Antibodies that identify only the active conformation of G_i 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 G_i family G protein α subunits. FASEB Journal, 22, pp. 1924-1932. (doi: 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 Gα_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 Gα_i1. This profile extended to other closely related G_i family G proteins but not to the less closely related Gα_s and Gα_q/Gα₁₁ 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 Gα_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.

• 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 > School of Psychology & Neuroscience
• Journal Name: FASEB Journal
• ISSN: 0892-6638