Quantitative Single-Cell Analysis of Signaling Pathways Activated Immediately Downstream of Histamine Receptor Subtypes

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.