Functional Modulation of a G Protein-Coupled Receptor Conformational Landscape in a Lipid Bilayer

Casiraghi, Marina; Damian, Marjorie; Lescop, Ewen; Point, Elodie; Moncoq, Karine; Morellet, Nelly; Lévy, Daniel; Marie, Jacky; Guittet, Éric; Banères, Jean-Louis; Catoire, Laurent J.

doi:10.1021/jacs.6b04432

Cited by 80 publications

(104 citation statements)

References 36 publications

(72 reference statements)

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“…Achieving conformational homogeneity is the key to crystallize membrane proteins (Loll, 2014). In agreement with our results, a recent experimental study showed that CHS impacts the conformational dynamics of a GPCR leading to a restricted conformational space (Casiraghi et al, 2016). Earlier it was experimentally reported that cholesterol induces a more compact conformational state of the oxytocin receptor (Muth et al, 2011).…”

Section: Discussionsupporting

confidence: 93%

“…The literature reporting on the specific functional role of cholesterol and other lipids is extensive (Pucadyil and Chattopadhyay, 2006; Gimpl, 2016). It has been experimentally shown that cholesterol affects the conformation (Muth et al, 2011; Casiraghi et al, 2016) and function (Gimpl et al, 1997; Paila et al, 2011; Pucadyil and Chattopadhyay, 2006; Casiraghi et al, 2016; Jafurulla et al, 2014) of many GPCRs. Based on X-ray crystal structures cholesterol has specific contacts with β 2 AR (Cherezov et al, 2007; Hanson et al, 2008), suggesting that β 2 AR has binding sites for cholesterol.…”

Section: Introductionmentioning

confidence: 99%

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Mechanism of allosteric regulation of β2-adrenergic receptor by cholesterol

Manna

Niemelä

Tynkkynen

et al. 2016

eLife

130

149

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There is evidence that lipids can be allosteric regulators of membrane protein structure and activation. However, there are no data showing how exactly the regulation emerges from specific lipid-protein interactions. Here we show in atomistic detail how the human β2-adrenergic receptor (β2AR) – a prototypical G protein-coupled receptor – is modulated by cholesterol in an allosteric fashion. Extensive atomistic simulations show that cholesterol regulates β2AR by limiting its conformational variability. The mechanism of action is based on the binding of cholesterol at specific high-affinity sites located near the transmembrane helices 5–7 of the receptor. The alternative mechanism, where the β2AR conformation would be modulated by membrane-mediated interactions, plays only a minor role. Cholesterol analogues also bind to cholesterol binding sites and impede the structural flexibility of β2AR, however cholesterol generates the strongest effect. The results highlight the capacity of lipids to regulate the conformation of membrane receptors through specific interactions.DOI: http://dx.doi.org/10.7554/eLife.18432.001

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Section: Discussionsupporting

confidence: 93%

Section: Introductionmentioning

confidence: 99%

Mechanism of allosteric regulation of β2-adrenergic receptor by cholesterol

Manna

Niemelä

Tynkkynen

et al. 2016

eLife

130

149

View full text Add to dashboard Cite

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“…Prior to biophysical investigation of the GHS receptor, a detailed pharmacological characterization was carried out using homogenous fluorescence assays as described in the literature29. Figure 1A shows the dose-dependent atto520-ghrelin ligand binding plots of the GHS receptor.…”

Section: Resultsmentioning

confidence: 99%

“…Functionality of GHS receptor preparations was tested by a homogeneous fluorescence binding assay using [Dpr 3 -Oct; Dpr 16 -atto520]-ghrelin (ghrelin-atto520), detecting fluorescence enhancement upon receptor binding29. Functionality of the fluorescent tracer ghrelin-atto520 was verified in 3 H-inositol phosphate accumulation assays as described before displaying an EC 50 value of 1.7 nM (pEC 50 = 8.8 ± 0.10; ghrelin: pEC 50 = 9.0 ± 0.15).…”

Section: Methodsmentioning

confidence: 99%

Expression, Functional Characterization, and Solid-State NMR Investigation of the G Protein-Coupled GHS Receptor in Bilayer Membranes

Schrottke

Kaiser

Vortmeier

et al. 2017

Sci Rep

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The expression, functional reconstitution and first NMR characterization of the human growth hormone secretagogue (GHS) receptor reconstituted into either DMPC or POPC membranes is described. The receptor was expressed in E. coli. refolded, and reconstituted into bilayer membranes. The molecule was characterized by 15N and 13C solid-state NMR spectroscopy in the absence and in the presence of its natural agonist ghrelin or an inverse agonist. Static 15N NMR spectra of the uniformly labeled receptor are indicative of axially symmetric rotational diffusion of the G protein-coupled receptor in the membrane. In addition, about 25% of the 15N sites undergo large amplitude motions giving rise to very narrow spectral components. For an initial quantitative assessment of the receptor mobility, 1H-13C dipolar coupling values, which are scaled by molecular motions, were determined quantitatively. From these values, average order parameters, reporting the motional amplitudes of the individual receptor segments can be derived. Average backbone order parameters were determined with values between 0.56 and 0.69, corresponding to average motional amplitudes of 40–50° of these segments. Differences between the receptor dynamics in DMPC or POPC membranes were within experimental error. Furthermore, agonist or inverse agonist binding only insignificantly influenced the average molecular dynamics of the receptor.

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“…Assessment of the binding affinity of the Y 1 R was carried out using homogenous fluorescence assays as described in the literature 47 . The reconstituted receptor was incubated in increasing concentrations with 50 nM fluorescently labelled NPY (NPY-atto520) overnight at room temperature in 50 mM NaP, pH 7.0 and 0.1% BSA.…”

Section: Methodsmentioning

confidence: 99%

Structural basis of ligand binding modes at the neuropeptide Y Y1 receptor

Yang

Han

Keller

et al. 2018

Nature

108

213

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Neuropeptide Y (NPY) receptors belong to the G protein-coupled receptor (GPCR) superfamily and play important roles in food intake, anxiety and cancer regulation1,2. The NPY/Y receptor system has emerged as one of the most complex networks with three peptide ligands (NPY, peptide YY and pancreatic polypeptide) binding to four receptors in mammals, namely Y1, Y2, Y4 and Y5 receptors, with different affinity and selectivity3. NPY is the most powerful stimulant of food intake and this effect is primarily mediated by Y1 receptor (Y1R)4. A number of peptides and small-molecule compounds have been characterized as Y1R antagonists and have shown clinical potential in the treatment of obesity4, tumor1 and bone loss5. However, their clinical usage has been hampered by low potency and selectivity, poor brain penetration ability or lack of oral bioavailability6. Here we report crystal structures of the human Y1R bound to two selective antagonists UR-MK299 and BMS-193885 at 2.7 and 3.0 Å resolution, respectively. The structures combined with mutagenesis studies reveal binding modes of Y1R to several structurally diverse antagonists and determinants of ligand selectivity. The Y1R structure and molecular docking of the endogenous agonist NPY, together with nuclear magnetic resonance (NMR), photo-crosslinking and functional studies, provide insights into the binding behavior of the agonist and for the first time determine the interaction of its N terminus with the receptor. These insights into Y1R can enable structure-based drug discovery targeting NPY receptors.

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Functional Modulation of a G Protein-Coupled Receptor Conformational Landscape in a Lipid Bilayer

Cited by 80 publications

References 36 publications

Mechanism of allosteric regulation of β2-adrenergic receptor by cholesterol

Mechanism of allosteric regulation of β2-adrenergic receptor by cholesterol

Expression, Functional Characterization, and Solid-State NMR Investigation of the G Protein-Coupled GHS Receptor in Bilayer Membranes

Structural basis of ligand binding modes at the neuropeptide Y Y1 receptor

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