoprecipitation of G␥13 and PSD95 from brain and of G␥13 and SAP97 from taste tissue indicates that G␥13 interacts with these proteins endogenously. This is the first demonstration that PDZ domain proteins interact with heterotrimeric G proteins via the CAAX tail of G␥ subunits. The interaction of G␥13 with PDZ domain-containing proteins may provide a means to target particular G␤␥ subunits to specific subcellular locations and/or macromolecular complexes involved in signaling pathways.A sophisticated and ordered protein network is essential to the proper functioning of cells. Precise assembly of individual components, through targeting and anchoring of proteins within designated subcellular compartments, ensures the integrity of these networks (1, 2). Specific protein-protein interactions are important for accomplishing this complex task. For example, PSD95 (postsynaptic density protein 95, also called SAP90), a member of the MAGUK (membrane-associated guanylate kinase) protein family (3), helps to assemble a complex postsynaptic protein network via its interactions with several different proteins. Some of these interactions rely on three PDZ domains (named after PSD95, Disc-large, and ZO-1) located in the N-terminal half of PSD95. PDZ domains function as protein-protein interaction modules and consist of about 90 amino acids (4 -6).PDZ domains typically bind to the extreme C terminus of a target protein in a sequence-specific manner. The PDZ domains of PSD95 recognize a canonical -X(S/T)XA motif (where X represents any amino acid and A represents an aliphatic amino acid). PDZ domains have been identified in proteins in bacteria, yeast, Drosophila, metazoans, and plants and comprise the most common protein module identified in the sequenced genome (4, 6). In addition to their affinity for C-terminal motifs, PDZ domains can also bind to internal sequences that mimic free C termini. Finally, PDZ domains c...

The PDZ Protein Mupp1 Promotes Gi Coupling and Signaling of the Mt1 Melatonin Receptor

“…However, more complex mechanisms cannot be excluded, since PDZ9 -13, which is able to restore high affinity binding of G i to MT 1 , is insufficient to reconstitute functional coupling to AC. Some PDZ proteins are indeed able to physically interact with heterotrimeric G proteins, as recently reported for PSD95, SAP97, and Veli2, which interact with G␥ 13 (39). Furthermore, ␣-syntrophin has been shown to bind to G ␤ ␥ through its PDZ domain (40).…”

Analysis of G Protein βγ Dimer Formation in Live Cells Using Multicolor Bimolecular Fluorescence Complementation Demonstrates Preferences of β1for Particular γ Subunits

“…Because ␤␥ complexes do not dissociate in the absence of denaturants (Clapham and Neer, 1997), except for ␤ 5 ␥ complexes (Jones et al, 2004), the relative abilities of CFP-N-␥ and YFP-N-␥ subunits to compete for interaction with CFP-C-␤ 1 presumably reflect their abilities to associate with this ␤ subunit. Differences in association could reflect variations in the affinities of the ␥ subunits for ␤ 1 , but other factors such as differential targeting to distinct cellular compartments or differences in interactions with chaperonins or other associated proteins (Clapham and Neer, 1997;Lukov et al, 2005;Li et al, 2006) could also regulate complex formation in vivo.…”

“…Based on studies using the yeast twohybrid system (Yan et al, 1996) and reticulocyte lysates (Schmidt et al, 1992;Ray et al, 1995;Dingus et al, 2005), most ␤ and ␥ subunits can form complexes. However, because the cellular environment can affect ␤␥ assembly (Clapham and Neer, 1997;Lukov et al, 2005;Li et al, 2006), it would be optimal to study dimerization in vivo. In addition, because cells express multiple isoforms of ␤ and ␥ subunits, determining the relative preferences of the subunits for each other in intact cells would help to predict which complexes are most likely to form in vivo.…”

Understanding Molecular Recognition by G protein βγ Subunits on the Path to Pharmacological Targeting

“…Proteomic methods and yeast two-hybrid screening have revealed multiple novel G␤␥ binding proteins. These include PDZ domain containing proteins (Li et al, 2006); guanine exchange factors (GEFs) for small G proteins such as P-Rex1 (Mayeenuddin et al, 2006), FLJ00018, also known as pleckstrin homology domain containing family G member 2 (a G␤␥-activated Rac and Cdc42 guanine nucleotide exchange factor) (Ueda et al, 2008), and p114-RhoGEF (Niu et al, 2003); protein kinase D (PKD) (Jamora et al, 1999); receptor for activated C kinase 1 (RACK1) (Dell et al, 2002); soluble NSF attachment protein (SNAP) receptor (SNARE) complex (Yoon et al, 2007); and a Radil-Rap1A complex (Ahmed et al, 2010). A striking observation for all of these G␤␥ binders is that no readily apparent consensus sequence or structure mediates binding of these proteins to G␤␥.…”