Scribble co-operatively binds multiple α_1D-adrenergic receptor C-terminal PDZ ligands

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_1D-adrenergic receptors (α_1D-ARs) - key regulators of cardiovascular and central nervous system function - form homodimeric, modular PDZ protein complexes with cell-type specificity. Towards mapping α_1D-AR complex architecture, biolayer interferometry (BLI) revealed the α_1D-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_1D-AR PDZ ligand interaction sites. SNAP-GST pull-down assays demonstrate SCRIB binds multiple α_1D-AR PDZ ligands via a co-operative mechanism. Structure-function analyses pinpoint R1110^PDZ4 as a unique, critical residue dictating SCRIB:α_1D-AR binding specificity. The crystal structure of SCRIB PDZ4 R1110G predicts spatial shifts in the SCRIB PDZ4 carboxylate binding loop dictate α_1D-AR binding specificity. Thus, the findings herein identify SCRIB PDZ domains 1 and 4 as high affinity α_1D-AR interaction sites, and potential drug targets to treat diseases associated with aberrant α_1D-AR signaling.