Nicole Merten, University of Bonn, Institute for Pharmaceutical Biology, Molecular-, Cellular- and Pharmacobiology Section, Bonn
It is now well accepted that G protein-coupled receptors (GPCRs) simultaneously engage distinct G proteins leading to the activation of multiple downstream effectors, potentially with different potencies, efficacies and in vivo relevance. While traditional second messenger assays are intended to portray precisely the activation of defined signaling cascades, label-free technology platforms capture complex phenotypic cell responses in a G protein-unbiased manner and are therefore ideally suited for investigations of orphan receptors. We took advantage of label-free assays, based on dynamic mass redistribution (DMR) of cellular constituents, to investigate the signaling of GPR17, initially in recombinant cells.
This receptor has been proposed as dualistic GPCR responding to cysteinyl-leukotrienes as well as uracil-nucleotides and represents according to genetic studies a negative regulator of oligodendrocyte maturation and myelination. We show that GPR17 is completely insensitive towards the putative endogenous ligands but can reliably be activated with the synthetic small molecule MDL29,951. Application of signaling pathway-specific inhibitors allowed us to attribute obtained holistic DMR responses to the engagement of Gαi and Gαq. Due to the high sensitivity of label-free detection, the relevance of these two GPR17-induced G protein pathways could be verified in the endogenous environment of primary rat oligodendrocytes, identifying Gαi proteins as the major contributor to global activity in these cells. Finally, it has been disclosed that activation of GPR17 with MDL29,951 has a negative impact on the oligodendroglial expression of the myelination marker myelin basic protein which largely depends on Gαi activation, emphasizing the predictive power of label-free technology for the relevance of distinct GPCR pathways in physiological processes.
