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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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Single-reactive cysteine mutants are fully functional. The affinity of the agonist isoproterenol (A) and the inverse agonist ICI 118,551 (B) was measured for all three single-cysteine mutants (Δ5-T66C, Δ5-A265C and Δ5-R333C) and wild-type receptor by competitive binding of [3H]-DHA. Results are expressed as percent of radio-ligand bound in the absence of competitor. (C) Functionality of the three single-cysteine mutants, unlabelled or labelled with Cy5, and wild-type receptor was determined by GTPγS binding as described in the Supplementary data. [35S]-GTPγS-specific binding induced by 10 μM isoproterenol (agonist response) or by 10 μM ICI 118,551 (inverse agonist response) is shown as fold over basal. All functional data represent the mean±s.e.m. of three independent experiments performed in triplicate.
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f4: Single-reactive cysteine mutants are fully functional. The affinity of the agonist isoproterenol (A) and the inverse agonist ICI 118,551 (B) was measured for all three single-cysteine mutants (Δ5-T66C, Δ5-A265C and Δ5-R333C) and wild-type receptor by competitive binding of [3H]-DHA. Results are expressed as percent of radio-ligand bound in the absence of competitor. (C) Functionality of the three single-cysteine mutants, unlabelled or labelled with Cy5, and wild-type receptor was determined by GTPγS binding as described in the Supplementary data. [35S]-GTPγS-specific binding induced by 10 μM isoproterenol (agonist response) or by 10 μM ICI 118,551 (inverse agonist response) is shown as fold over basal. All functional data represent the mean±s.e.m. of three independent experiments performed in triplicate.

Mentions: We performed saturation binding on purified, reconstituted receptor to determine the affinity of all three single-cysteine mutants for the antagonist [3H]-dihydroalprenolol (DHA). We observed no significant difference between the three modified β2ARs and wild-type β2ARs (Table I and Supplementary Figure 3). Competition binding studies with [3H]-DHA were used to determine the Ki values for the agonist isoproterenol (Iso) and the inverse agonist ICI 118,551 (ICI). As shown in Table I and Figure 4, the values for the single-cysteine mutants are comparable to those obtained for wild-type β2AR, suggesting that introduction of the single-reactive cysteines and reconstitution of purified β2AR into lipid vesicles does not alter the pharmacology of the receptor.


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)

Single-reactive cysteine mutants are fully functional. The affinity of the agonist isoproterenol (A) and the inverse agonist ICI 118,551 (B) was measured for all three single-cysteine mutants (Δ5-T66C, Δ5-A265C and Δ5-R333C) and wild-type receptor by competitive binding of [3H]-DHA. Results are expressed as percent of radio-ligand bound in the absence of competitor. (C) Functionality of the three single-cysteine mutants, unlabelled or labelled with Cy5, and wild-type receptor was determined by GTPγS binding as described in the Supplementary data. [35S]-GTPγS-specific binding induced by 10 μM isoproterenol (agonist response) or by 10 μM ICI 118,551 (inverse agonist response) is shown as fold over basal. All functional data represent the mean±s.e.m. of three independent experiments performed in triplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Single-reactive cysteine mutants are fully functional. The affinity of the agonist isoproterenol (A) and the inverse agonist ICI 118,551 (B) was measured for all three single-cysteine mutants (Δ5-T66C, Δ5-A265C and Δ5-R333C) and wild-type receptor by competitive binding of [3H]-DHA. Results are expressed as percent of radio-ligand bound in the absence of competitor. (C) Functionality of the three single-cysteine mutants, unlabelled or labelled with Cy5, and wild-type receptor was determined by GTPγS binding as described in the Supplementary data. [35S]-GTPγS-specific binding induced by 10 μM isoproterenol (agonist response) or by 10 μM ICI 118,551 (inverse agonist response) is shown as fold over basal. All functional data represent the mean±s.e.m. of three independent experiments performed in triplicate.
Mentions: We performed saturation binding on purified, reconstituted receptor to determine the affinity of all three single-cysteine mutants for the antagonist [3H]-dihydroalprenolol (DHA). We observed no significant difference between the three modified β2ARs and wild-type β2ARs (Table I and Supplementary Figure 3). Competition binding studies with [3H]-DHA were used to determine the Ki values for the agonist isoproterenol (Iso) and the inverse agonist ICI 118,551 (ICI). As shown in Table I and Figure 4, the values for the single-cysteine mutants are comparable to those obtained for wild-type β2AR, suggesting that introduction of the single-reactive cysteines and reconstitution of purified β2AR into lipid vesicles does not alter the pharmacology of the receptor.

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