Duplexed label-free G protein--coupled receptor assays for high-throughput screening.

This article describes duplexed label-free optical biosensor cellular assays for simultaneously assaying 2 endogenous receptors, the G(q)-coupled histamine receptor (H( 1)) and the G(s)-coupled beta(2)-adrenergic receptor (beta(2)AR), in A431 cells. The biosensor cellular assays consist of 2 sequential steps-an initial agonist screening using Sigma LOPAC (Library of Pharmaceutically Active Compounds) and a subsequent antagonist screening using a solution mixture containing the H(1) agonist histamine and the beta(2)AR agonist epinephrine. Results showed that costimulating A431 cells with histamine and epinephrine led to an optical response additive to individual responses. The agonist screening not only identified all full agonists for both the H(1) and beta(2) receptors, but also detected pathway-biased ligands for the beta(2)AR. Furthermore, the succeeding antagonist screening documented all known antagonists in the library for either the H(1) or beta(2) receptors. This is the 1st demonstration of a single cellular assay that is capable of screening ligands against 2 GPCRs coupled to distinct G proteins, and highlights the power of pathway-unbiased and label-free biosensor cellular assays for GPCR screens.