Glucocorticoid Receptor Activation Confirms Steroid Handling via Cell-based Luciferase Assay.

We recently published a study in Science Signaling establishing the G protein coupling profile of the adhesion GPCR, GPR97/ADGRG3 and demonstrated that the receptor is activated by its tethered peptide agonist to facilitate ex vivo human and mouse neutrophil polarization and chemokinetic migration (1). Prior to our work, the only agonists posited to activate GPR97/ADGRG3 were beclomethasone-related corticosteroids (2, 3). We tested beclomethasone alongside a GPR97/ADGRG3 tethered agonist (TA) peptidomimetic and an established ADGRG subfamily small molecule agonist (3-a-DOG) in multiple orthogonal signaling assays and neutrophil functional tests (1). Beclomethasone provided no ability to activate the receptor in any of these assays, whereas the TA peptidomimetic and 3-a-DOG did so consistently. The validity of our work was called into question with a claim that our laboratory may not have properly solubilized steroids and handled them incorrectly in the aqueous biochemical and cell-based assays. Our procedures for handling 3-a-DOG and the more difficult to solubilize TA peptidomimetic were identical to the way that we handled the steroids. To dispel this aspersion, we present a post-publication HEK293T cell-based Glucocorticoid Response Element Luciferase (GRE-LUC) assay demonstrating glucocorticoid activation of the endogenous glucocorticoid receptor (GR) that is present in the cells (4). This assay format is highly similar to the HEK293T cell-based serum response element (SRE)- and cyclic AMP response element (CRE)-Luciferase assays that we used to assign the TA peptidomimetic and 3-a-DOG, but not beclomethasone as GPR97/ADGRG3 agonists (1).