Screening of GPCR C-Terminal Tails for Interactions with PSD-95 - A Quantitative Approach to Identify and Characterize GPCR-PDZ Interactions

Abstract

Scaffolding proteins containing PDZ domains are among the most abundant interaction partners of G protein-coupled receptors (GPCRs). Discovery and characterization of GPCR-PDZ interactions are important steps in the understanding of these interactions and how they affect the function of GPCRs. We have used the prototypical PDZ domain scaffold postsynaptic density protein 95 (PSD-95) to develop a generic high-throughput compatible approach to accelerate the description of GPCR-PDZ interactions. By screening of two libraries of GPCR C-terminal tails, we have identified a number of novel GPCR interactions with PSD-95, e.g. four of the somatostatin receptors (SSTRs), the neuropeptide Y receptor Y2 and the chemokine receptor CXCR2. These in vitro findings correlated well with the interactions in HEK293 cells, which shows the potential for discovery of new interactions. We show that a fluorescence polarization-based assay has higher sensitivity than a pull-down assay for primary screening of GPCR-PDZ interactions. Quantitative characterization showed inhibition constants (Ki values) around 100 μM or lower for known GPCR-PSD-95 interactions, and Ki values ranging from below 100 μM to the detection limit of 1000 μM for the identified interactions. Quantitative characterization is useful to evaluate the significance of an interaction and to compare the results with other studies. The results obtained with different lengths of the receptor (full-length GPCR, the full cytosolic C-terminal tail and peptides containing only the PDZ motif) and of the PDZ protein (full-length PSD-95 and isolated PDZ domains) were generally in close agreement.