Functional characterization of naturally expressed G-protein-coupled receptors in mammalian cells using the automated high-throughput pharmacological system HT-PS 100

Ilya Okun; Alex Okun; Vellareddy Anantharam; Mark E. Goldman; Michael Otto; Gregory V. Kaler

doi:10.1117/12.346752

21 April 1999 Functional characterization of naturally expressed G-protein-coupled receptors in mammalian cells using the automated high-throughput pharmacological system HT-PS 100

Ilya Okun, Alex Okun, Vellareddy Anantharam, Mark E. Goldman, Michael Otto, Gregory V. Kaler

Author Affiliations +

Proceedings Volume 3603, Systems and Technologies for Clinical Diagnostics and Drug Discovery II; (1999) https://doi.org/10.1117/12.346752
Event: BiOS '99 International Biomedical Optics Symposium, 1999, San Jose, CA, United States

Abstract

In studying the molecular mechanics of stimulation of a receptor and mechanisms of the receptor's interaction with ligands, the widely sued approach is to characterize dose- dependent functional responses of stimulation or inhibition of the receptor. Many GPCRs respond to the stimulation by transient changes in cytoplasmic calcium. A time trace of the ligand-evoked 'calcium signal' visualizes the sequence of signaling events taking place after the initial receptor stimulation. It is more important to know how those events depend on the ligand concentration. This type of data provides ligand affinity profiles together with information about mechanisms of the receptor/ligand interaction - competitive, non-competitive antagonism, full or partial stimulation. We have developed an automated system, HT-PS 100, for registering continuous concentration-dependent functional responses in real time at the rate of 2 min per dose-response curve. The flow-through fluidics prepares a concentration gradient of the compound and sequentially mixes it with another reagent, agonist or antagonist, and finally with cells. The resulting 'real time' concentration dependent signal is registered with a fluorescence detector. By monitoring calcium mobilization with Fura-2, we have functionally and mechanistically characterized a variety of G protein-coupled receptors, cholinergic, histaminergic, purinergic, endothelin, and bradykinin, endogenously expressed in different cell lines, SK-N-MC, TE671 and DDT₁MF-2.

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Ilya Okun, Alex Okun, Vellareddy Anantharam, Mark E. Goldman, Michael Otto, and Gregory V. Kaler "Functional characterization of naturally expressed G-protein-coupled receptors in mammalian cells using the automated high-throughput pharmacological system HT-PS 100", Proc. SPIE 3603, Systems and Technologies for Clinical Diagnostics and Drug Discovery II, (21 April 1999); https://doi.org/10.1117/12.346752

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