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Negative cooperativity across β1-adrenoceptor homodimers provides insights into the nature of the secondary low-affinity CGP 12177 β1-adrenoceptor binding conformation.

Gherbi K, May LT, Baker JG, Briddon SJ, Hill SJ - FASEB J. (2015)

Bottom Line: At the β1-adrenoceptor, CGP 12177 potently antagonizes agonist responses at the primary high-affinity catecholamine conformation while also exerting agonist effects of its own through a secondary low-affinity conformation.These effects on the BODIPY-TMR-CGP dissociation rate were markedly enhanced in β1-adrenoceptor homodimers constrained by bimolecular fluorescence complementation (9.8- and 9.9-fold for 1 µM CGP 12177 and 1 µM propranolol, respectively) and abolished in β1-adrenoceptors containing TM4 mutations vital for the second conformation pharmacology.This study suggests that negative cooperativity across a β1-adrenoceptor homodimer may be responsible for generating the low-affinity pharmacology of the secondary β1-adrenoceptor conformation.

View Article: PubMed Central - PubMed

Affiliation: Cell Signalling Research Group, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.

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Cell surface expression of BiFC-constrained β1-adrenoceptor homodimers. Confocal images show binding of 3 nM BODIPY-TMR-CGP following 4-min association to CHO-K1 cells transiently expressing BiFC-constrained wild-type β1-adrenoceptor homodimers (β1YFPN/β1YFPC; upper panel) and BiFC-constrained β1-adrenoceptor homodimers containing 1 wild-type and 1 nonligand-binding protomer (β1YFPN/β1D138AYFPC; lower panel). The fluorescence of the complimented YFP was measured simultaneously to confirm the cell surface expression of β1-adrenoceptor homodimers. Images are representative of 5 separate experiments. Scale bars, 50 μm. The binding of BODIPY-TMR-CGP in the lower right confirms that the β1YFPN/β1D138AYFPC dimer pairs contain a viable single orthosteric binding site.
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Figure 7: Cell surface expression of BiFC-constrained β1-adrenoceptor homodimers. Confocal images show binding of 3 nM BODIPY-TMR-CGP following 4-min association to CHO-K1 cells transiently expressing BiFC-constrained wild-type β1-adrenoceptor homodimers (β1YFPN/β1YFPC; upper panel) and BiFC-constrained β1-adrenoceptor homodimers containing 1 wild-type and 1 nonligand-binding protomer (β1YFPN/β1D138AYFPC; lower panel). The fluorescence of the complimented YFP was measured simultaneously to confirm the cell surface expression of β1-adrenoceptor homodimers. Images are representative of 5 separate experiments. Scale bars, 50 μm. The binding of BODIPY-TMR-CGP in the lower right confirms that the β1YFPN/β1D138AYFPC dimer pairs contain a viable single orthosteric binding site.

Mentions: BiFC uses 2 nonfluorescent fragments of a fluorescent protein, which reconstitute the functional (i.e., fluorescent) full-length fluorescent protein when in close proximity to one another (32). The N-terminal fragment and the C-terminal fragment of the YFP (YFPN and YFPC, respectively) were fused to the C-terminal end of the wild-type or D138A β1-adrenoceptor to generate the β1YFPN, β1YFPC, and β1D138AYFPC receptor constructs. The β1YFPN/β1YFPC and β1YFPN/β1D138AYFPC constructs were transiently cotransfected into CHO-K1 cells, and clear membrane fluorescence of reconstituted YFP and BODIPY-TMR-CGP binding could be seen (Fig. 7), confirming cell surface expression of wild-type/wild-type and wild-type/D138A β1-adrenoceptor homodimers that each contain at least 1 BODIPY-TMR-CGP binding conformation. To confirm that the D138A mutation abolished ligand binding to the β1-adrenoceptor, we examined the binding of 3 nM BODIPY-TMR-CGP to a SNAP-tagged D138A β1-adrenoceptor. Indeed, no binding of 3 nM BODIPY-TMR-CGP could be seen in CHO-K1 cells transiently transfected with the SNAP-β1D138A construct, but clear membrane fluorescence was observed following labeling of the SNAP-tag with 1 μM BG-488, confirming the expression of the non–ligand-binding receptor at the cell surface (Fig. 8, lower left panel). A SNAP-tagged wild-type β1-adrenoceptor was transiently transfected as a positive control, and clear fluorescence of the BG-488 labeled SNAP-tag and 3 nM BODIPY-TMR-CGP binding to the wild-type receptor can be seen (Fig. 8, upper panel). This indicates that the lack of BODIPY-TMR-CGP fluorescence seen for the SNAP-β1D138A–transfected cells is caused by the D138A mutation introduced into the β1-adrenoceptor.


Negative cooperativity across β1-adrenoceptor homodimers provides insights into the nature of the secondary low-affinity CGP 12177 β1-adrenoceptor binding conformation.

Gherbi K, May LT, Baker JG, Briddon SJ, Hill SJ - FASEB J. (2015)

Cell surface expression of BiFC-constrained β1-adrenoceptor homodimers. Confocal images show binding of 3 nM BODIPY-TMR-CGP following 4-min association to CHO-K1 cells transiently expressing BiFC-constrained wild-type β1-adrenoceptor homodimers (β1YFPN/β1YFPC; upper panel) and BiFC-constrained β1-adrenoceptor homodimers containing 1 wild-type and 1 nonligand-binding protomer (β1YFPN/β1D138AYFPC; lower panel). The fluorescence of the complimented YFP was measured simultaneously to confirm the cell surface expression of β1-adrenoceptor homodimers. Images are representative of 5 separate experiments. Scale bars, 50 μm. The binding of BODIPY-TMR-CGP in the lower right confirms that the β1YFPN/β1D138AYFPC dimer pairs contain a viable single orthosteric binding site.
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Related In: Results  -  Collection

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Figure 7: Cell surface expression of BiFC-constrained β1-adrenoceptor homodimers. Confocal images show binding of 3 nM BODIPY-TMR-CGP following 4-min association to CHO-K1 cells transiently expressing BiFC-constrained wild-type β1-adrenoceptor homodimers (β1YFPN/β1YFPC; upper panel) and BiFC-constrained β1-adrenoceptor homodimers containing 1 wild-type and 1 nonligand-binding protomer (β1YFPN/β1D138AYFPC; lower panel). The fluorescence of the complimented YFP was measured simultaneously to confirm the cell surface expression of β1-adrenoceptor homodimers. Images are representative of 5 separate experiments. Scale bars, 50 μm. The binding of BODIPY-TMR-CGP in the lower right confirms that the β1YFPN/β1D138AYFPC dimer pairs contain a viable single orthosteric binding site.
Mentions: BiFC uses 2 nonfluorescent fragments of a fluorescent protein, which reconstitute the functional (i.e., fluorescent) full-length fluorescent protein when in close proximity to one another (32). The N-terminal fragment and the C-terminal fragment of the YFP (YFPN and YFPC, respectively) were fused to the C-terminal end of the wild-type or D138A β1-adrenoceptor to generate the β1YFPN, β1YFPC, and β1D138AYFPC receptor constructs. The β1YFPN/β1YFPC and β1YFPN/β1D138AYFPC constructs were transiently cotransfected into CHO-K1 cells, and clear membrane fluorescence of reconstituted YFP and BODIPY-TMR-CGP binding could be seen (Fig. 7), confirming cell surface expression of wild-type/wild-type and wild-type/D138A β1-adrenoceptor homodimers that each contain at least 1 BODIPY-TMR-CGP binding conformation. To confirm that the D138A mutation abolished ligand binding to the β1-adrenoceptor, we examined the binding of 3 nM BODIPY-TMR-CGP to a SNAP-tagged D138A β1-adrenoceptor. Indeed, no binding of 3 nM BODIPY-TMR-CGP could be seen in CHO-K1 cells transiently transfected with the SNAP-β1D138A construct, but clear membrane fluorescence was observed following labeling of the SNAP-tag with 1 μM BG-488, confirming the expression of the non–ligand-binding receptor at the cell surface (Fig. 8, lower left panel). A SNAP-tagged wild-type β1-adrenoceptor was transiently transfected as a positive control, and clear fluorescence of the BG-488 labeled SNAP-tag and 3 nM BODIPY-TMR-CGP binding to the wild-type receptor can be seen (Fig. 8, upper panel). This indicates that the lack of BODIPY-TMR-CGP fluorescence seen for the SNAP-β1D138A–transfected cells is caused by the D138A mutation introduced into the β1-adrenoceptor.

Bottom Line: At the β1-adrenoceptor, CGP 12177 potently antagonizes agonist responses at the primary high-affinity catecholamine conformation while also exerting agonist effects of its own through a secondary low-affinity conformation.These effects on the BODIPY-TMR-CGP dissociation rate were markedly enhanced in β1-adrenoceptor homodimers constrained by bimolecular fluorescence complementation (9.8- and 9.9-fold for 1 µM CGP 12177 and 1 µM propranolol, respectively) and abolished in β1-adrenoceptors containing TM4 mutations vital for the second conformation pharmacology.This study suggests that negative cooperativity across a β1-adrenoceptor homodimer may be responsible for generating the low-affinity pharmacology of the secondary β1-adrenoceptor conformation.

View Article: PubMed Central - PubMed

Affiliation: Cell Signalling Research Group, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.

Show MeSH
Related in: MedlinePlus