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

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology
J Robert LaneG Milligan

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...Continue Reading

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Citations

Jul 22, 2014·Nature Communications·Inmaculada Lopez-SanchezPradipta Ghosh
Sep 5, 2014·Molecular Biology of the Cell·Changsheng LinPradipta Ghosh
Feb 26, 2015·Proceedings of the National Academy of Sciences of the United States of America·Krishna K MiddePradipta Ghosh
Jul 12, 2014·Journal of the American Society of Nephrology : JASN·Honghui WangMarilyn G Farquhar
Aug 18, 2020·The Journal of Cell Biology·Andre Landin MaltXiaowei Lu
Nov 4, 2020·Proceedings of the National Academy of Sciences of the United States of America·Nicholas A KalogriopoulosPradipta Ghosh

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