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

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ABSTRACT

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.

No MeSH data available.


(A) Saturation binding of atto520-labeled ghrelin (c = 100 nM) to increasing amounts of GHS receptor-containing bicelles or empty bicelles. As a control, atto520-labeled NPY was used, which did not display enhanced fluorescence in the presence of GHS receptor-loaded or empty bicelles. Data reflect fluorescence enhancement upon binding. The inflection point (EC50 = 28 nM) for GHS receptor binding is approximately at the limit of the assay of Ltotal/2 = 50 nM; demonstrating high functionality of the system. (B) Displacement of atto520-ghrelin binding to the GHS receptor by unlabeled ghrelin. In contrast, neuropeptide Y (NPY) was not able to displace the bound atto520-ghrelin ligand. Results represent mean +/− SEM of three independent assays each performed in triplicates.
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f1: (A) Saturation binding of atto520-labeled ghrelin (c = 100 nM) to increasing amounts of GHS receptor-containing bicelles or empty bicelles. As a control, atto520-labeled NPY was used, which did not display enhanced fluorescence in the presence of GHS receptor-loaded or empty bicelles. Data reflect fluorescence enhancement upon binding. The inflection point (EC50 = 28 nM) for GHS receptor binding is approximately at the limit of the assay of Ltotal/2 = 50 nM; demonstrating high functionality of the system. (B) Displacement of atto520-ghrelin binding to the GHS receptor by unlabeled ghrelin. In contrast, neuropeptide Y (NPY) was not able to displace the bound atto520-ghrelin ligand. Results represent mean +/− SEM of three independent assays each performed in triplicates.

Mentions: 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.


Expression, Functional Characterization, and Solid-State NMR Investigation of the G Protein-Coupled GHS Receptor in Bilayer Membranes
(A) Saturation binding of atto520-labeled ghrelin (c = 100 nM) to increasing amounts of GHS receptor-containing bicelles or empty bicelles. As a control, atto520-labeled NPY was used, which did not display enhanced fluorescence in the presence of GHS receptor-loaded or empty bicelles. Data reflect fluorescence enhancement upon binding. The inflection point (EC50 = 28 nM) for GHS receptor binding is approximately at the limit of the assay of Ltotal/2 = 50 nM; demonstrating high functionality of the system. (B) Displacement of atto520-ghrelin binding to the GHS receptor by unlabeled ghrelin. In contrast, neuropeptide Y (NPY) was not able to displace the bound atto520-ghrelin ligand. Results represent mean +/− SEM of three independent assays each performed in triplicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC5384189&req=5

f1: (A) Saturation binding of atto520-labeled ghrelin (c = 100 nM) to increasing amounts of GHS receptor-containing bicelles or empty bicelles. As a control, atto520-labeled NPY was used, which did not display enhanced fluorescence in the presence of GHS receptor-loaded or empty bicelles. Data reflect fluorescence enhancement upon binding. The inflection point (EC50 = 28 nM) for GHS receptor binding is approximately at the limit of the assay of Ltotal/2 = 50 nM; demonstrating high functionality of the system. (B) Displacement of atto520-ghrelin binding to the GHS receptor by unlabeled ghrelin. In contrast, neuropeptide Y (NPY) was not able to displace the bound atto520-ghrelin ligand. Results represent mean +/− SEM of three independent assays each performed in triplicates.
Mentions: 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.

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.