Ligand-Specific Factors Influencing GLP-1 Receptor Post-Endocytic Trafficking and Degradation in Pancreatic Beta Cells
Abstract
:1. Introduction
2. Results
2.1. GLP-1R Degradation in Beta Cells is More Prominent after Treatment with Exendin-4 versus GLP-1
2.2. GLP-1R Degradation Rates are Associated with Ligand Sensitivity to ECE-1
2.3. Peptide Agonist N-Terminal Substitutions Influence GLP-1R Degradation
2.4. A Natural GLP-1R Variant Shows Altered Trafficking Response
3. Discussion
4. Materials and Methods
4.1. Reagents
4.2. Plasmids
4.3. Cell Culture
4.4. Fluorescence Imaging of Fixed Samples
4.5. Electron Microscopy
4.6. Fluorescent GLP-1R Ligand Uptake Assay in Primary Dispersed Pancreatic Islets
4.7. High Content Imaging Internalisation and Recycling Assay
4.8. SNAP-GLP-1R Degradation Assay by Microscopy
4.9. SNAP-GLP-1R Degradation Assay by Immunoblotting
4.10. Measurement of Binding Affinities
4.11. NanoBiT Assays
4.12. Cyclic AMP Assay
4.13. Measurement of ECE-1 Activity
4.14. Measurement of Peptide Stability by HPLC
4.15. Structural Analysis
4.16. Data Analysis and Statistics
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Assay | N-Terminus | GLP-1 | Chi1 | Chi2 | Chi3 | Ex4(1-30) | Ex4 | GLP-1-G2 |
---|---|---|---|---|---|---|---|---|
Log Ki (M) | H1 | −7.6 ± 0.1 | −8.2 ± 0.1 | −8.4 ± 0.1 | −9.1 ± 0.0 | −9.0 ± 0.0 | −9.5 ± 0.1 | −7.0 ± 0.1 |
F1 | −6.4 ± 0.1 | −6.4 ± 0.1 | −6.5 ± 0.0 | −7.8 ± 0.1 | −7.5 ± 0.0 | −7.3 ± 0.1 | −6.1 ± 0.0 | |
D3 | −8.0 ± 0.1 | −8.5 ± 0.1 | −9.0 ± 0.1 | 8.6 ± 0.1 | −9.2 ± 0.1 | −9.2 ± 0.1 | −7.6 ± 0.1 | |
β-arrestin-2 recruitment (AUC) | H1 | 6.2 ± 0.9 | 7.0 ± 1.6 | 8.4 ± 2.3 | 8.9 ± 2.1 | 4.0 ± 0.5 | 10.2 ± 1.6 | 1.3 ± 0.5 |
F1 | 2.0 ± 1.3 | 2.0 ± 0.8 | 4.6 ± 1.6 | 4.8 ± 1.4 | 0.8 ± 0.2 | 1.3 ± 1 | 0.9 ± 0.5 | |
D3 | 4.9 ± 0.9 | 9.4 ± 0.5 | 8.1 ± 1.2 | 8.9 ± 1.4 | 8.8 ± 0.3 | 8.3 ± 0.3 | 4.0 ± 0.7 | |
Internalisation (%) | H1 | 69.6 ± 14.1 | 88.9 ± 12.7 | 97.3 ± 14.3 | 93 ± 13.7 | 84.5 ± 17.4 | 110.7 ± 9.9 | 58.9 ± 11.4 |
F1 | 28.1 ± 11.1 | 18.5 ± 8.7 | 24.4 ± 9.3 | 40.6 ± 19.4 | 29.1 ± 11.3 | 25.5 ± 11.8 | 12.7 ± 7.8 | |
D3 | 88.5 ± 16.9 | 97.7 ± 14.1 | 78.6 ± 15.7 | 101.5 ± 12.2 | 98 ± 12.3 | 105.4 ± 11.7 | 60.3 ± 17 | |
Recycling (% internalised GLP-1R) | H1 | 44.1 ± 3.1 | 35.3 ± 2.8 | 30.9 ± 3.2 | 21.8 ± 3.8 | 26.4 ± 2.8 | 20.8 ± 3.6 | 44.4 ± 3.8 |
F1 | 55.2 ± 3.4 | 48.7 ± 4.8 | 48.5 ± 5.2 | 40.7 ± 2.7 | 45.9 ± 3.6 | 49.9 ± 3.2 | 62.7 ± 17.9 | |
D3 | 33.2 ± 4.1 | 28.6 ± 4.0 | 22.9 ± 5.1 | 19.0 ± 4.3 | 19.2 ± 3.2 | 19.0 ± 3.5 | 35.4 ± 5.6 | |
Degradation (% remaining GLP-1R) | H1 | 80.4 ± 2.7 | 72.9 ± 1.6 | 69.1 ± 5.7 | 52.8 ± 5.9 | 57.0 ± 5.3 | 52.6 ± 6.6 | 86.2 ± 5.5 |
F1 | 96.4 ± 6.7 | 95.6 ± 2 | 92.8 ± 4.5 | 77.1 ± 6.6 | 91.8 ± 6.4 | 99.3 ± 5.0 | 102.8 ± 4.7 | |
D3 | 58.9 ± 4.4 | 62.9 ± 3.8 | 51.3 ± 8.1 | 51.8 ± 7.7 | 51.9 ± 7.1 | 55.0 ± 4.2 | 62.2 ± 7.7 |
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Fang, Z.; Chen, S.; Manchanda, Y.; Bitsi, S.; Pickford, P.; David, A.; Shchepinova, M.M.; Corrêa Jr, I.R.; Hodson, D.J.; Broichhagen, J.; et al. Ligand-Specific Factors Influencing GLP-1 Receptor Post-Endocytic Trafficking and Degradation in Pancreatic Beta Cells. Int. J. Mol. Sci. 2020, 21, 8404. https://doi.org/10.3390/ijms21218404
Fang Z, Chen S, Manchanda Y, Bitsi S, Pickford P, David A, Shchepinova MM, Corrêa Jr IR, Hodson DJ, Broichhagen J, et al. Ligand-Specific Factors Influencing GLP-1 Receptor Post-Endocytic Trafficking and Degradation in Pancreatic Beta Cells. International Journal of Molecular Sciences. 2020; 21(21):8404. https://doi.org/10.3390/ijms21218404
Chicago/Turabian StyleFang, Zijian, Shiqian Chen, Yusman Manchanda, Stavroula Bitsi, Philip Pickford, Alessia David, Maria M. Shchepinova, Ivan R. Corrêa Jr, David J. Hodson, Johannes Broichhagen, and et al. 2020. "Ligand-Specific Factors Influencing GLP-1 Receptor Post-Endocytic Trafficking and Degradation in Pancreatic Beta Cells" International Journal of Molecular Sciences 21, no. 21: 8404. https://doi.org/10.3390/ijms21218404