title: Dynamic mass redistribution reveals diverging importance of PDZ-ligands for G protein-coupled receptor pharmacodynamics.
abstract: 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.
authors.affiliation: Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA 98195, USA.
authors.forename: Nathan D
authors.initials: ND
authors.lastname: Camp
authors.affiliation: Department of Pharmacology, University of Washington School of Medicine, Seattle, WA 98195, USA.
authors.forename: Kyung-Soon
authors.initials: KS
authors.lastname: Lee
authors.affiliation: Department of Pharmacology, University of Washington School of Medicine, Seattle, WA 98195, USA.
authors.forename: Allison
authors.initials: A
authors.lastname: Cherry
authors.affiliation: Department of Pharmacology, University of Washington School of Medicine, Seattle, WA 98195, USA.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Wacker-Mhyre
authors.affiliation: Department of Pharmacology, University of Washington School of Medicine, Seattle, WA 98195, USA.
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Kountz
authors.affiliation: Department of Pharmacology, University of Washington School of Medicine, Seattle, WA 98195, USA.
authors.forename: Ji-Min
authors.initials: JM
authors.lastname: Park
authors.affiliation: Department of Pharmacology, University of Washington School of Medicine, Seattle, WA 98195, USA.
authors.forename: Dorathy-Ann
authors.initials: DA
authors.lastname: Harris
authors.affiliation: Department of Pharmacology, University of Washington School of Medicine, Seattle, WA 98195, USA.
authors.forename: Marianne
authors.initials: M
authors.lastname: Estrada
authors.affiliation: Department of Pharmacology, University of Washington School of Medicine, Seattle, WA 98195, USA.
authors.forename: Aaron
authors.initials: A
authors.lastname: Stewart
authors.affiliation: Department of Pharmacology, University of Washington School of Medicine, Seattle, WA 98195, USA.
authors.forename: Nephi
authors.initials: N
authors.lastname: Stella
authors.affiliation: Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA 98195, USA.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Wolf-Yadlin
authors.affiliation: Department of Pharmacology, University of Washington School of Medicine, Seattle, WA 98195, USA. Electronic address: chague@uw.edu.
authors.forename: Chris
authors.initials: C
authors.lastname: Hague
mesh_terms: D055808:Drug Discovery / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D054731:PDZ Domains; D054730:Protein Interaction Domains and Motifs; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000032:analysis / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D008024:Ligands; D043562:Receptors, G-Protein-Coupled
keywords: G protein-coupled receptor; Label-free signaling; PDZ domain; Pharmacology