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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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β2ARs are homogenously distributed in lipid vesicles. (A) To determine the distribution of β2ARs in lipid vesicles, sucrose density gradients of samples containing 0.4% NBD–phosphocholine and Cy5–β2ARs reconstituted at a lipid-to-receptor ratio of 1000:1 were performed as described in the Supplementary data. Detection of lipid fractions was performed by following NBD fluorescence (λex=460 nm) and receptor fractions by following Cy5 fluorescence (λex=649 nm). (B) Reconstituted β2ARs were imaged using a negative staining protocol as described in the Supplementary data to determine the size distribution of vesicles and the number of receptors per vesicle. Scale bar length represents 200 nm. Data are representative of three independent experiments.
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f3: β2ARs are homogenously distributed in lipid vesicles. (A) To determine the distribution of β2ARs in lipid vesicles, sucrose density gradients of samples containing 0.4% NBD–phosphocholine and Cy5–β2ARs reconstituted at a lipid-to-receptor ratio of 1000:1 were performed as described in the Supplementary data. Detection of lipid fractions was performed by following NBD fluorescence (λex=460 nm) and receptor fractions by following Cy5 fluorescence (λex=649 nm). (B) Reconstituted β2ARs were imaged using a negative staining protocol as described in the Supplementary data to determine the size distribution of vesicles and the number of receptors per vesicle. Scale bar length represents 200 nm. Data are representative of three independent experiments.

Mentions: In studying oligomerization, it is important to avoid forcing protein together by inhomogeneous reconstitution, that is, trapping the majority of the receptor molecules in a minor population of lipid vesicles. For instance, it has been shown previously that 90% of rhodopsin molecules were incorporated into only 10% of vesicles (Mansoor et al, 2006). We used isopycnic density centrifugation to assess the distribution of β2ARs in lipid vesicles as previously described for rhodopsin (Mansoor et al, 2006). Cy5-labelled β2ARs were reconstituted at a lipid-to-receptor ratio of 1000:1 in lipids containing NBD–phosphocholine (at a final of 0.4% of total lipid content). This allowed us to analyze samples subjected to a discontinuous sucrose density gradient by following Cy5 fluorescence (for the presence of β2AR) and NBD fluorescence (for the presence of lipid vesicles). Our results show nearly perfect correlation between Cy5 fluorescence and NBD fluorescence at every fraction analyzed, suggesting that β2AR molecules are uniformly distributed in these vesicles (Figure 3A). Similar results were obtained with β2AR reconstituted at a 10 000:1 lipid-to-receptor ratio (Supplementary Figure 2).


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)

β2ARs are homogenously distributed in lipid vesicles. (A) To determine the distribution of β2ARs in lipid vesicles, sucrose density gradients of samples containing 0.4% NBD–phosphocholine and Cy5–β2ARs reconstituted at a lipid-to-receptor ratio of 1000:1 were performed as described in the Supplementary data. Detection of lipid fractions was performed by following NBD fluorescence (λex=460 nm) and receptor fractions by following Cy5 fluorescence (λex=649 nm). (B) Reconstituted β2ARs were imaged using a negative staining protocol as described in the Supplementary data to determine the size distribution of vesicles and the number of receptors per vesicle. Scale bar length represents 200 nm. Data are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: β2ARs are homogenously distributed in lipid vesicles. (A) To determine the distribution of β2ARs in lipid vesicles, sucrose density gradients of samples containing 0.4% NBD–phosphocholine and Cy5–β2ARs reconstituted at a lipid-to-receptor ratio of 1000:1 were performed as described in the Supplementary data. Detection of lipid fractions was performed by following NBD fluorescence (λex=460 nm) and receptor fractions by following Cy5 fluorescence (λex=649 nm). (B) Reconstituted β2ARs were imaged using a negative staining protocol as described in the Supplementary data to determine the size distribution of vesicles and the number of receptors per vesicle. Scale bar length represents 200 nm. Data are representative of three independent experiments.
Mentions: In studying oligomerization, it is important to avoid forcing protein together by inhomogeneous reconstitution, that is, trapping the majority of the receptor molecules in a minor population of lipid vesicles. For instance, it has been shown previously that 90% of rhodopsin molecules were incorporated into only 10% of vesicles (Mansoor et al, 2006). We used isopycnic density centrifugation to assess the distribution of β2ARs in lipid vesicles as previously described for rhodopsin (Mansoor et al, 2006). Cy5-labelled β2ARs were reconstituted at a lipid-to-receptor ratio of 1000:1 in lipids containing NBD–phosphocholine (at a final of 0.4% of total lipid content). This allowed us to analyze samples subjected to a discontinuous sucrose density gradient by following Cy5 fluorescence (for the presence of β2AR) and NBD fluorescence (for the presence of lipid vesicles). Our results show nearly perfect correlation between Cy5 fluorescence and NBD fluorescence at every fraction analyzed, suggesting that β2AR molecules are uniformly distributed in these vesicles (Figure 3A). Similar results were obtained with β2AR reconstituted at a 10 000:1 lipid-to-receptor ratio (Supplementary Figure 2).

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