title: Quantitative Single-Cell Analysis of Signaling Pathways Activated Immediately Downstream of Histamine Receptor Subtypes.
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.
authors.affiliation: Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Jakobus
authors.initials: J
authors.lastname: van Unen
authors.affiliation: Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Ali
authors.initials: A
authors.lastname: Rashidfarrokhi
authors.affiliation: Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Eelco
authors.initials: E
authors.lastname: Hoogendoorn
authors.affiliation: Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Marten
authors.initials: M
authors.lastname: Postma
authors.affiliation: Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Theodorus W J
authors.initials: TW
authors.lastname: Gadella
authors.affiliation: Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, Amsterdam, The Netherlands j.goedhart@uva.nl.
authors.forename: Joachim
authors.initials: J
authors.lastname: Goedhart
mesh_terms: D015374:Biosensing Techniques; D020013:Calcium Signaling; D000242:Cyclic AMP / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008954:Models, Biological; D011968:Receptors, Histamine / Q000378:metabolism*; D015398:Signal Transduction; D059010:Single-Cell Analysis / Q000379:methods*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D008164:Luminescent Proteins; D011968:Receptors, Histamine; D000242:Cyclic AMP; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020742:rhoA GTP-Binding Protein