RT Journal Article
SR Electronic
T1 Quantitative Single-Cell Analysis of Signaling Pathways Activated Immediately Downstream of Histamine Receptor Subtypes
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 162
OP 176
DO 10.1124/mol.116.104505
VO 90
IS 3
A1 van Unen, Jakobus
A1 Rashidfarrokhi, Ali
A1 Hoogendoorn, Eelco
A1 Postma, Marten
A1 Gadella, Theodorus W. J.
A1 Goedhart, Joachim
YR 2016
UL http://molpharm.aspetjournals.org/content/90/3/162.abstract
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.