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

Lane, J.R., Henderson, D., Powney, B., Wise, A., Rees, S., Daniels, D., Plumpton, C., Kinghorn, I. and Milligan, G. (2008) Antibodies that identify only the active conformation of Gi family G protein α subunits. FASEB Journal, 22, pp. 1924-1932. (doi: 10.1096/fj.07-100388)

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Publisher's URL: http://dx.doi.org/10.1096/fj.07-100388

Abstract

Production of antisera able to recognize individual heterotrimeric G proteinαsubunits 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 Gαi1 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 Gαi1. This profile extended to other closely related Gi family G proteins but not to the less closely related Gαs and Gαq/Gα11 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 Gαi 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.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Milligan, Professor Graeme
Authors: Lane, J.R., Henderson, D., Powney, B., Wise, A., Rees, S., Daniels, D., Plumpton, C., Kinghorn, I., and Milligan, G.
Subjects:Q Science > QH Natural history > QH345 Biochemistry
College/School:College of Medical Veterinary and Life Sciences > Institute of Neuroscience and Psychology
Journal Name:FASEB Journal
ISSN:0892-6638

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