Ligand-regulated oligomerization of beta(2)-adrenoceptors in a model lipid bilayer.
The beta(2)-adrenoceptor (beta(2)AR) was one of the first Family A G protein-coupled receptors (GPCRs) shown to form oligomers in cellular membranes, yet we still know little about the number and arrangement of protomers in oligomers, the influence of ligands on the organization or stability of oligomers, or the requirement for other proteins to promote oligomerization. We used fluorescence resonance energy transfer (FRET) to characterize the oligomerization of purified beta(2)AR site-specifically labelled at three different positions with fluorophores and reconstituted into a model lipid bilayer. Our results suggest that the beta(2)AR is predominantly tetrameric following reconstitution into phospholipid vesicles. Agonists and antagonists have little effect on the relative orientation of protomers in oligomeric complexes. In contrast, binding of inverse agonists leads to significant increases in FRET efficiencies for most labelling pairs, suggesting that this class of ligand promotes tighter packing of protomers and/or the formation of more complex oligomers by reducing conformational fluctuations in individual protomers. The results provide new structural insights into beta(2)AR oligomerization and suggest a possible mechanism for the functional effects of inverse agonists.
Fung
Juan José
JJ
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA.
Deupi
Xavier
X
Pardo
Leonardo
L
Yao
Xiao Jie
XJ
Velez-Ruiz
Gisselle A
GA
Devree
Brian T
BT
Sunahara
Roger K
RK
Kobilka
Brian K
BK
D003545:Cysteine / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008051:Lipid Bilayers / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008958:Models, Molecular; D017354:Point Mutation; D011485:Protein Binding; D055503:Protein Multimerization; D018343:Receptors, Adrenergic, beta-2 / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
D008024:Ligands; D008051:Lipid Bilayers; D008081:Liposomes; D018343:Receptors, Adrenergic, beta-2; D019204:GTP-Binding Proteins; D003545:Cysteine