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.