Journal: Nature Communications

Article Title: Allosteric modulation of ghrelin receptor signaling by lipids

doi: 10.1038/s41467-021-23756-y

Figure Lengend Snippet: a FRET-monitored proximity assay with nanodiscs containing increasing amounts in Bodipy-FL PIP2 or Bodipy-FL PA and GHSR labeled with Lumi-4 Tb on C255 6.27 . b FRET-monitored competition between Bodipy-FL PIP2 (2.5 molar%) and unlabeled lipids. The latter were used at a limited concentration (2.5 molar%) not to affect the general physicochemical properties of the bilayer. The signal was normalized to that in the absence of competing lipids (first lane). Data in ( b ) are mean ± SD of three experiments. Source data are provided as a Source data file.

Article Snippet: Bodipy-FL PI (Echelon), Bodipy-FL PIP2 (Echelon), Bodipy-FL PA (Thermofisher), or C11 TopFluor GM3 (Thermofisher) were mixed with the unlabeled lipids at the desired molar ratio before the chloroform evaporation step.

Techniques: Proximity Assay, Labeling, Concentration Assay

Journal: Nature Communications

Article Title: Allosteric modulation of ghrelin receptor signaling by lipids

doi: 10.1038/s41467-021-23756-y

Figure Lengend Snippet: a – c Representative snapshots showing the position of the lipid(s) in the three sites (active conformation) that were most occupied by PIP2 ( a site 1; b site 2; c site 3). The residues interacting with the lipid within each of these sites are indicated (blue), the PIP2 molecules are reported in sticks colored according to the CG beads and the receptor backbone (active conformation) as a white surface. d FRET signal between Bodipy-FL PIP2 (2.5 molar%) and Lumi-4 Tb attached to C255 6.27 of the wild-type receptor or of the putative PIP2-binding sites mutants. The FRET signal obtained with negative control labeled lipid Bodipy-PA is given for comparison. The signal was normalized to that measured with wild-type GHSR. Data in ( d ) are mean ± SD of three experiments and is provided as a Source data file.

Article Snippet: Bodipy-FL PI (Echelon), Bodipy-FL PIP2 (Echelon), Bodipy-FL PA (Thermofisher), or C11 TopFluor GM3 (Thermofisher) were mixed with the unlabeled lipids at the desired molar ratio before the chloroform evaporation step.

Techniques: Binding Assay, Negative Control, Labeling

Journal: Nature Communications

Article Title: Allosteric modulation of ghrelin receptor signaling by lipids

doi: 10.1038/s41467-021-23756-y

Figure Lengend Snippet: a MB emission spectra of GHSR in POPC or POPC:PIP2 (2.5% PIP2) nanodiscs in the absence of ligand, in the presence of 10 µM ghrelin, in the presence of 10 µM LEAP2(1-14) or in the presence of 10 µM ghrelin and the Gα q β 1 γ 2 trimer (1:5 receptor-to-G protein molar ratio). b , c Changes in λ max for the wild-type receptor and the mutants of the PIP2-binding sites. Data in ( b ) and ( c ) are mean ± SD of three experiments. Statistical values were obtained by means of unpaired Student’s t test **0.001 < p < 0.01, ***0.0001 < p < 0.001). d Intramolecular sensitized-emission decays from isolated ghrelin-loaded GHSR (10 µM ligand) in the absence of G protein and in the absence or the presence of PIP2 (2.5% molar ratio), with the donor and acceptor fluorophores in the cytoplasmic end of TM6 and TM1. Data are presented as normalized fluorescence intensity as a function of time and represent the average of three measurements. Source data are provided as a Source data file.

Article Snippet: Bodipy-FL PI (Echelon), Bodipy-FL PIP2 (Echelon), Bodipy-FL PA (Thermofisher), or C11 TopFluor GM3 (Thermofisher) were mixed with the unlabeled lipids at the desired molar ratio before the chloroform evaporation step.

Techniques: Binding Assay, Isolation, Fluorescence

Journal: Nature Communications

Article Title: Allosteric modulation of ghrelin receptor signaling by lipids

doi: 10.1038/s41467-021-23756-y

Figure Lengend Snippet: Lifetimes of Alexa Fluor 488 sensitized emission and corresponding molecular fractions.

Article Snippet: Bodipy-FL PI (Echelon), Bodipy-FL PIP2 (Echelon), Bodipy-FL PA (Thermofisher), or C11 TopFluor GM3 (Thermofisher) were mixed with the unlabeled lipids at the desired molar ratio before the chloroform evaporation step.

Techniques:

Journal: Nature Communications

Article Title: Allosteric modulation of ghrelin receptor signaling by lipids

doi: 10.1038/s41467-021-23756-y

Figure Lengend Snippet: a FRET signal between Bodipy-FL PIP2 (2.5 molar%) and Lumi-4 Tb attached to C255 6.27 of the wild-type receptor or of the GHSR-A204E mutant. The signal obtained with the S1,2,3 mutant is given for comparison. The signal was normalized to that measured with wild-type GHSR. b Changes in MB emission λ max for wild-type GHSR, the A204E, and the S1,2,3 mutant in the absence or in the presence of 10 μM ghrelin. Data are mean ± SD of three experiments. Statistical values were obtained by means of unpaired Student’s t test (**0.001 < p < 0.01, **** p < 0.0001). Source data are provided as a Source data file.

Article Snippet: Bodipy-FL PI (Echelon), Bodipy-FL PIP2 (Echelon), Bodipy-FL PA (Thermofisher), or C11 TopFluor GM3 (Thermofisher) were mixed with the unlabeled lipids at the desired molar ratio before the chloroform evaporation step.

Techniques: Mutagenesis

Journal: Nature Communications

Article Title: Allosteric modulation of ghrelin receptor signaling by lipids

doi: 10.1038/s41467-021-23756-y

Figure Lengend Snippet: GTP turnover for Gq ( a , c ) and Gi2 ( b , d ) catalyzed by wild-type GHSR ( a , b ) or its PIP2-binding sites mutant ( c , d ) in nanodiscs containing different amounts in PIP2 in the absence of ligand, in the presence of 10 µM ghrelin or in the presence of 10 µM LEAP2(1-14). In all cases, the signal was normalized to that obtained for the G protein in the absence of receptor, and data are mean ± SD of five experiments. Statistical values were obtained by means of unpaired Student’s t test (*0.01 < p < 0.05, **0.001 < p < 0.01). e , f Intermolecular sensitized-emission decays from Gα q β 1 γ 2 ( e ) or Gα i2 β 1 γ 2 ( f ) and ghrelin-loaded GHSR (10 µM ligand) assembled into nanodiscs containing or not PIP2 (2.5% molar ratio), with the Lumi-4 Tb donor on the Gα N-terminus and the Alexa Fluor 488 acceptor on the cytoplasmic end of GHSR TM1. In each case, data are presented as normalized fluorescence intensity as a function of time and represent the average of three measurements. Source data are provided as a Source data file.

Article Snippet: Bodipy-FL PI (Echelon), Bodipy-FL PIP2 (Echelon), Bodipy-FL PA (Thermofisher), or C11 TopFluor GM3 (Thermofisher) were mixed with the unlabeled lipids at the desired molar ratio before the chloroform evaporation step.

Techniques: Binding Assay, Mutagenesis, Fluorescence

Journal: Nature Communications

Article Title: Allosteric modulation of ghrelin receptor signaling by lipids

doi: 10.1038/s41467-021-23756-y

Figure Lengend Snippet: a FRET signal between C11 TopFluor GM3 (GM3*; 5 molar%) and Lumi-4 Tb attached to either C255 6.27 or C304 7.34 of GHSR in the absence of competing lipid or in the presence of 5% (molar ratio) unlabeled GM3, PIP2, or POPG. b Changes in MB emission λ max for the wild-type receptor and the PIP2-binding sites mutant assembled into nanodiscs containing 5% GM3, PIP2 or GM3 and PIP2 in equimolar amounts in the absence or presence of 10 µM ghrelin. Data are mean ± SD of three experiments. c – e GTP turnover for Gq and Gi2 catalyzed by wild-type GHSR ( c , d ) or its PIP2-binding sites mutant ( e , f ) in nanodiscs containing 5% GM3, PIP2 or GM3 and PIP2 in equimolar amounts, in the absence or presence of 10 µM ghrelin. The signal was normalized to that obtained for the G protein in the absence of receptor, and data are mean ± SD of five experiments. In all cases, statistical values were obtained by means of unpaired Student’s t test (*0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001, **** p < 0.0001). Source data are provided as a Source data file.

Article Snippet: Bodipy-FL PI (Echelon), Bodipy-FL PIP2 (Echelon), Bodipy-FL PA (Thermofisher), or C11 TopFluor GM3 (Thermofisher) were mixed with the unlabeled lipids at the desired molar ratio before the chloroform evaporation step.

Techniques: Binding Assay, Mutagenesis