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

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

Pharmacological research (2016-01-17)
Nathan D Camp, Kyung-Soon Lee, Allison Cherry, Jennifer L Wacker-Mhyre, Timothy S Kountz, Ji-Min Park, Dorathy-Ann Harris, Marianne Estrada, Aaron Stewart, Nephi Stella, Alejandro Wolf-Yadlin, Chris Hague
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
IL-8 (77aa) (CXCL8) human, recombinant, expressed in E. coli, ≥98% (SDS-PAGE), ≥98% (HPLC), suitable for cell culture
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(−)-Isoproterenol hydrochloride
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S-Nitroso-N-acetyl-DL-penicillamine, ≥97%, powder