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Ligand-biased signaling is starting to have significant impact on drug discovery programs in the pharmaceutical industry and has reinvigorated our understanding of pharmacological efficacy. As such, many investigators and screening campaigns are now being directed at a larger section of the signaling responses downstream of an individual G protein-coupled receptor. Many biosensor-based platforms have been developed to capture signaling signatures. Despite our growing ability to use such signaling signatures, we remain hampered by the fact that signaling signatures may be particular to an individual cell type and thus our platforms may not be portable from cell to cell, necessitating further cell-specific biosensor development. Here, we provide a complementary strategy based on capturing receptor-proximal conformational profiles using intra-molecular BRET-based sensors composed of a Renilla luciferase donor engineered into the carboxy-terminus and CCPGCC motifs which bind fluorescent hairpin arsenical dyes engineered into different positions in intracellular loop 3 of FP, the receptor for PGF2α. We discuss the design and optimization of such sensors for orthosteric and allosteric ligands., (Copyright © 2015 Elsevier Inc. All rights reserved.)

G protein-coupled receptors (GPCRs) represent one of the largest families of cell surface receptors as key targets for pharmacological manipulation. G proteins have long been recognized as allosteric modulators of GPCR ligand binding. More recently, small molecule allosteric modulators have now been widely characterized for a number of GPCRs, and some are now used clinically. Many studies have also underscored the importance of GPCR dimerization or higher-order oligomerization in the control of the physiological responses they modulate. Thus, allosterism can also, between monomer equivalents in the context of a dimer, oligomer, or receptor mosaic, influence signaling pathways downstream. It therefore becomes essential to characterize both small molecule allosteric ligands and allosteric interactions between receptors modulated by canonical orthosteric ligands, in a pathway-specific manner. Here, we describe a simple, radioligand-binding method, which is designed to probe for allosteric modulation mediated by any GPCR interactor, from small molecules to interacting proteins. It can also detect allosteric asymmetries within a GPCR heterodimer, via orthosteric or allosteric ligands. This assay measures time-dependent ligand occupancy of radiolabeled orthosteric or (with adaptations) allosteric ligands as modulated by either small molecules or receptor dimer partners bound or unbound with their own ligands., (Copyright © 2013 Elsevier Inc. All rights reserved.)