Antibodies that identify only the active conformation of Gi family G protein alpha subunits

Production of antisera able to recognize individual heterotrimeric G protein I? subunits resulted in rapid expansion of information on their distribution and function. However, no antibodies that specifically recognize the active state have been available. Four-way primary screening of 763 hybridomas generated from mice immunized with guanosine 5 -O-(3-thio)triphosphate-loaded GI?i1 and isolated using an automated robotic colony picker identified three antibodies that interacted with the constitutively active, Q204L, mutant but neither the constitutively inactive, G203A, mutant nor wild-type GI?i1. This profile extended to other closely related Gi family G proteins but not to the less closely related GI?s and GI?q/GI?11 families. Each antibody was, however, also able to identify wild-type, GDP-bound Gi family G proteins in the presence of fluoroaluminate, which mimics the presence of the terminal phosphate of GTP and hence generates an active/transition state conformation. Stimulation of cells coexpressing a wild-type GI?i subunit and the dopamine D2 receptor with the agonist ligand nor-apomorphine also allowed these conformationally selective antibodies to bind the G protein. Such reagents allow the specific identification of activated G proteins in a native environment and may allow the development of label-free screening assays for G protein-coupled receptor-mediated activation of Gi family G proteins