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
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.