Antibodies that identify only the active conformation of G(i) family G protein alpha subunits

Lane, J Robert; Henderson, David; Powney, Ben; Wise, Alan; Rees, Stephen; Daniels, Dion; Plumpton, Chris; Kinghorn, Ian; Milligan, Graeme

Abstract

Production of antisera able to recognize individual heterotrimeric G protein alpha 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 alpha(i1) and isolated using an automated robotic colony picker identified three antibodies that interacted with the constitutively active, Q(204)L, mutant but neither the constitutively inactive, G(203)A, mutant nor wild-type G alpha(i1). This profile extended to other closely related G(i) family G proteins but not to the less closely related G alpha(s) and G alpha(q)/G alpha(11) 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 alpha(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.

Citation

Lane, J. R., Henderson, D., Powney, B., Wise, A., Rees, S., Daniels, D., …Milligan, G. (2008). Antibodies that identify only the active conformation of G(i) family G protein alpha subunits. FASEB Journal, 22(6), 1924-1932. https://doi.org/10.1096/fj.07-100388

• Journal Article Type: Article
• Acceptance Date: Dec 6, 2007
• Online Publication Date: Jan 16, 2008
• Publication Date: 2008-06
• Deposit Date: Apr 22, 2020
• Print ISSN: 0892-6638
• Publisher: Federation of American Society of Experimental Biology
• Peer Reviewed: Peer Reviewed
• Volume: 22
• Issue: 6
• Pages: 1924-1932
• DOI: https://doi.org/10.1096/fj.07-100388
• Public URL: http://www.fasebj.org/cgi/doi/10.1096/fj.07-100388
• Publisher URL: https://faseb.onlinelibrary.wiley.com/doi/full/10.1096/fj.07-100388