Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of the G protein Gs on FRET saturation of Cy5- and Cy3-labelled β2AR. FRET saturation was performed by varying the ratio of Cy5- to Cy3-labelled β2AR-R333C over a range of 1:4 to 4:1 (Cy5:Cy3), while the overall β2AR concentration was kept constant. Purified Gs heterotrimer was added at a molar ratio of 3 Gs:1 β2AR before reconstitution. (A) The inclusion of Gs in the reconstitution did not alter the orientation of β2AR in vesicles as determined by the susceptibility of reconstituted β2AR to PNGase F (see Figure 3C). FRET saturation was significantly lower in the presence of Gs compared with β2AR alone (B) or β2AR and Gs with 10 μM GTPγS (C). (D) β2AR was labelled on C265 at the cytoplasmic end of TM6 with mBBr–β2AR and reconstituted with Gs under the same conditions that were used for FRET saturation experiments. Gs induced a decrease in intensity and a 4-nm shift in λMAX of mBBr–β2AR relative to the same reconstitution in the presence of GTPγS. A two-way ANOVA was used to compare FRET values for β2AR, β2AR+Gs and β2AR+Gs+GTPγS at the different Cy5:Cy3 ratios. A posteriori statistical analysis showed significant decrease in FRET between β2AR and β2AR+Gs (P<0.008), and a significant increase in FRET between β2AR+Gs and β2AR+Gs+GTPγS (P<0.04) for all Cy5:Cy3 ratios except 2 and 4. No statistical differences are found between β2AR and β2AR+Gs+GTPγS.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2748299&req=5

f8: Effect of the G protein Gs on FRET saturation of Cy5- and Cy3-labelled β2AR. FRET saturation was performed by varying the ratio of Cy5- to Cy3-labelled β2AR-R333C over a range of 1:4 to 4:1 (Cy5:Cy3), while the overall β2AR concentration was kept constant. Purified Gs heterotrimer was added at a molar ratio of 3 Gs:1 β2AR before reconstitution. (A) The inclusion of Gs in the reconstitution did not alter the orientation of β2AR in vesicles as determined by the susceptibility of reconstituted β2AR to PNGase F (see Figure 3C). FRET saturation was significantly lower in the presence of Gs compared with β2AR alone (B) or β2AR and Gs with 10 μM GTPγS (C). (D) β2AR was labelled on C265 at the cytoplasmic end of TM6 with mBBr–β2AR and reconstituted with Gs under the same conditions that were used for FRET saturation experiments. Gs induced a decrease in intensity and a 4-nm shift in λMAX of mBBr–β2AR relative to the same reconstitution in the presence of GTPγS. A two-way ANOVA was used to compare FRET values for β2AR, β2AR+Gs and β2AR+Gs+GTPγS at the different Cy5:Cy3 ratios. A posteriori statistical analysis showed significant decrease in FRET between β2AR and β2AR+Gs (P<0.008), and a significant increase in FRET between β2AR+Gs and β2AR+Gs+GTPγS (P<0.04) for all Cy5:Cy3 ratios except 2 and 4. No statistical differences are found between β2AR and β2AR+Gs+GTPγS.

Mentions: To investigate the effect of G protein coupling on oligomerization, we performed FRET saturation experiments by reconstituting β2AR with a three-fold molar excess of purified Gs heterotrimer (Figure 8). This concentration of G protein was chosen to ensure that sufficient G protein would be incorporated into vesicles while having a minimal effect on the reconstitution. The inclusion of Gs did not alter the orientation of the β2AR as determined by the susceptibility to PNGase F (Figure 8A). We observed a statistically significant (P<0.008) decrease in FRET saturation in the presence of Gs as compared with reconstitutions in the absence of Gs (Figure 8B) for Cy5/Cy3 of 0.25, 0.5 and 1. To determine whether the effect of Gs on FRET saturation was due to nonspecific effects of reconstituting with another membrane-associated protein, we performed FRET saturation of β2AR in the presence of Gs and GTPγS, which uncouples β2AR and Gs. As shown in Figure 8C, the presence of GTPγS increases, in a statistically significant manner (P<0.04), FRET saturation to the values observed for β2AR alone. To estimate the fraction of β2AR that couples to Gs under these reconstitution conditions, we labelled C265 at the cytoplasmic end of TM6 with mBBr, an environmentally sensitive fluorescent probe. We previously showed that maximal coupling of Gs to β2AR reconstituted into HDL particles results in a decrease in the fluorescence intensity and an 18-nm shift in the maximal emission wavelength (λMAX) of mBBr–β2AR (mBBr–β2AR; Yao et al, 2009). As shown in Figure 8D, under reconstitution conditions used for FRET saturation experiments, Gs induced a decrease in intensity and a 4-nm shift in λMAX of mBBr–β2AR relative to the same reconstitution in the presence of GTPγS. Based on the shift of λMAX we estimate that approximately 20% of the reconstituted β2AR is coupled to Gs.


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)

Effect of the G protein Gs on FRET saturation of Cy5- and Cy3-labelled β2AR. FRET saturation was performed by varying the ratio of Cy5- to Cy3-labelled β2AR-R333C over a range of 1:4 to 4:1 (Cy5:Cy3), while the overall β2AR concentration was kept constant. Purified Gs heterotrimer was added at a molar ratio of 3 Gs:1 β2AR before reconstitution. (A) The inclusion of Gs in the reconstitution did not alter the orientation of β2AR in vesicles as determined by the susceptibility of reconstituted β2AR to PNGase F (see Figure 3C). FRET saturation was significantly lower in the presence of Gs compared with β2AR alone (B) or β2AR and Gs with 10 μM GTPγS (C). (D) β2AR was labelled on C265 at the cytoplasmic end of TM6 with mBBr–β2AR and reconstituted with Gs under the same conditions that were used for FRET saturation experiments. Gs induced a decrease in intensity and a 4-nm shift in λMAX of mBBr–β2AR relative to the same reconstitution in the presence of GTPγS. A two-way ANOVA was used to compare FRET values for β2AR, β2AR+Gs and β2AR+Gs+GTPγS at the different Cy5:Cy3 ratios. A posteriori statistical analysis showed significant decrease in FRET between β2AR and β2AR+Gs (P<0.008), and a significant increase in FRET between β2AR+Gs and β2AR+Gs+GTPγS (P<0.04) for all Cy5:Cy3 ratios except 2 and 4. No statistical differences are found between β2AR and β2AR+Gs+GTPγS.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f8: Effect of the G protein Gs on FRET saturation of Cy5- and Cy3-labelled β2AR. FRET saturation was performed by varying the ratio of Cy5- to Cy3-labelled β2AR-R333C over a range of 1:4 to 4:1 (Cy5:Cy3), while the overall β2AR concentration was kept constant. Purified Gs heterotrimer was added at a molar ratio of 3 Gs:1 β2AR before reconstitution. (A) The inclusion of Gs in the reconstitution did not alter the orientation of β2AR in vesicles as determined by the susceptibility of reconstituted β2AR to PNGase F (see Figure 3C). FRET saturation was significantly lower in the presence of Gs compared with β2AR alone (B) or β2AR and Gs with 10 μM GTPγS (C). (D) β2AR was labelled on C265 at the cytoplasmic end of TM6 with mBBr–β2AR and reconstituted with Gs under the same conditions that were used for FRET saturation experiments. Gs induced a decrease in intensity and a 4-nm shift in λMAX of mBBr–β2AR relative to the same reconstitution in the presence of GTPγS. A two-way ANOVA was used to compare FRET values for β2AR, β2AR+Gs and β2AR+Gs+GTPγS at the different Cy5:Cy3 ratios. A posteriori statistical analysis showed significant decrease in FRET between β2AR and β2AR+Gs (P<0.008), and a significant increase in FRET between β2AR+Gs and β2AR+Gs+GTPγS (P<0.04) for all Cy5:Cy3 ratios except 2 and 4. No statistical differences are found between β2AR and β2AR+Gs+GTPγS.
Mentions: To investigate the effect of G protein coupling on oligomerization, we performed FRET saturation experiments by reconstituting β2AR with a three-fold molar excess of purified Gs heterotrimer (Figure 8). This concentration of G protein was chosen to ensure that sufficient G protein would be incorporated into vesicles while having a minimal effect on the reconstitution. The inclusion of Gs did not alter the orientation of the β2AR as determined by the susceptibility to PNGase F (Figure 8A). We observed a statistically significant (P<0.008) decrease in FRET saturation in the presence of Gs as compared with reconstitutions in the absence of Gs (Figure 8B) for Cy5/Cy3 of 0.25, 0.5 and 1. To determine whether the effect of Gs on FRET saturation was due to nonspecific effects of reconstituting with another membrane-associated protein, we performed FRET saturation of β2AR in the presence of Gs and GTPγS, which uncouples β2AR and Gs. As shown in Figure 8C, the presence of GTPγS increases, in a statistically significant manner (P<0.04), FRET saturation to the values observed for β2AR alone. To estimate the fraction of β2AR that couples to Gs under these reconstitution conditions, we labelled C265 at the cytoplasmic end of TM6 with mBBr, an environmentally sensitive fluorescent probe. We previously showed that maximal coupling of Gs to β2AR reconstituted into HDL particles results in a decrease in the fluorescence intensity and an 18-nm shift in the maximal emission wavelength (λMAX) of mBBr–β2AR (mBBr–β2AR; Yao et al, 2009). As shown in Figure 8D, under reconstitution conditions used for FRET saturation experiments, Gs induced a decrease in intensity and a 4-nm shift in λMAX of mBBr–β2AR relative to the same reconstitution in the presence of GTPγS. Based on the shift of λMAX we estimate that approximately 20% of the reconstituted β2AR is coupled to Gs.

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