Antibodies that identify only the active conformation of G(i) family G protein alpha subunits.
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.
Lane
J Robert
JR
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK.
Henderson
David
D
Powney
Ben
B
Wise
Alan
A
Rees
Stephen
S
Daniels
Dion
D
Plumpton
Chris
C
Kinghorn
Ian
I
Milligan
Graeme
G
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D044385:GTP-Binding Protein alpha Subunits; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006825:Hybridomas; D051379:Mice; D020125:Mutation, Missense / Q000276:immunology; D011487:Protein Conformation; D017448:Receptors, Dopamine D2
D000906:Antibodies; D044385:GTP-Binding Protein alpha Subunits; D017448:Receptors, Dopamine D2; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D019206:GTP-Binding Protein alpha Subunits, Gi-Go