Abstract

Genetically encoded biosensors based on Förster resonance energy transfer (FRET) can visualize responses of individual cells in real time. Here, we evaluated whether FRET-based biosensors provide sufficient contrast and specificity to measure activity of G-protein–coupled receptors. The four histamine receptor subtypes (H₁R, H₂R, H₃R, and H₄R) respond to the ligand histamine by activating three canonical heterotrimeric G-protein–mediated signaling pathways with a reported high degree of specificity. Using FRET-based biosensors, we demonstrate that H₁R activates Gαq. We also observed that H₁R activates Gαi, albeit at a 10-fold lower potency. In addition to increasing cAMP levels, most likely via Gαs, we found that the H₂R induces Gαq-mediated calcium release. The H₃R and H₄R activated Gαi with high specificity and a high potency. We demonstrate that a number of FRET sensors provide sufficient contrast to: 1) analyze the specificity of the histamine receptor subtypes for different heterotrimeric G-protein families with single-cell resolution, 2) probe for antagonist specificity, and 3) allow the measurement of single-cell concentration-response curves.