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Ligand-regulated oligomerization of beta(2)-adrenoceptors in a model lipid bilayer.

Fung JJ, Deupi X, Pardo L, Yao XJ, Velez-Ruiz GA, Devree BT, Sunahara RK, Kobilka BK - EMBO J. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA.

ABSTRACT
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.

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Schematic representation of possible β2AR oligomers. (A) Cytoplasmic view of the 3D structure of the β2AR (left) and cartoon of this footprint (right), with the centre of mass of Cy3 depicted as spheres: T66C (green), A265C (blue) and R333C (red). TM6 is depicted in the inactive conformation (top) and in the proposed active conformation as observed in the structure of opsin (bottom). (B) Receptor oligomerization involving the surfaces of TMs 4 or/and 5 is not compatible with our FRET results and might sterically prevent movement of TM6. (C) Our FRET results suggest an arrangement of protomers involving the TM1 interface. (D) The movement of the cytoplasmic end of TM6 upon agonist binding repositions the fluorophore outwards in two of the four protomers, and towards H8 in the other two. (E) Treatment with the inverse agonist ICI probably reduces conformational fluctuations responsible for basal activity, increasing the packing of the oligomers. (F) ICI may stabilize higher-order oligomers where TM6 is packed into the core of the oligomer, contributing to increase FRET efficiency and possibly the inactive state of the receptor.
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f9: Schematic representation of possible β2AR oligomers. (A) Cytoplasmic view of the 3D structure of the β2AR (left) and cartoon of this footprint (right), with the centre of mass of Cy3 depicted as spheres: T66C (green), A265C (blue) and R333C (red). TM6 is depicted in the inactive conformation (top) and in the proposed active conformation as observed in the structure of opsin (bottom). (B) Receptor oligomerization involving the surfaces of TMs 4 or/and 5 is not compatible with our FRET results and might sterically prevent movement of TM6. (C) Our FRET results suggest an arrangement of protomers involving the TM1 interface. (D) The movement of the cytoplasmic end of TM6 upon agonist binding repositions the fluorophore outwards in two of the four protomers, and towards H8 in the other two. (E) Treatment with the inverse agonist ICI probably reduces conformational fluctuations responsible for basal activity, increasing the packing of the oligomers. (F) ICI may stabilize higher-order oligomers where TM6 is packed into the core of the oligomer, contributing to increase FRET efficiency and possibly the inactive state of the receptor.

Mentions: Our reconstitution system, as well as current cell-based methods, is limited in the ability to precisely define the structure and stoichiometry of oligomers. Nevertheless, recent evidence from resonance energy transfer studies, cysteine cross-linking and fluorescence recovery after photobleaching (FRAP) studies suggest that the D2 dopamine receptor and the β2AR form higher-order oligomers (tetramers or greater) (Guo et al, 2008; Dorsch et al, 2009). Data from FRET studies can provide insight into the orientation of protomers within these oligomers. However, the following facts should be taken into account. First, while Cy3 and Cy5 are small relative to fluorescent and luminescent proteins, they are still large compared with amino acids and have relatively long flexible linkers tethering them to cysteine (Figure 9A). As such, their precise orientation in the protein is not known and has been estimated by computational techniques. Second, both the distance between fluorophores and the orientation of fluorophores influence FRET. The orientation factor becomes a concern for highly constrained fluorophores; however, this probably is not the case here, since the anisotropy values that we observe experimentally for Cy3 and Cy5 are similar to those reported for free fluorophores in solution (Kobitski et al, 2007), and these values are not influenced by ligands. Third, as discussed below, the oligomers may be dynamic. While the predominant form may be a tetramer, these may exist in equilibrium with monomers, dimers and higher-order oligomers. Finally, in a tetramer several combinations of donor/acceptor pairs (0 donors, 4 acceptors; 1 donor, 3 acceptors; etc…) are possible. In such a system, the measured FRET signal will be the combination of all the individual energy transfers between every possible donor/acceptor pair, which complicates the analysis of the measured FRET signal, and thus, the estimation of protomer orientation in the tetramer.


Ligand-regulated oligomerization of beta(2)-adrenoceptors in a model lipid bilayer.

Fung JJ, Deupi X, Pardo L, Yao XJ, Velez-Ruiz GA, Devree BT, Sunahara RK, Kobilka BK - EMBO J. (2009)

Schematic representation of possible β2AR oligomers. (A) Cytoplasmic view of the 3D structure of the β2AR (left) and cartoon of this footprint (right), with the centre of mass of Cy3 depicted as spheres: T66C (green), A265C (blue) and R333C (red). TM6 is depicted in the inactive conformation (top) and in the proposed active conformation as observed in the structure of opsin (bottom). (B) Receptor oligomerization involving the surfaces of TMs 4 or/and 5 is not compatible with our FRET results and might sterically prevent movement of TM6. (C) Our FRET results suggest an arrangement of protomers involving the TM1 interface. (D) The movement of the cytoplasmic end of TM6 upon agonist binding repositions the fluorophore outwards in two of the four protomers, and towards H8 in the other two. (E) Treatment with the inverse agonist ICI probably reduces conformational fluctuations responsible for basal activity, increasing the packing of the oligomers. (F) ICI may stabilize higher-order oligomers where TM6 is packed into the core of the oligomer, contributing to increase FRET efficiency and possibly the inactive state of the receptor.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2748299&req=5

f9: Schematic representation of possible β2AR oligomers. (A) Cytoplasmic view of the 3D structure of the β2AR (left) and cartoon of this footprint (right), with the centre of mass of Cy3 depicted as spheres: T66C (green), A265C (blue) and R333C (red). TM6 is depicted in the inactive conformation (top) and in the proposed active conformation as observed in the structure of opsin (bottom). (B) Receptor oligomerization involving the surfaces of TMs 4 or/and 5 is not compatible with our FRET results and might sterically prevent movement of TM6. (C) Our FRET results suggest an arrangement of protomers involving the TM1 interface. (D) The movement of the cytoplasmic end of TM6 upon agonist binding repositions the fluorophore outwards in two of the four protomers, and towards H8 in the other two. (E) Treatment with the inverse agonist ICI probably reduces conformational fluctuations responsible for basal activity, increasing the packing of the oligomers. (F) ICI may stabilize higher-order oligomers where TM6 is packed into the core of the oligomer, contributing to increase FRET efficiency and possibly the inactive state of the receptor.
Mentions: Our reconstitution system, as well as current cell-based methods, is limited in the ability to precisely define the structure and stoichiometry of oligomers. Nevertheless, recent evidence from resonance energy transfer studies, cysteine cross-linking and fluorescence recovery after photobleaching (FRAP) studies suggest that the D2 dopamine receptor and the β2AR form higher-order oligomers (tetramers or greater) (Guo et al, 2008; Dorsch et al, 2009). Data from FRET studies can provide insight into the orientation of protomers within these oligomers. However, the following facts should be taken into account. First, while Cy3 and Cy5 are small relative to fluorescent and luminescent proteins, they are still large compared with amino acids and have relatively long flexible linkers tethering them to cysteine (Figure 9A). As such, their precise orientation in the protein is not known and has been estimated by computational techniques. Second, both the distance between fluorophores and the orientation of fluorophores influence FRET. The orientation factor becomes a concern for highly constrained fluorophores; however, this probably is not the case here, since the anisotropy values that we observe experimentally for Cy3 and Cy5 are similar to those reported for free fluorophores in solution (Kobitski et al, 2007), and these values are not influenced by ligands. Third, as discussed below, the oligomers may be dynamic. While the predominant form may be a tetramer, these may exist in equilibrium with monomers, dimers and higher-order oligomers. Finally, in a tetramer several combinations of donor/acceptor pairs (0 donors, 4 acceptors; 1 donor, 3 acceptors; etc…) are possible. In such a system, the measured FRET signal will be the combination of all the individual energy transfers between every possible donor/acceptor pair, which complicates the analysis of the measured FRET signal, and thus, the estimation of protomer orientation in the tetramer.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus