Cross-Linking and Functional Analyses for Dimerization of a Cysteine Mutant of Glycine Transporter 1
Abstract
:1. Introduction
2. Results
2.1. Association of FLAG-GlyT1 with GlyT1-HA
2.2. Cross-Linking of GlyT1 L288C
2.3. Functional Consequences of CuP Cross-Linking of L288C
2.4. Influence of Intramolecular K286-E289 Ion Pair on CuP Cross-Linking of L288C
2.5. Influence of Zn2+ on CuP Cross-Linking of L288C
2.6. Influence of GlyT1 Conformation on CuP Cross-Linking of L288C
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Mutagenesis
4.3. Lentivirus and Stable Cell Line Preparation
4.4. Transport and CHIBA-3007 Binding Assays
4.5. Co-Immunoprecipitation and Immunoblotting
4.6. Biotinylation
4.7. Oxidative Cross-Linking of GlyT1b
4.8. Homology Modeling
4.9. Dimer Docking of GlyT1b
4.10. Data Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Km (μM) | Vmax (pmol/mg/min) | Cell Surface Expression (% of WT) | Kd (nM) | Bmax (pmol/mg) | |
---|---|---|---|---|---|
WT | 38.6 ± 2.6 | 1413 ± 40 | 100 | 5.70 ± 0.47 | 2.76 ± 0.09 |
L288C | 43.6 ± 2.5 | 918.5 ± 20 * | 68.3 ± 5.3 * | 7.38 ± 0.43 | 1.73 ± 0.07 * |
L288C + CuP | 54.1 ± 1.8 | 598.8 ± 17 # | nd | 20.6 ± 1.4 # | 1.88 ± 0.07 |
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Wang, J.; Liu, H.; Zhang, Y.-W. Cross-Linking and Functional Analyses for Dimerization of a Cysteine Mutant of Glycine Transporter 1. Int. J. Mol. Sci. 2022, 23, 16157. https://doi.org/10.3390/ijms232416157
Wang J, Liu H, Zhang Y-W. Cross-Linking and Functional Analyses for Dimerization of a Cysteine Mutant of Glycine Transporter 1. International Journal of Molecular Sciences. 2022; 23(24):16157. https://doi.org/10.3390/ijms232416157
Chicago/Turabian StyleWang, Jingru, Hanhe Liu, and Yuan-Wei Zhang. 2022. "Cross-Linking and Functional Analyses for Dimerization of a Cysteine Mutant of Glycine Transporter 1" International Journal of Molecular Sciences 23, no. 24: 16157. https://doi.org/10.3390/ijms232416157
APA StyleWang, J., Liu, H., & Zhang, Y. -W. (2022). Cross-Linking and Functional Analyses for Dimerization of a Cysteine Mutant of Glycine Transporter 1. International Journal of Molecular Sciences, 23(24), 16157. https://doi.org/10.3390/ijms232416157