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
APA StyleFang, Z., Chen, S., Manchanda, Y., Bitsi, S., Pickford, P., David, A., Shchepinova, M. M., Corrêa Jr, I. R., Hodson, D. J., Broichhagen, J., Tate, E. W., Reimann, F., Salem, V., Rutter, G. A., Tan, T., Bloom, S. R., Tomas, A., & Jones, B. (2020). Ligand-Specific Factors Influencing GLP-1 Receptor Post-Endocytic Trafficking and Degradation in Pancreatic Beta Cells. International Journal of Molecular Sciences, 21(21), 8404. https://doi.org/10.3390/ijms21218404