Thromboxane A2 (TxA2), a potent platelet aggregator and vasoconstrictor, is produced by cyclooxygenase-mediated cleavage of arachidonic acid. Its cellular effects are mediated through a Gq protein-coupled receptor (TP), leading to protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activation. However, the mechanism of the latter effect has been poorly characterized. Relying on previous studies, which suggest that epidermal growth factor receptor (EGFR) is the link between Gq/Gi protein-coupled receptors and MAPK activation, Gao et al.1xThe Thromboxane A2 receptor activates mitogen-activated protein kinase via protein kinase C-dependent Gi coupling and Src-dependent phosphorylation of the epidermal growth factor receptor. Gao, Y. et al. J. Pharm. Exp. Ther. 2001; 296: 426–433PubMedSee all References1 embark on a ‘pharmacological and molecular’ dissection of the TxA2–MAPK signaling pathway. Specifically, this study illuminates a network of interactions between a G protein-coupled receptor, phosphoinositide, and a growth factor receptor pathway. More importantly, however, the methods illustrate the principles of chemical genetics, and portend the promise of this new field.Armed with an arsenal of activators and inhibitors of TP, Gi, PKC, EGFR, and Src tyrosine kinase, the authors investigate the effects of these modulators on MAPK phosphorylation. Starting with the TxA2 agonist IBOP, they show that treatment of a human bladder cancer cell line with certain inhibitors attenuates IBOP-stimulated MAPK phosphorylation. Working backwards toward the physiological stimulus, they demonstrate that IBOP-mediated phosphorylation of EGFR is suppressed by each inhibitor, suggesting that the EGFR is upstream of MAPK but downstream of Gi, PKC, and Src. The role of Src in TP-dependent activation of MAPK was further tested using a dominant negative Src construct. The authors demonstrate that, in response to IBOP, both Src kinase activity and MAPK phosphorylation are stimulated either in the presence or the absence of an inhibitor of EGFR, confirming a place for Src upstream of the EGFR in this network. Using the agonists LPA and PMA, Gao et al. investigate the roles of Gi and PKC, respectively, in MAPK phosphorylation. Calphostin C, a PKC inhibitor, has no effect on LPA stimulation, whereas each other inhibitor reduces MAPK phosphorylation, albeit slightly. Pursuing the role of PKC in TP-Gi signaling, the authors note an inhibitor-mediated decrease in TP-G?i interaction, as measured by immunoprecipitation and Western blot. Gao et al. close by modeling the PKC most proximal to the TxA2 receptor itself and as the primary facilitator of Src activity, EGFR autophosphorylation and MAPK phosphorylation.In classical genetics, the relationships between the elements of a signal transduction network are studied using epistatic analysis, whereby a sequence of biological events is inferred by comparing single and multiple gene deletions with the wild-type state. By analogy, chemical epistatic analysis is the process of establishing such a relationship using small molecules, rather than mutations, to alter protein function. Small molecules, as well as temperature-sensitive mutations, provide the additional advantage of temporal control over protein function. Gao et al. have successfully applied these principles to shed new light on the interaction between a G protein-coupled receptor and the MAPK cascade. In particular, the positioning of Src upstream of EGFR represents an important departure from conventional wisdom. As the number of ligands that modulate protein function increases, we will probably see similar studies that employ chemical epistatic analysis to define network relationships in signal transduction.