Characterizing GPCR Signaling
Our experiments have shown that label-free confirms the functional pharmacological profile (i.e., agonist, antagonist, inverse or partial agonist or allosteric modulator) of a particular ligand receptor interaction as shown by comparable labeled assays, regardless of exact potencies. For well-studied, canonical ligands and receptor targets, a ligand showing agonist behavior in a labeled system will present agonist behavior in label-free.
There is no reason to expect exact correlation between potencies in labeled and label-free analysis. In fact, there will probably be variation due to major differences in how the two platforms record cellular behavior. One of three things can happen when studying a ligand’s behavior using labeled and label-free assays: potency determined by the label-free is higher; potency determined by the labeled assay is higher; they are equivalent.
While ligand potency values may differ from label-free to labeled systems, the rank ordering of potencies between the two should be consistent. This has been shown in many publications.
Each outcome gives important information about both the pathway-dependent and global events upon receptor activation and is an important value-add for researchers. In two studies, a label-free assay pointed to activation of the Gq signaling pathway, which the labeled calcium assay confirmed. In another two studies, label-free pointed to activation of the Gi pathway, which was then confirmed by the labeled AlphaScreen™ cAMP assay (PerkinElmer). Importantly, a single label-free assay will indicate if no activation has occurred, which a labeled assay may not.