Regulation of the G

[摘要] Transmembrane signaling through Gαq-coupled receptors is linked to physiological processes such as cardiovascular development and smooth muscle function.Crystallographic studies have shown that Gαq forms an effector like interaction with G protein-coupled receptor kinase 2 (GRK2) in a manner that does not appear to sterically overlap with the binding site for regulators of G protein signaling (RGS) proteins.We confirm the formation of higher order RGS-Gαq-effector complexes using a flow cytometry protein interaction assay (FCPIA).Our data further supports the notion that GRK2 is a bona fide effector of Gαq.Small molecule inhibitors that could specifically inhibit the binding of Gαq to GRK2 would serve as powerful pharmacological tools that could be used to better understand the full significance of this interaction.We developed a high-throughput screening assay using FCPIA to identify small molecule inhibitors of the Gαq-GRK2 interaction.While our initial screening efforts failed to yield any discernable lead compounds, modifications of our assay could be used to screen additional chemical libraries that are better designed towards inhibiting protein-protein interactions. The canonical role of GRK2 is to phosphorylate the cytoplasmic tails/loops of activated G protein-coupled receptors (GPCRs).Phosphorylated receptors can then be uncoupled from G proteins, thereby desensitizing the signaling pathway.Since the discovery of a linkage between the over-expression of GRK2 and heart failure, GRK2 has become a pharmaceutical target for the treatment of heart disease.Takeda Pharmaceuticals have discovered a class of selective GRK2 inhibitors.We have solved the crystal structures of two of these compounds bound to GRK2-Gβγ.Our crystal structures reveal that these compounds bind to an inactive kinase conformation, in a manner similar to the cancer drug imatinib.Recently, an RNA aptamer has been discovered that is also capable of selectively inhibiting GRK2.Crystallographic data indicates that the aptamer stabilizes an inactive conformation similar to that recognized by the Takeda compounds.We have developed an aptamer displacement assay using FCPIA that is currently in use to screen for small molecule inhibitors that bind to this inactive kinase conformation.

[发布机构] University of Michigan

[效力级别] G Alpha Q Signaling

[关键词] G Protein-Coupled Receptor Kinase 2;G Alpha Q Signaling;High Throughput Screening;Protein-Protein Interactions;Biological Chemistry;Science;Chemical Biology