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

J. Robert Lane^*, David Henderson^*, Ben Powney, Alan Wise, Stephen Rees, Dion Daniels, Chris Plumpton, Ian Kinghorn and Graeme Milligan^*,1

^* Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK;

Screening and Compound Profiling, GlaxoSmithKline Research and Development, New Frontiers Science Park, Harlow, Essex, UK; and

Biological Reagents and Assay Development, GlaxoSmithKline Research and Development, Medicines Research Centre, Stevenage, Herts, UK

¹Correspondence: Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK. E-mail: g.milligan{at}bio.gla.ac.uk

Production of antisera able to recognize individual heterotrimeric G protein 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 G_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 G_i1. This profile extended to other closely related G_i family G proteins but not to the less closely related G_s and G_q/G₁₁ families. Each antibody was, however, also able to identify wild-type, GDP-bound G_i 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 G_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 G_i family G proteins.—Lane, J. R., Henderson, D., Powney, B., Wise, A., Rees, S., Daniels, D., Plumpton, C., Kinghorn, I., Milligan, G. Antibodies that identify only the active conformation of G_i family G protein subunits.

Key Words: G protein-coupled receptor • ligand screening