Agonists induce conformational changes in transmembrane domains III and VI of the beta 2 adrenoceptor

Gether, Ulrik; Lin, Sansan; Ghanouni, Pejman; Ballesteros, Juan A.; Weinstein, Harel; Kobilka, Brian K.

doi:10.1093/emboj/16.22.6737

Cited by 360 publications

(347 citation statements)

References 57 publications

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“…In earlier biophysical studies with ␤ 2 AR labeled at multiple TM cysteines, we obtained evidence for movement of TM3 and TM6 on agonist activation (23). Recent f luorescence spectroscopic analysis of mutant ␤ 2 ARs labeled on the cytoplasmic side of TM6 suggests that a rigid body motion occurs in this region during agonist-induced activation (34), whereas additional support for movement of TM3 and TM6 in the ␤ 2 AR comes from zinc crosslinking studies (35) and chemical reactivity measurements in constitutively active ␤ 2 AR mutants (36,37).…”

Section: Discussionmentioning

confidence: 79%

Agonist-induced conformational changes in the G-protein-coupling domain of the β ₂ adrenergic receptor

Ghanouni

Steenhuis

Farrens

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

314

271

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The majority of extracellular physiologic signaling molecules act by stimulating GTP-binding protein (G-protein)-coupled receptors (GPCRs). To monitor directly the formation of the active state of a prototypical GPCR, we devised a method to site specifically attach fluorescein to an endogenous cysteine (Cys-265) at the cytoplasmic end of transmembrane 6 (TM6) of the ␤2 adrenergic receptor (␤2AR), adjacent to the G-protein-coupling domain. We demonstrate that this tag reports agonist-induced conformational changes in the receptor, with agonists causing a decline in the fluorescence intensity of fluorescein-␤ 2AR that is proportional to the biological efficacy of the agonist. We also find that agonists alter the interaction between the fluorescein at Cys-265 and fluorescence-quenching reagents localized to different molecular environments of the receptor. These observations are consistent with a rotation and͞or tilting of TM6 on agonist activation. Our studies, when compared with studies of activation in rhodopsin, indicate a general mechanism for GPCR activation; however, a notable difference is the relatively slow kinetics of the conformational changes in the ␤2AR, which may reflect the different energetics of activation by diffusible ligands.D espite diverse physiologic roles, the majority of GTPbinding protein (G-protein)-coupled receptors (GPCRs) are thought to share a common activation mechanism. Briefly, agonists induce conformational changes in receptors, which then stimulate heterotrimeric G proteins. Activated G proteins influence cellular physiology by modulating specific effector enzymes and ion channels involved in cardiovascular, neural, endocrine, and sensory signaling systems (1). GPCRs share a common structural motif consisting of seven TM helices with an extracellular amino terminus. For all known GPCRs, the site of ligand binding is distant from the site of G-protein regulation (2). Therefore, the overall structural effects of agonists binding to extracellular sequences or TM domains must physically converge at the cytoplasmic interface between the receptor and its G protein.We chose to characterize the conformational changes involved in G-protein activation by studying the human ␤ 2 adrenergic receptor (␤ 2 AR). The ␤ 2 AR is an important pharmaceutical target for pulmonary and cardiovascular diseases, and a wide spectrum of pharmacologically well-characterized agonists and antagonists is readily available (3). Moreover, extensive mutagenesis studies have identified amino acids involved in ligand binding and G-protein coupling (Fig. 1 A), thereby providing a basis for focusing our studies to domains likely to report conformational changes involved in receptor activation (4). We devised a means of labeling purified detergent-solubilized ␤ 2 AR at Cys-265 in the carboxyl-terminal region of IC3 with the sulfhydryl-reactive f luorescent probe f luorescein maleimide (FM). Cys-265 is found in the native receptor, and labeling at this position did not require alteration of the receptor's amino acid se...

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Section: Discussionmentioning

confidence: 79%

Agonist-induced conformational changes in the G-protein-coupling domain of the β ₂ adrenergic receptor

Ghanouni

Steenhuis

Farrens

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

314

271

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“…A number of CAM forms of GPCRs appear to be physically destabilized compared to the wild-type receptors (MacEwan & Milligan, 1996;Lee et al, 1997;Gether et al, 1997b …”

Section: Discussionmentioning

confidence: 99%

“…Such e ects of antagonist/inverse agonist ligands on CAM GPCRs have been examined using approaches as diverse as alterations in¯uorescence of a conformation-sensitive reporter in the puri®ed receptor (Gether et al, 1997b) and analysis of increased levels of receptor binding sites present in cells following treatment with appropriate ligands (MacEwan & Milligan, 1996;Lee et al, 1997).…”

Section: British Journal Of Pharmacology Vol 133 (2)mentioning

confidence: 99%

“…As such ligands are thought to favour the presence of inactive or ground states of GPCRs they are generally now referred to as`inverse' agonists because they mirror the ability of agonist ligands to favour the enrichment of GPCRs in active conformational states (Milligan et al, 1995;Chen et al, 2000).signi®cantly greater levels of cyclic AMP in an agonistindependent manner than the wild-type b 2 -adrenoceptor, this mutant displays a substantially higher a nity to bind agonist but not antagonist/inverse agonist ligands. Furthermore, a Cys residue in transmembrane helix VI which can provide a monitor of agonist binding (Gether et al, 1997b) is closer to the ligand binding pocket in the CAM b 2 -adrenoceptor than in the wild-type receptor (Javitch et al, 1997). This CAM receptor has also been reported to be more structurally unstable and to denature more easily than the wild-type receptor (Gether et al, 1997a).…”

Section: Introductionmentioning

confidence: 99%

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Detection of receptor ligands by monitoring selective stabilization of aRenillaluciferase‐tagged, constitutively active mutant, G‐protein‐coupled receptor

Ramsay

Bevan²,

Rees³

et al. 2001

British J Pharmacology

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1 The wild-type b 2 -adrenoceptor and a constitutively active mutant of this receptor were Cterminally tagged with luciferase from the sea pansy Renilla reniformis. 2 C-terminal addition of Renilla luciferase did not substantially alter the levels of expression of either form of the receptor, the elevated constitutive activity of the mutant b 2 -adrenoceptor nor the capacity of isoprenaline to elevate cyclic AMP levels in intact cells expressing these constructs. 3 Treatment of cells expressing constitutively active mutant b 2 -adrenoceptor-Renilla luciferase with antagonist/inverse agonist ligands resulted in upregulation of levels of this polypeptide which could be monitored by the elevated luciferase activity. 4 The pEC 50 for ligand-induced luciferase upregulation and ligand a nity to bind the receptor were highly correlated. 5 Similar upregulation could be observed following sustained treatment with agonist ligands. 6 These e ects were only observed at a constitutively active mutant of the b 2 -adrenoceptor. Coexpression of the wild-type b 2 -adrenoceptor C-terminally tagged with the luciferase from Photinus pyralis did not result in ligand-induced upregulation of the levels of activity of this luciferase. 7 Co-expression of the constitutively active mutant b 2 -adrenoceptor-Renilla luciferase and an equivalent mutant of the a 1b -adrenoceptor C-terminally tagged with green¯uorescent protein allowed pharmacological selectivity of adrenoceptor antagonists to be demonstrated. 8 This approach o ers a sensitive and convenient means, which is amenable to high throughput analysis, to monitor ligand binding to a constitutively active mutant receptor. 9 As no prior knowledge of receptor ligands is required this approach may be suitable to identify ligands at orphan G protein-coupled receptors.

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“…In many cases the necessary conformational change is at least initiated by subtle alterations in one or more transmembranespanning regions of the protein. For example, fluorescence spectroscopy reveals that agonist binding to the ␤2 adrenoreceptor causes conformational changes in two of the seven transmembrane helices that comprise the membrane domain (Gether et al, 1997). These changes are then believed to alter cytoplasmic regions of the protein, allowing interactions with the associated G-protein ␣ subunit necessary for signal transduction.…”

Section: Discussionmentioning

confidence: 99%

The Role of the 3-Hydroxy 3-Methylglutaryl Coenzyme A Reductase Cytosolic Domain in Karmellae Biogenesis

Profant¹,

Roberts

Koning

et al. 1999

MBoC

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In all cells examined, specific endoplasmic reticulum (ER) membrane arrays are induced in response to increased levels of the ER membrane protein 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase. In yeast, expression of Hmg1p, one of two yeast HMG-CoA reductase isozymes, induces assembly of nuclear-associated ER stacks called karmellae. Understanding the features of HMG-CoA reductase that signal karmellae biogenesis would provide useful insights into the regulation of membrane biogenesis. The HMG-CoA reductase protein consists of two domains, a multitopic membrane domain and a cytosolic catalytic domain. Previous studies had indicated that the HMG-CoA reductase membrane domain was exclusively responsible for generation of ER membrane proliferations. Surprisingly, we discovered that this conclusion was incorrect: sequences at the carboxyl terminus of HMG-CoA reductase can profoundly affect karmellae biogenesis. Specifically, truncations of Hmg1p that removed or shortened the carboxyl terminus were unable to induce karmellae assembly. This result indicated that the membrane domain of Hmg1p was not sufficient to signal for karmellae assembly. Using β-galactosidase fusions, we demonstrated that the carboxyl terminus was unlikely to simply serve as an oligomerization domain. Our working hypothesis is that a truncated or misfolded cytosolic domain prevents proper signaling for karmellae by interfering with the required tertiary structure of the membrane domain.

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Agonists induce conformational changes in transmembrane domains III and VI of the beta 2 adrenoceptor

Abstract: Agonist binding to G protein-coupled receptors is

Cited by 360 publications

References 57 publications

Agonist-induced conformational changes in the G-protein-coupling domain of the β ₂ adrenergic receptor

Agonist-induced conformational changes in the G-protein-coupling domain of the β ₂ adrenergic receptor

Detection of receptor ligands by monitoring selective stabilization of aRenillaluciferase‐tagged, constitutively active mutant, G‐protein‐coupled receptor

The Role of the 3-Hydroxy 3-Methylglutaryl Coenzyme A Reductase Cytosolic Domain in Karmellae Biogenesis

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Agonists induce conformational changes in transmembrane domains III and VI of the beta 2 adrenoceptor

Abstract: Agonist binding to G protein-coupled receptors is

Cited by 360 publications

References 57 publications

Agonist-induced conformational changes in the G-protein-coupling domain of the β 2 adrenergic receptor

Agonist-induced conformational changes in the G-protein-coupling domain of the β 2 adrenergic receptor

Detection of receptor ligands by monitoring selective stabilization of aRenillaluciferase‐tagged, constitutively active mutant, G‐protein‐coupled receptor

The Role of the 3-Hydroxy 3-Methylglutaryl Coenzyme A Reductase Cytosolic Domain in Karmellae Biogenesis

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Agonist-induced conformational changes in the G-protein-coupling domain of the β ₂ adrenergic receptor

Agonist-induced conformational changes in the G-protein-coupling domain of the β ₂ adrenergic receptor