Assay Development and Screening

Leveraging label-free dynamic mass redistribution technology to study G protein-coupled receptor ligand pharmacodynamics

Monday, February 5
3:00 PM - 3:30 PM
Location: 6C

Sponsored By 

Label-free dynamic mass redistribution (DMR) technology represents a powerful approach to studying G protein-coupled receptor (GPCR) signaling in cultured cells. Recently, our laboratory has leveraged DMR to study multiple facets of human adrenergic receptor biology, including:

1) Deconvoluting the α1D-adrenergic receptor (ADRA1D) PDZ-protein macromolecular complex. Tandem-affinity purification/mass spectrometry identified novel PDZ-protein interactors syntrophin and scribble for the ADRA1D in human cells. DMR assays subsequently revealed syntrophin and scribble differentially enhance agonist efficacy.
2) Investigating the importance of PDZ-ligands for GPCR agonist pharmacodynamics. DMR screens were used to assess the importance of PDZ-protein interactions for agonist pharmacodynamics of 24 human GPCRs containing PDZ-ligands in their distal C-termini. DMR agonist concentration response-curves were generated for full length and PDZ-ligand truncated GPCRs expressed in human cells.
3) Structure-function analysis of GPCR structural domains. SNAP-technology revealed the ADRA1D undergoes constitutive N-terminal domain proteolytic cleavage in human cells. DMR assays indicate this N-terminal cleavage event enhances ADRA1D signaling properties.
4) Identification and pharmacological characterization of endogenous adrenergic receptors in human cancer cell lines. DMR assays examining subtype-selective adrenergic receptor drugs revealed previously undetectable adrenergic receptors in SW480 human colon carcinoma cells. Schild plot analysis with adrenergic receptor subtype-selective antagonists permitted pharmacological characterization of functional adrenergic receptors expressed in SW480 cells. DMR data facilitated subsequent examination of adrenergic receptor stimulation on SW480 cell fate.

To conclude, label-free DMR technology is a diverse, powerful tool that can be used to study both transfected and endogenous GPCRs in cultured cells; to deconvolute functional modules of GPCR macromolecular complexes; to address the importance of specific structural domains for GPCR function; and can be combined with traditional analytical methods to facilitate pharmacological characterization of ligand-receptor interactions.

Chris Hague

Associate Professor
University of Washington - Pharmacology

Dr. Hague's research focuses on the molecular pharmacology of G protein-coupled receptors (GPCRs). The Hague lab currently focuses on proteomic identification of GPCR macromolecular complexes in human cells, and molecular deconvolution of functional roles of de novo interacting proteins. The long-term goal of the Hague lab is to identify novel GPCR protein-protein interaction interfaces that have the potential to be targeted by novel small molecules to modulate GPCR function. Dr. Hague received his B.Sc from McMaster University (Hamilton, ON, Canada) in Pharmacology, his Ph.D. from Creighton University (Omaha, NE) in Pharmacology, and performed his post-doctoral fellowship in the Department of Pharmacology at Emory University (Atlanta, GA). Dr. Hague has been a faculty member in the Department of Pharmacology at the University of Washington since 2005.

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Leveraging label-free dynamic mass redistribution technology to study G protein-coupled receptor ligand pharmacodynamics



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