Journal: eLife

Article Title: Molecular mapping and functional validation of GLP-1R cholesterol binding sites in pancreatic beta cells

doi: 10.7554/eLife.101011

Figure Lengend Snippet: ( A ) Representative images of INS-1 832/3 SNAP/FLAG-hGLP-1R WT or V229A sublines labelled with SNAP-Surface Alexa Fluor 647; size bars, 100 µm. ( B ) Surface expression of SNAP/FLAG-hGLP-1R WT vs V229A; n=6. ( C ) Schematic diagram of the GLP-1R PhotoClick cholesterol binding assay. ( D ) SNAP/FLAG-hGLP-1R-bound cholesterol normalised to receptor levels in INS-1 832/3 SNAP/FLAG-hGLP-1R WT or V229A treated with vehicle (Veh) or 100 nM exendin-4 (Ex-4) for 2 min; n=4. ( E ) Representative images of INS-1 832/3 SNAP/FLAG-hGLP-1R WT vs V229A cells labelled with SNAP-Surface 488 (green) and stimulated with Veh vs Ex-4 for 2 min prior to fixation and labelling with D4H*-mCherry (red); size bars, 5 µm. ( F ) Quantification of co-localisation (Mander’s tM1) between SNAP/FLAG-hGLP-1R WT or V229A and D4H*-mCherry in cells from ( E ); n=5. ( G ) Ex-4 over Veh co-localisation fold change for WT vs V229A SNAP/FLAG-hGLP-1R; n=5. Data is mean +/- SEM, ns, non-significant, *p<0.05, **p<0.01 by paired t-test or one-way ANOVA with Sidak’s multiple comparison test.

Article Snippet: INS-1 832/3 SNAP/FLAG-hGLP-1R WT vs V229A cells seeded onto glass bottom MatTek dishes, or pancreatic islets, were transduced overnight with the Green Up cADDis biosensor in a BacMam vector (#U0200G, Montana Molecular), according to the manufacturer’s instructions.

Techniques: Expressing, Binding Assay, Comparison

Journal: eLife

Article Title: Molecular mapping and functional validation of GLP-1R cholesterol binding sites in pancreatic beta cells

doi: 10.7554/eLife.101011

Figure Lengend Snippet: ( A ) Representative images of bound PhotoClick cholesterol and corresponding SNAP Western blot from SNAP/FLAG-hGLP-1R WT or V229A immunoprecipitation samples; vehicle (Veh), exendin-4 (Ex-4). ( B ) Schematic diagram of GLP-1R conformational shift assay. ( C ) Average conformational shift in response to 100 nM Ex-4 stimulation in INS-1 832/3 SNAP/FLAG-hGLP-1R WT vs V229A; area under the curve (AUC) of conformational shift response shown; n=6. ( D ) Binding affinity to exendin-asp3-TMR (Ex-D3-TMR) in INS-1 832/3 SNAP/FLAG-hGLP-1R WT vs V229A; Kd values shown; n=5. Data is mean +/- SEM; ns, not significant using paired t-test. Figure 3—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 3—figure supplement 1—source data 2. Original files for western blot analysis displayed in .

Article Snippet: INS-1 832/3 SNAP/FLAG-hGLP-1R WT vs V229A cells seeded onto glass bottom MatTek dishes, or pancreatic islets, were transduced overnight with the Green Up cADDis biosensor in a BacMam vector (#U0200G, Montana Molecular), according to the manufacturer’s instructions.

Techniques: Western Blot, Immunoprecipitation, Shift Assay, Binding Assay

Journal: eLife

Article Title: Molecular mapping and functional validation of GLP-1R cholesterol binding sites in pancreatic beta cells

doi: 10.7554/eLife.101011

Figure Lengend Snippet: ( A ) Representative images from GLP-1R WT vs V229A raster image correlation spectroscopy (RICS) analysis of plasma membrane lateral diffusion in INS-1 832/3 SNAP/FLAG-hGLP-1R WT or V229A cells labelled with SNAP-Surface Alexa Fluor 647 before stimulation with vehicle (Veh) or 100 nM exendin-4 (Ex-4). ( B ) Average RICS diffusion coefficients from GLP-1R WT vs V229A from ( A ); n=4. ( C ) TIRF-SPT analysis of average total displacement (top) and speed (bottom) of GLP-1R WT vs V229A under Veh or Ex-4-stimulated conditions in INS-1 832/3 GLP-1R KO cells expressing hGLP-1R-mEGFP WT vs V229A; n=4 for total displacement and n=5 for speed. ( D ) Average RICS diffusion coefficients of plasma membrane lipid-ordered nanodomains (measured as changes in membrane fluidity/lipid packing assessed by Laurdan GP values) in INS-1 832/3 SNAP/FLAG-hGLP-1R WT vs V229A cells under Veh or Ex-4-stimulated conditions; n=5. ( E ) Average diffusion coefficient from RICCS analysis of SNAP-Surface Alexa Fluor 647-labelled SNAP/FLAG-hGLP-1R WT vs V229A with lipid-ordered nanodomains assessed as in ( D ) under Veh or Ex-4-stimulated conditions in INS-1 832/3 SNAP/FLAG-hGLP-1R WT vs V229A cells; n=5. Data is mean +/- SEM; ns, non-significant, **p<0.01, ***p<0.001 by one-way ANOVA with Sidak’s multiple comparison test.

Article Snippet: INS-1 832/3 SNAP/FLAG-hGLP-1R WT vs V229A cells seeded onto glass bottom MatTek dishes, or pancreatic islets, were transduced overnight with the Green Up cADDis biosensor in a BacMam vector (#U0200G, Montana Molecular), according to the manufacturer’s instructions.

Techniques: Spectroscopy, Clinical Proteomics, Membrane, Diffusion-based Assay, Expressing, Comparison

Journal: eLife

Article Title: Molecular mapping and functional validation of GLP-1R cholesterol binding sites in pancreatic beta cells

doi: 10.7554/eLife.101011

Figure Lengend Snippet: Representative images from GLP-1R WT vs V229A raster image cross-correlation spectroscopy (RICCS) analysis of plasma membrane lateral diffusion in INS-1 832/3 SNAP/FLAG-hGLP-1R WT or V229A cells labelled with SNAP-Surface Alexa Fluor 647 (red) and Laurdan (blue) before stimulation with vehicle (Veh) or 100 nM exendin-4 (Ex-4).

Article Snippet: INS-1 832/3 SNAP/FLAG-hGLP-1R WT vs V229A cells seeded onto glass bottom MatTek dishes, or pancreatic islets, were transduced overnight with the Green Up cADDis biosensor in a BacMam vector (#U0200G, Montana Molecular), according to the manufacturer’s instructions.

Techniques: Spectroscopy, Clinical Proteomics, Membrane, Diffusion-based Assay

Journal: eLife

Article Title: Molecular mapping and functional validation of GLP-1R cholesterol binding sites in pancreatic beta cells

doi: 10.7554/eLife.101011

Figure Lengend Snippet: ( A ) N&B estimation of average number of pixels for the different oligomerisation states of the GLP-1R, either as monomers-dimers, dimers-hexamers, hexamers-decamers, or higher order oligomers, calculated at different time frames after stimulation with either vehicle (Veh) or 100 nM exendin-4 (Ex-4) from INS-1 832/3 SNAP/FLAG-hGLP-1R WT or V229A cells; n=4. ( B ) GLP-1R WT vs V229A levels at lipid raft fractions purified from INS-1 832/3 SNAP/FLAG-hGLP-1R WT vs V229A cells under Veh or Ex-4-stimulated conditions. Results represent SNAP levels assessed by Western blotting normalised to flotillin as a marker of lipid raft enrichment; n=5–6. ( C ) Left: representative TIRF images of INS-1 832/3 SNAP/FLAG-hGLP-1R WT or V229A cells co-expressing clathrin light chain-GFP (CLC-GFP) labelled with SNAP-Surface Alexa Fluor 647 prior to Veh or 100 nM Ex-4 stimulation; right: quantification of association (ΔF/S, see Methods) of SNAP/FLAG-hGLP1-R WT or V229A with clathrin puncta; n=29 and 127 cells for WT Veh vs Ex-4, and n=31 and 106 cells for V229A Veh vs Ex-4, respectively; each data point represents mean of 3 cells, data collated from three separate experiments. Data is mean +/-SEM; ns, non-significant, *p<0.05, **p<0.01, ****p<0.0001 by unpaired t-test, one- or two-way ANOVA with Sidak’s multiple comparison test.

Article Snippet: INS-1 832/3 SNAP/FLAG-hGLP-1R WT vs V229A cells seeded onto glass bottom MatTek dishes, or pancreatic islets, were transduced overnight with the Green Up cADDis biosensor in a BacMam vector (#U0200G, Montana Molecular), according to the manufacturer’s instructions.

Techniques: Purification, Western Blot, Marker, Expressing, Comparison

Journal: eLife

Article Title: Molecular mapping and functional validation of GLP-1R cholesterol binding sites in pancreatic beta cells

doi: 10.7554/eLife.101011

Figure Lengend Snippet: ( A ) Representative images of INS-1 832/3 SNAP/FLAG-hGLP-1R WT or V229A cells labelled with SNAP-Surface Alexa Fluor 647 probe under vehicle (Veh) conditions or following stimulation with 100 nM exendin-4 (Ex-4) for 10 min; size bars, 5 µm. ( B ) Schematic diagram of agonist-mediated SNAP/FLAG-hGLP-1R internalisation assay. ( C ) Percentage of internalised SNAP/FLAG-hGLP-1R WT vs V229A at the indicated time points after stimulation with 100 nM Ex-4; corresponding area under the curve (AUC) also shown; n=4. ( D ) Schematic diagram of SNAP/FLAG-hGLP-1R plasma membrane recycling assay. ( E ) Percentage of recycled SNAP/FLAG-hGLP-1R WT vs V229A at the indicated time points after stimulation with 100 nM Ex-4; corresponding AUC also shown; n=3. ( F ) Schematic diagram of agonist-mediated SNAP/FLAG-hGLP-1R degradation assay. ( G ) Percentage of SNAP/FLAG-hGLP-1R WT vs V229A degradation at the indicated time points after stimulation with 100 nM Ex-4; corresponding AUC also shown; n=4. Data is mean +/- SEM; ns, non-significant, *p<0.05, **p<0.01, ***p<0.001 by paired t-test or two-way ANOVA with Sidak’s multiple comparison test.

Article Snippet: INS-1 832/3 SNAP/FLAG-hGLP-1R WT vs V229A cells seeded onto glass bottom MatTek dishes, or pancreatic islets, were transduced overnight with the Green Up cADDis biosensor in a BacMam vector (#U0200G, Montana Molecular), according to the manufacturer’s instructions.

Techniques: Internalisation Assay, Clinical Proteomics, Membrane, Degradation Assay, Comparison

Journal: eLife

Article Title: Molecular mapping and functional validation of GLP-1R cholesterol binding sites in pancreatic beta cells

doi: 10.7554/eLife.101011

Figure Lengend Snippet: ( A ) Mini-G s recruitment dose response curves and log 10 (Emax/EC 50 ) following stimulation with the indicated concentrations of exendin-4 (Ex-4) in INS-1 832/3 GLP-1R KO cells transiently transfected with GLP-1R-SmBiT WT or V229A and LgBiT-mini-G s ; n=5. ( B ) Mini-G q recruitment dose response curves and log 10 (Emax/EC 50 ) after stimulation with the indicated concentrations of Ex-4 in INS-1 832/3 GLP-1R KO cells transiently transfected with GLP-1R-SmBiT WT or V229A and LgBiT-mini-G q ; n=6. ( C ) β-arrestin 2 (βarr2) recruitment dose response curves and log 10 (Emax/EC 50 ) after stimulation with the indicated concentrations of Ex-4 in INS-1 832/3 GLP-1R KO cells transiently transfected with GLP-1R-SmBiT WT or V229A and LgBiT-βarr2; n=5. ( D ) Mini-G s over βarr2 bias calculation for GLP-1R V229A vs WT. ( E ) GLP-1R WT vs V229A plasma membrane activation after stimulation with 100 nM Ex-4 in INS-1 832/3 GLP-1R KO cells co-transfected with Nb37-SmBiT, LgBiT-CAAX and SNAP/FLAG-hGLP-1R WT or V229A, measured by NanoBiT bystander complementation assay; area under the curve (AUC) also shown; n=6. ( F ) As in ( E ) but for GLP-1R WT vs V229A endosomal activation in INS-1 832/3 GLP-1R KO cells co-transfected with Nb37-SmBiT, Endofin-LgBiT and SNAP/FLAG-hGLP-1R WT or V229A; n=6. Data is mean +/- SEM; ns, non-significant, *p<0.05, **p<0.01 by paired t-test or two-way ANOVA with Sidak’s multiple comparison test.

Article Snippet: INS-1 832/3 SNAP/FLAG-hGLP-1R WT vs V229A cells seeded onto glass bottom MatTek dishes, or pancreatic islets, were transduced overnight with the Green Up cADDis biosensor in a BacMam vector (#U0200G, Montana Molecular), according to the manufacturer’s instructions.

Techniques: Transfection, Clinical Proteomics, Membrane, Activation Assay, Comparison

Journal: eLife

Article Title: Molecular mapping and functional validation of GLP-1R cholesterol binding sites in pancreatic beta cells

doi: 10.7554/eLife.101011

Figure Lengend Snippet: ( A ) cAMP responses of INS-1 832/3 SNAP/FLAG-hGLP-1R WT vs V229A cells transduced with the Green Up Global cAMP cADDis biosensor before stimulation with 100 nM Exendin-4 (Ex-4) followed by 100 μM isobutyl methylxanthine (IBMX) +10 µM forskolin (FSK) for maximal response. ( B ) AUC and maximal response for the Ex-4 period from ( A ); n=5. ( C ) Insulin secretion from INS-1 832/3 SNAP/FLAG-hGLP-1R WT vs V229A cells following stimulation with 11 mM glucose ( G11 ) +/-100 nM Ex-4; n=5. ( D ) Insulin secretion Ex-4-fold increase over G11 calculated from data in ( C ). ( E ) Representative images of GLP-1R KO mouse islets transduced with adenoviruses expressing SNAP/FLAG-hGLP-1R WT or V229A, labelled with SNAP-Surface Alexa Fluor 647 prior to stimulation with vehicle (Veh) or 100 nM Ex-4 for 5 min; size bars, 100 µm. ( F ) Surface expression of SNAP/FLAG-hGLP-1R WT vs V229A expressed in GLP-1R KO mouse islets from ( E ); n=3. ( G ) Percentage of SNAP/FLAG-hGLP-1R WT vs V229A internalisation in GLP-1R KO mouse islets following stimulation with 100 nM Ex-4 for 5 min; n=3. ( H ) Insulin secretion responses from GLP-1R KO islets transduced with SNAP/FLAG-hGLP-1R WT vs V229A adenoviruses following stimulation with G11 +/-100 nM Ex-4; n=3. ( I ) Insulin secretion Ex-4-fold increase over G11 calculated from data in ( H ). Data is mean +/- SEM; ns, non-significant, *p<0.05, **p<0.01 by paired t-test or one-way ANOVA with Sidak’s multiple comparison test.

Article Snippet: INS-1 832/3 SNAP/FLAG-hGLP-1R WT vs V229A cells seeded onto glass bottom MatTek dishes, or pancreatic islets, were transduced overnight with the Green Up cADDis biosensor in a BacMam vector (#U0200G, Montana Molecular), according to the manufacturer’s instructions.

Techniques: Transduction, Expressing, Comparison

Journal: eLife

Article Title: Molecular mapping and functional validation of GLP-1R cholesterol binding sites in pancreatic beta cells

doi: 10.7554/eLife.101011

Figure Lengend Snippet: ( A ) Percentage of insulin secretion from GLP-1R KO islets transduced with SNAP/FLAG-hGLP-1R WT vs V229A adenoviruses preincubated with Veh or methyl-β-cyclodextrin (MβCD) loaded with 20 mM cholesterol (MβCD/chol) for 1 hr before stimulation with G11 +/-100 nM exendin-4 (Ex-4); n=4. ( B ) Insulin secretion Ex-4-fold increase over G11 calculated from data in ( A ). Data is mean +/- SEM; ns, non-significant by paired t-test.

Article Snippet: INS-1 832/3 SNAP/FLAG-hGLP-1R WT vs V229A cells seeded onto glass bottom MatTek dishes, or pancreatic islets, were transduced overnight with the Green Up cADDis biosensor in a BacMam vector (#U0200G, Montana Molecular), according to the manufacturer’s instructions.

Techniques: Transduction

Journal: eLife

Article Title: Molecular mapping and functional validation of GLP-1R cholesterol binding sites in pancreatic beta cells

doi: 10.7554/eLife.101011

Figure Lengend Snippet: Overview of effects of glucagon-like peptide-1 receptor (GLP-1R) V229A in inactive and active states compared with GLP-1R wild-type (WT). (↑ increased, ↓ decreased, ≈ unchanged).

Article Snippet: INS-1 832/3 SNAP/FLAG-hGLP-1R WT vs V229A cells seeded onto glass bottom MatTek dishes, or pancreatic islets, were transduced overnight with the Green Up cADDis biosensor in a BacMam vector (#U0200G, Montana Molecular), according to the manufacturer’s instructions.

Techniques: Expressing, Binding Assay, Clinical Proteomics, Membrane, Diffusion-based Assay, Activation Assay

Journal: eLife

Article Title: Molecular mapping and functional validation of GLP-1R cholesterol binding sites in pancreatic beta cells

doi: 10.7554/eLife.101011

Figure Lengend Snippet: Site-directed mutagenesis primers for SNAP/FLAG-hGLP-1R cholesterol binding mutant generation.

Article Snippet: INS-1 832/3 SNAP/FLAG-hGLP-1R WT vs V229A cells seeded onto glass bottom MatTek dishes, or pancreatic islets, were transduced overnight with the Green Up cADDis biosensor in a BacMam vector (#U0200G, Montana Molecular), according to the manufacturer’s instructions.

Techniques: Mutagenesis, Binding Assay

Journal: eLife

Article Title: Molecular mapping and functional validation of GLP-1R cholesterol binding sites in pancreatic beta cells

doi: 10.7554/eLife.101011

Figure Lengend Snippet: Transfected plasmid amounts for Nb37 bystander NanoBiT assays.

Article Snippet: INS-1 832/3 SNAP/FLAG-hGLP-1R WT vs V229A cells seeded onto glass bottom MatTek dishes, or pancreatic islets, were transduced overnight with the Green Up cADDis biosensor in a BacMam vector (#U0200G, Montana Molecular), according to the manufacturer’s instructions.

Techniques: Transfection, Plasmid Preparation, Concentration Assay

Journal: bioRxiv

Article Title: Binding Kinetics, Bias, Receptor Internalization and Effects on Insulin Secretion in vitro and in vivo of a Novel GLP-1R/GIPR Dual Agonist, HISHS-2001

doi: 10.1101/2025.01.13.632834

Figure Lengend Snippet: ( A ) Schematic showing HISHS-2001 sequence and other formula details. ( B ) cAMP dose responses for the indicated agonists in HEK293-SNAP-GLP-1R cells, n=5. ( C ) Potency (pEC50) and maximal response estimates from (B). ( D ) cAMP dose responses for the indicated agonists in HEK293-SNAP-GIPR cells, n=4. ( E ) Potency (pEC50) and maximal response estimates from (D). ( F ) cAMP responses in DiscoverX GLP-1R-β-arrestin 2 cells, n=4. ( G ) β-arrestin 2 recruitment responses in DiscoverX GLP-1R-β-arrestin 2 cells, n=4. ( H ) Bias factor calculated using from transduction ratios ( τ / K A ) method for HISHS-2021 and tirzepatide compared to semaglutide using data from (F) and (G). Data is shown as mean +/− SEM; ns, non-significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 by one-way ANOVA with Dunnett’s post-hoc test.

Article Snippet: The cell lines used were INS-1 832/3 GLP-1R −/− and INS-1 832/3 GIPR −/− (kind gifts from Dr Jackeline Naylor, MedImmune/AstraZeneca ), and INS-1 832/3 stably expressing SNAP-tagged human GLP-1R (generated in house and previously described ).

Techniques: Sequencing, Transduction

Journal: bioRxiv

Article Title: Binding Kinetics, Bias, Receptor Internalization and Effects on Insulin Secretion in vitro and in vivo of a Novel GLP-1R/GIPR Dual Agonist, HISHS-2001

doi: 10.1101/2025.01.13.632834

Figure Lengend Snippet: ( A ) K on calculated from competitive kinetics assay in HEK293-SNAP-GLP-1R cells, n=4. ( B ) K off calculated from competitive kinetics assay in HEK293-SNAP-GLP-1R cells, n=4. ( C ) Calculated affinity from data shown in (A) and (B). Data is shown as mean +/− SEM; ns, non-significant; ***p<0.001 by paired t-test.

Article Snippet: The cell lines used were INS-1 832/3 GLP-1R −/− and INS-1 832/3 GIPR −/− (kind gifts from Dr Jackeline Naylor, MedImmune/AstraZeneca ), and INS-1 832/3 stably expressing SNAP-tagged human GLP-1R (generated in house and previously described ).

Techniques:

Journal: bioRxiv

Article Title: Binding Kinetics, Bias, Receptor Internalization and Effects on Insulin Secretion in vitro and in vivo of a Novel GLP-1R/GIPR Dual Agonist, HISHS-2001

doi: 10.1101/2025.01.13.632834

Figure Lengend Snippet: ( A ) GLP-1R G αs recruitment dose response to HISHS-2001 and tirzepatide at indicated doses by NanoBiT complementation in INS-1 832–3 GLP-1R −/− cells transfected with GLP-1R-SmBiT and G αs -LgBiT. ( B ) Potency (pEC50) and maximal response from (A); n=6. ( C ) GIPR G αs recruitment dose response to HISHS-2001 and tirzepatide at indicated doses by NanoBiT complementation in INS-1 832–3 GIPR −/− cells transfected with GIPR-SmBiT and G αs -LgBiT. ( D ) Potency (pEC50) and maximal response from (C); n=7. Data is shown as mean +/− SEM; ns, non-significant; *p<0.05 by paired t-test.

Article Snippet: The cell lines used were INS-1 832/3 GLP-1R −/− and INS-1 832/3 GIPR −/− (kind gifts from Dr Jackeline Naylor, MedImmune/AstraZeneca ), and INS-1 832/3 stably expressing SNAP-tagged human GLP-1R (generated in house and previously described ).

Techniques: Transfection

Journal: bioRxiv

Article Title: Binding Kinetics, Bias, Receptor Internalization and Effects on Insulin Secretion in vitro and in vivo of a Novel GLP-1R/GIPR Dual Agonist, HISHS-2001

doi: 10.1101/2025.01.13.632834

Figure Lengend Snippet: ( A ) Representative images of SNAP-GLP-1R subcellular localisation at 0- and 10-minutes post-stimulation with 100 nM HISHS-2001, tirzepatide, or semaglutide in INS-1 832/3 SNAP-GLP-1R cells. ( B ) Percentage of SNAP-GLP-1R internalization with the indicated agonist calculated from (A); n=3–7. ( C ) Percentage of SNAP-GLP-1R recycling to the plasma membrane in INS-1 832/3 SNAP-GLP-1R cells in response to 100 nM HISHS-2001, tirzepatide, or semaglutide, with corresponding AUCs shown; n=2–3. ( D ) Cholesterol-rich lipid nanodomain segregation of SNAP-GLP-1R in INS-1 832/3 SNAP-GLP-1R cells under vehicle conditions or in response to 100 nM HISHS-2001, tirzepatide, semaglutide, or semaglutide analogue GL0034 ; DRM, detergent-resistant membrane fractions; DMS, detergent-soluble membrane fractions; flotillin indicates cholesterol-rich lipid nanodomain enrichment. ( E ) Quantification of SNAP-GLP-1R/flotillin from (D); n=7. Data is shown as mean +/− SEM; ns, non-significant; *p<0.05; **p<0.01 by one-way ANOVA with Dunnett’s post-hoc test.

Article Snippet: The cell lines used were INS-1 832/3 GLP-1R −/− and INS-1 832/3 GIPR −/− (kind gifts from Dr Jackeline Naylor, MedImmune/AstraZeneca ), and INS-1 832/3 stably expressing SNAP-tagged human GLP-1R (generated in house and previously described ).

Techniques: Clinical Proteomics, Membrane