Scribble co-operatively binds multiple α1D-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 α1D-adrenergic receptors (α1D-ARs) - key regulators of cardiovascular and central nervous system function - form homodimeric, modular PDZ protein complexes with cell-type specificity. Towards mapping α1D-AR complex architecture, biolayer interferometry (BLI) revealed the α1D-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 α1D-AR PDZ ligand interaction sites. SNAP-GST pull-down assays demonstrate SCRIB binds multiple α1D-AR PDZ ligands via a co-operative mechanism. Structure-function analyses pinpoint R1110PDZ4 as a unique, critical residue dictating SCRIB:α1D-AR binding specificity. The crystal structure of SCRIB PDZ4 R1110G predicts spatial shifts in the SCRIB PDZ4 carboxylate binding loop dictate α1D-AR binding specificity. Thus, the findings herein identify SCRIB PDZ domains 1 and 4 as high affinity α1D-AR interaction sites, and potential drug targets to treat diseases associated with aberrant α1D-AR signaling.

Label-Free Dynamic Mass Redistribution Reveals Low-Density, Prosurvival α1B-Adrenergic Receptors in Human SW480 Colon Carcinoma Cells.

Small molecules that target the adrenergic family of G protein-coupled receptors (GPCRs) show promising therapeutic efficacy for the treatment of various cancers. In this study, we report that human colon cancer cell line SW480 expresses low-density functional α1B-adrenergic receptors (ARs) as revealed by label-free dynamic mass redistribution (DMR) signaling technology and confirmed by quantitative reverse-transcriptase polymerase chain reaction analysis. Remarkably, although endogenous α1B-ARs are not detectable via either [3H]-prazosin-binding analysis or phosphoinositol hydrolysis assays, their activation leads to robust DMR and enhanced cell viability. We provide pharmacological evidence that stimulation of α1B-ARs enhances SW480 cell viability without affecting proliferation, whereas stimulating ß-ARs diminishes both viability and proliferation of SW480 cells. Our study illustrates the power of label-free DMR technology for identifying and characterizing low-density GPCRs in cells and suggests that drugs targeting both α1B- and ß-ARs may represent valuable small-molecule therapeutics for the treatment of colon cancer.

Endogenous N-terminal Domain Cleavage Modulates α1D-Adrenergic Receptor Pharmacodynamics.

The α1D-adrenergic receptor (ADRA1D) is a key regulator of cardiovascular, prostate, and central nervous system functions. This clinically relevant G protein-coupled receptor has proven difficult to study, as it must form an obligate modular homodimer containing the PDZ proteins scribble and syntrophin or become retained in the endoplasmic reticulum as non-functional protein. We previously determined that targeted removal of the N-terminal (NT) 79 amino acids facilitates ADRA1D plasma membrane expression and agonist-stimulated functional responses. However, whether such an event occurs in physiological contexts was unknown. Herein, we report the ADRA1D is subjected to innate NT processing in cultured human cells. SNAP near-infrared imaging and tandem-affinity purification revealed the ADRA1D is expressed as both full-length and NT truncated forms in multiple human cell lines. Serial truncation mapping identified the cleavage site as Leu(90)/Val(91) in the 95-amino acid ADRA1D NT domain, suggesting human cells express a Δ1-91 ADRA1D species. Tandem-affinity purification MS/MS and co-immunoprecipitation analysis indicate NT processing of ADRA1D is not required to form scribble-syntrophin macromolecular complexes. Yet, label-free dynamic mass redistribution signaling assays demonstrate that Δ1-91 ADRA1D agonist responses were greater than WT ADRA1D. Mutagenesis of the cleavage site nullified the processing event, resulting in ADRA1D agonist responses less than the WT receptor. Thus, we propose that processing of the ADRA1D NT domain is a physiological mechanism employed by cells to generate a functional ADRA1D isoform with optimal pharmacodynamic properties.

Dynamic mass redistribution reveals diverging importance of PDZ-ligands for G protein-coupled receptor pharmacodynamics.

G protein-coupled receptors (GPCRs) are essential membrane proteins that facilitate cell-to-cell communication and co-ordinate physiological processes. At least 30 human GPCRs contain a Type I PSD-95/DLG/Zo-1 (PDZ) ligand in their distal C-terminal domain; this four amino acid motif of X-[S/T]-X-[φ] sequence facilitates interactions with PDZ domain-containing proteins. Because PDZ protein interactions have profound effects on GPCR ligand pharmacology, cellular localization, signal-transduction effector coupling and duration of activity, we analyzed the importance of Type I PDZ ligands for the function of 23 full-length and PDZ-ligand truncated (ΔPDZ) human GPCRs in cultured human cells. SNAP-epitope tag polyacrylamide gel electrophoresis revealed most Type I PDZ GPCRs exist as both monomers and multimers; removal of the PDZ ligand played minimal role in multimer formation. Additionally, SNAP-cell surface staining indicated removal of the PDZ ligand had minimal effects on plasma membrane localization for most GPCRs examined. Label-free dynamic mass redistribution functional responses, however, revealed diverging effects of the PDZ ligand. While no clear trend was observed across all GPCRs tested or even within receptor families, a subset of GPCRs displayed diminished agonist efficacy in the absence of a PDZ ligand (i.e. HT2RB, ADRB1), whereas others demonstrated enhanced agonist efficacies (i.e. LPAR2, SSTR5). These results demonstrate the utility of label-free functional assays to tease apart the contributions of conserved protein interaction domains for GPCR signal-transduction coupling in cultured cells.

Individual protomers of a G protein-coupled receptor dimer integrate distinct functional modules.

Recent advances in proteomic technology reveal G-protein-coupled receptors (GPCRs) are organized as large, macromolecular protein complexes in cell membranes, adding a new layer of intricacy to GPCR signaling. We previously reported the α1D-adrenergic receptor (ADRA1D)-a key regulator of cardiovascular, urinary and CNS function-binds the syntrophin family of PDZ domain proteins (SNTA, SNTB1, and SNTB2) through a C-terminal PDZ ligand interaction, ensuring receptor plasma membrane localization and G-protein coupling. To assess the uniqueness of this novel GPCR complex, 23 human GPCRs containing Type I PDZ ligands were subjected to TAP/MS proteomic analysis. Syntrophins did not interact with any other GPCRs. Unexpectedly, a second PDZ domain protein, scribble (SCRIB), was detected in ADRA1D complexes. Biochemical, proteomic, and dynamic mass redistribution analyses indicate syntrophins and SCRIB compete for the PDZ ligand, simultaneously exist within an ADRA1D multimer, and impart divergent pharmacological properties to the complex. Our results reveal an unprecedented modular dimeric architecture for the ADRA1D in the cell membrane, providing unexpected opportunities for fine-tuning receptor function through novel protein interactions in vivo, and for intervening in signal transduction with small molecules that can stabilize or disrupt unique GPCR:PDZ protein interfaces.