PT  - JOURNAL ARTICLE
AU  - van Unen, Jakobus
AU  - Rashidfarrokhi, Ali
AU  - Hoogendoorn, Eelco
AU  - Postma, Marten
AU  - Gadella, Theodorus W. J.
AU  - Goedhart, Joachim
TI  - Quantitative Single-Cell Analysis of Signaling Pathways Activated Immediately Downstream of Histamine Receptor Subtypes
AID  - 10.1124/mol.116.104505
DP  - 2016 Sep 01
TA  - Molecular Pharmacology
PG  - 162--176
VI  - 90
IP  - 3
4099  - http://molpharm.aspetjournals.org/content/90/3/162.short
4100  - http://molpharm.aspetjournals.org/content/90/3/162.full
SO  - Mol Pharmacol2016 Sep 01; 90
AB  - 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.