Single-cell measurements of GPCR activity

G-protein coupled receptors (GPCR) are a class of transmembrane receptor proteins that are involved in a wide range of physiological processes. Central to the classification of the GPCRs is the ability to activate G-proteins following binding of an agonist to receptor. Frizzleds (FZDs) are a ubiquitously expressed type of transmembrane receptor that share many features with GPCRs, but whose inclusion into that category is disputed due to the lack of evidence for functional G-protein activation. In the paper FZD5 is a Gαq-coupled receptor that exhibits the functional hallmarks of prototypical GPCRs, Hoffmann and co-workers use the BioPen to study single cells that express the receptor FZD₅ and show that the protein exhibits the functional hallmarks of a GPCR.

 Both the activation of FZD₅ and of a G-protein was investigated using fluorescence resonance energy transfer (FRET). BioPen was used to stimulate individual live-cells with the FZD₅ agonist WNT-5A. Because BioPen allows rapid exchange of the microenvironment around the cell, the authors were able to record the time-resolved activation of FZD₅ in single cells, which occurs 10-20 s after addition of WNT-5A and which appears as a dequenching of the FRET donor. A similar assay was used for G-protein activation, where dissociation of the G-protein subunits was used as a marker. Here, a dequenching of the FRET donor following ligand addition was again observed.

Together, this together shows that WNT-5A not only activates FZD₅, but that receptor activation by the agonist also leads to subsequent activation of the G-protein. This completed the link between ligand, receptor and G-protein activation and confirms that FZD₅ can be classified as a GPCR.