Conformational changes in G‐protein‐coupled receptors—the quest for functionally selective conformations is open

Hoffmann, Carsten; Zürn, Alexander; Bünemann, Moritz

doi:10.1038/sj.bjp.0707615

Cited by 75 publications

(57 citation statements)

References 80 publications

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“…To date, by introducing FRET fluorophores at the intracellular loop and the C terminus of receptor protomer, several studies have clarified the activation mechanism in family A and B receptors (14 -16). All of them demonstrated FRET changes upon ligand applications, consistent with previously proposed intra-protomer conformational changes, thus validating the usefulness of this methodology (17,18). As for family C, the above approach has also been applied for homodimeric mGluR1␣ by our group, revealing the intersubunit rearrangement upon the receptor activation by glutamate application (19).…”

supporting

confidence: 62%

Ligand-induced Rearrangements of the GABAB Receptor Revealed by Fluorescence Resonance Energy Transfer

Matsushita

Nakata

Kubo

et al. 2010

Journal of Biological Chemistry

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The ␥-aminobutyric acid type B receptor (GABA B R), one of the family C G-protein-coupled receptor members, exists as a heterodimer comprised of subunits GB1 and GB2. To clarify the ligand-induced activation mechanism of the GABA B R, each subunit was fused with either Cerulean or enhanced yellow fluorescent protein at its intracellular loop, and fluorescence resonance energy transfer (FRET) changes upon agonist application were monitored. As a result, FRET decreases were observed between GB1a loop 2 and GB2 loop 2 and between GB1a loop 2 and GB2 loop 1, suggesting the dissociation of intracellular domains during the receptor activation. Both intersubunit FRET pairs were expected to faithfully capture the activation of the original receptor as their pharmacological properties were highly similar to that of the wild-type receptor. However, the intrasubunit data suggest that the receptor activation does not involve major structural changes within the transmembrane domain of each subunit. By combining the results obtained from two different levels, it was concluded that the GABA B R activation by agonist is associated with an asymmetrical intersubunit rearrangement of GB1a and GB2 on the membrane. This type of activation mode, an intersubunit rearrangement without apparent intrahelical structural changes, appears commonly shared by the GABA B R and the metabotropic glutamate receptor 1␣, another family C G-protein-coupled receptor previously studied by our group. Nevertheless, the directions of intracellular domain movements and its asymmetry observed here highlight the qualitative difference between the two receptors. G-protein-coupled receptors (GPCRs)2 mediate various physiological responses in cells of organisms. They are expressed on the cell membrane and are known to have extracellular N and intracellular C termini and seven-transmembrane domain (7TMD) as a commonly shared motif. These GPCRs can be categorized into three groups, family A, B, and C (1). Family A includes rhodopsin, adrenergic receptors, adenosine receptors, and muscarinic acetylcholine receptors (mAChRs), etc. Family B members are basically hormone receptors. The last group, family C, is represented by homodimeric metabotropic glutamate receptors (mGluRs) and heterodimeric GABA B receptor (GABA B R), both known for their extremely large extracellular domains, the so-called venus flytrap modules (VFTM) (2).Mechanisms of activation in GPCRs, how signals triggered by bound ligands are transmitted to their intracellular regions via 7TMD, are in general still poorly understood except for well studied family A members. The activation models proposed for family A GPCRs commonly suggest that the intracellular part of helix VI moves away from the bundle of other helices during activation (3-6). Moreover, understanding of the activation mechanism of this family is greatly aided by x-ray crystallography recently applied for several members (7-10). In contrast, not much is known for the activation mechanism of family C. This is partly because there are no...

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supporting

confidence: 62%

Ligand-induced Rearrangements of the GABAB Receptor Revealed by Fluorescence Resonance Energy Transfer

Matsushita

Nakata

Kubo

et al. 2010

Journal of Biological Chemistry

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“…Several recent studies suggest that GPCR activation is a multistep process and that it may occur via multiple active conformations of the receptors (19,20,24). It is very plausible that such distinct conformations underlie the phenomenon of biased signaling, i.e.…”

Section: Discussionmentioning

confidence: 99%

Differential Signaling of the Endogenous Agonists at the β2-Adrenergic Receptor

Reiner

Ambrosio

Hoffmann

et al. 2010

Journal of Biological Chemistry

105

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The concept of "functional selectivity" or "biased signaling" suggests that a ligand can have distinct efficacies with regard to different signaling pathways. We have investigated the question of whether biased signaling may be related to distinct agonist-induced conformational changes in receptors using the ␤ 2 -adrenergic receptor (␤ 2 AR) and its two endogenous ligands epinephrine and norepinephrine as a model system. Agonist-induced conformational changes were determined in a fluorescently tagged ␤ 2 AR FRET sensor. In this ␤ 2 AR sensor, norepinephrine caused signals that amounted to only ≈50% of those induced by epinephrine and the standard "full" agonist isoproterenol. Furthermore, norepinephrine-induced changes in the ␤ 2 AR FRET sensor were slower than those induced by epinephrine (rate constants, 47 versus 128 ms). A similar partial ␤ 2 AR activation signal was revealed for the synthetic agonists fenoterol and terbutaline. However, norepinephrine was almost as efficient as epinephrine (and isoproterenol) in causing activation of G s and adenylyl cyclase. In contrast, fenoterol was quite efficient in triggering ␤-arrestin2 recruitment to the cell surface and its interaction with ␤ 2 AR, as well as internalization of the receptors, whereas norepinephrine caused partial and slow changes in these assays. We conclude that partial agonism of norepinephrine at the ␤ 2 AR is related to the induction of a different active conformation and that this conformation is efficient in signaling to G s and less efficient in signaling to ␤-arrestin2. These observations extend the concept of biased signaling to the endogenous agonists of the ␤ 2 AR and link it to distinct conformational changes in the receptor.

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“…Such conformational sensors are usually modified with CFP at the C terminus and a second fluorophore (YFP or a tetracysteine-motif capable of binding a small soluble fluorophore called FlAsH) within the third intracellular loop of the receptor (Hoffmann et al, 2008;Vilardaga et al, 2009). Upon agonist binding, a change in receptor conformation occurs, and, hence, these receptor sensors respond with an alteration in the observed FRET signal.…”

Section: Introductionmentioning

confidence: 99%

Comparison of the Activation Kinetics of the M₃Acetylcholine Receptor and a Constitutively Active Mutant Receptor in Living Cells

Hoffmann¹,

Nuber²,

Zabel³

et al. 2012

Mol Pharmacol

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Activation of G-protein-coupled receptors is the first step of the signaling cascade triggered by binding of an agonist. Here we compare the activation kinetics of the G q -coupled M 3 acetylcholine receptor (M 3 -AChR) with that of a constitutively active mutant receptor (M 3 -AChR-N514Y) using M 3 -AChR constructs that report receptor activation by changes in the fluorescence resonance energy transfer (FRET) signal. We observed a leftward shift in the concentration-dependent FRET response for acetylcholine and carbachol with M 3 -AChR-N514Y. Consistent with this result, at submaximal agonist concentrations, the activation kinetics of M 3 -AChR-N514Y were significantly faster, whereas at maximal agonist concentrations the kinetics of receptor activation were identical. Receptor deactivation was significantly faster with carbachol than with acetylcholine and was significantly delayed by the N514Y mutation. Receptor-Gprotein interaction was measured by FRET between M 3 -AChRyellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)-G␥ 2 . Agonist-induced receptor-G-protein coupling was of a time scale similar to that of receptor activation. As observed for receptor deactivation, receptor-G-protein dissociation was slower for acetylcholine than that for carbachol. Acetylcholine-stimulated increases in receptor-G-protein coupling of M 3 -AChR-N514Y reached only 12% of that of M 3 -AChR and thus cannot be kinetically analyzed. G-protein activation was measured using YFP-tagged G␣ q and CFP-tagged G␥ 2 . Activation of G q was significantly slower than receptor activation and indistinguishable for the two agonists. However, G q deactivation was significantly prolonged for acetylcholine compared with that for carbachol. Consistent with decreased agoniststimulated coupling to G q , agonist-stimulated G q activation by M 3 -AChR-N514Y was not detected. Taken together, these results indicate that the N514Y mutation produces constitutive activation of M 3 -AChR by decreasing the rate of receptor deactivation, while having minimal effect on receptor activation.

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Conformational changes in G‐protein‐coupled receptors—the quest for functionally selective conformations is open

Cited by 75 publications

References 80 publications

Ligand-induced Rearrangements of the GABAB Receptor Revealed by Fluorescence Resonance Energy Transfer

Ligand-induced Rearrangements of the GABAB Receptor Revealed by Fluorescence Resonance Energy Transfer

Differential Signaling of the Endogenous Agonists at the β2-Adrenergic Receptor

Comparison of the Activation Kinetics of the M₃Acetylcholine Receptor and a Constitutively Active Mutant Receptor in Living Cells

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Conformational changes in G‐protein‐coupled receptors—the quest for functionally selective conformations is open

Cited by 75 publications

References 80 publications

Ligand-induced Rearrangements of the GABAB Receptor Revealed by Fluorescence Resonance Energy Transfer

Ligand-induced Rearrangements of the GABAB Receptor Revealed by Fluorescence Resonance Energy Transfer

Differential Signaling of the Endogenous Agonists at the β2-Adrenergic Receptor

Comparison of the Activation Kinetics of the M3Acetylcholine Receptor and a Constitutively Active Mutant Receptor in Living Cells

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Comparison of the Activation Kinetics of the M₃Acetylcholine Receptor and a Constitutively Active Mutant Receptor in Living Cells