Gγ13 Interacts with PDZ Domain-containing Proteins

Robert F. Margolskee,Zairong Li,Outhiriaradjou Benard

doi:10.1074/jbc.m600113200

Robert F. Margolskee, Zairong Li + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m600113200

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Abstract

The G protein gamma13 subunit (Ggamma13) 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 Ggamma13 by yeast two-hybrid and pull-down assays. In two-hybrid assays, Ggamma13 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. Ggamma13 did not interact with the other PDZ domains of PSD95. Coexpression of Ggamma13 with its Gbeta1 partner did not interfere with these two-hybrid interactions. The physical interaction of Ggamma13 with PSD95 in the cellular milieu was confirmed in pull-down assays following heterologous expression in HEK293 cells. The interaction of Ggamma13 with the PDZ domain of PSD95 was via the C-terminal CAAX tail of Ggamma13 (where AA indicates the aliphatic amino acid); alanine substitution of the CTAL sequence at the C terminus of Ggamma13 abolished its interactions with PSD95 in two-hybrid and pull-down assays. Veli-2 and SAP97 were identified in taste tissue and in Ggamma13-expressing taste cells. Coimmunoprecipitation of Ggamma13 and PSD95 from brain and of Ggamma13 and SAP97 from taste tissue indicates that Ggamma13 interacts with these proteins endogenously. This is the first demonstration that PDZ domain proteins interact with heterotrimeric G proteins via the CAAX tail of Ggamma subunits. The interaction of Ggamma13 with PDZ domain-containing proteins may provide a means to target particular Gbetagamma subunits to specific subcellular locations and/or macromolecular complexes involved in signaling pathways.

Highlights

PDZ domains typically bind to the extreme C terminus of a target protein in a sequence-specific manner
Interaction of ␥13 subunit (G␥13) with PSD95 and Veli-2—The yeast two-hybrid system is a valuable tool for studying protein-protein interactions and for identifying interacting partners of known proteins [25], and it has been used to characterize the interaction between the G protein ␤ and ␥ subunits [26]
We have identified an interaction between the C terminus of G␥13 and three closely related PDZ domains present in PSD95, SAP97, and Veli-2 proteins

Summary

Introduction

PDZ domains typically bind to the extreme C terminus of a target protein in a sequence-specific manner. Neither of these fragments bound G␥13 in a two-hybrid assay (Fig. 2A), suggesting that the PDZ domain of Veli-2 must be intact for this protein to interact with G␥13. The PDZ domains of PSD95 and Veli recognize the canonical X(S/ T)XA motif at the C terminus of proteins (where X represents any amino acid; S/T is serine or threonine; A is any aliphatic amino acid).

Results

Conclusion