Transducin activation by nanoscale lipid bilayers containing one and two rhodopsins.
Nanodiscs are nanometer scale planar membranes of controlled size that are rendered soluble in aqueous solution via an encircling amphipathic membrane scaffold protein "belt" (Bayburt, T. H., Grinkova, Y. V., and Sligar, S. G. (2002) Nano. Lett. 2, 853-856). Integral membrane proteins can be self-assembled into the Nanodisc bilayer with defined stoichiometry, which allows an unprecedented opportunity to investigate the nature of the oligomerization state of a G-protein-coupled receptor and its coupling to heterotrimeric G-proteins. We generated Nanodiscs having one and two rhodopsins present in the 10-nm-diameter lipid bilayer domain. Efficient transducin activation and isolation of a high affinity transducin-metarhodopsin II complex was demonstrated for a monodisperse and monomeric receptor. A population of Nanodiscs containing two rhodopsins was generated using an increased ratio of receptor to membrane scaffold protein in the self-assembly mixture. The two-rhodopsin population was isolated and purified by density gradient centrifugation. Interestingly, in this case, only one of the two receptors present in the Nanodisc was able to form a stable metarhodopsin II-G-protein complex. Thus there is clear evidence that a monomeric rhodopsin is capable of full coupling to transducin. Importantly, presumably due to steric interactions, it appears that only a single receptor in the Nanodiscs containing two rhodopsins can interact with G-protein. These results have important implications for the stoichiometry of receptor-G-protein coupling and cross talk in signaling pathways.
Bayburt
Timothy H
TH
Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, USA.
Leitz
Andrew J
AJ
Xie
Guifu
G
Oprian
Daniel D
DD
Sligar
Stephen G
SG
D000818:Animals; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry*; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics; D053758:Nanoparticles / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics; D015293:Transducin / Q000737:chemistry* / Q000235:genetics
D008051:Lipid Bilayers; D046912:Multiprotein Complexes; D011994:Recombinant Proteins; C013110:metarhodopsins; D012243:Rhodopsin; D015293:Transducin