Journal Article

Scribble co-operatively binds multiple α 1D -adrenergic receptor C-terminal PDZ ligands

Hsu, Peter L, Dinh, Diana, Janezic, Eric M, Hague, Chris, Lee, Kyung-Soon, Zheng, Ning, Harris, Dorathy-Ann, Stewart, Aaron, Hinds, Thomas R

2019

Overview

Many G protein-coupled receptors (GPCRs) are organized as dynamic macromolecular complexes in human cells. Unraveling the structural determinants of unique GPCR complexes may identify unique protein:protein interfaces to be exploited for drug development. We previously reported α -adrenergic receptors (α -ARs) - key regulators of cardiovascular and central nervous system function - form homodimeric, modular PDZ protein complexes with cell-type specificity. Towards mapping α -AR complex architecture, biolayer interferometry (BLI) revealed the α -AR C-terminal PDZ ligand selectively binds the PDZ protein scribble (SCRIB) with >8x higher affinity than known interactors syntrophin, CASK and DLG1. Complementary in situ and in vitro assays revealed SCRIB PDZ domains 1 and 4 to be high affinity α -AR PDZ ligand interaction sites. SNAP-GST pull-down assays demonstrate SCRIB binds multiple α -AR PDZ ligands via a co-operative mechanism. Structure-function analyses pinpoint R1110 as a unique, critical residue dictating SCRIB:α -AR binding specificity. The crystal structure of SCRIB PDZ4 R1110G predicts spatial shifts in the SCRIB PDZ4 carboxylate binding loop dictate α -AR binding specificity. Thus, the findings herein identify SCRIB PDZ domains 1 and 4 as high affinity α -AR interaction sites, and potential drug targets to treat diseases associated with aberrant α -AR signaling.

• Subject: Binding Sites / Crystallography, X-Ray / HEK293 Cells / Humans / Immunoprecipitation / Interferometry / Membrane Proteins - metabolism / Molecular Docking Simulation / PDZ Domains / Receptors, Adrenergic, alpha-1 - metabolism / Structure-Activity Relationship / Tumor Suppressor Proteins - metabolism