Quantification of Receptor Occupancy by Ligand—An Understudied Class of Potential Biomarkers
Abstract
:Simple Summary
Abstract
1. Introduction
2. Soluble Ligand–Receptor Complexes
3. Signaling Mechanisms of Soluble Ligand–Receptor Complexes
4. Agonistic Activity
4.1. Initiation
4.2. Sensitization
5. Antagonistic Activity
6. Quantification of Ligand–Receptor Complexes and Fractional Occupancy
7. A Novel Assay Platform Based on LIRECAP
- Aptamer selection: A pair of RNA aptamers are identified, where one aptamer binds preferentially to the ligand–receptor complex and the second aptamer binds preferentially to the unoccupied receptor. Differences in their variable regions are responsible for this distinct binding. For the LIRECAP assay to be effective, such aptamers should be of the same length and have the same 5′ and 3′ primer-binding regions. The selected aptamers should not cross block each other, nor interfere with the binding of the ligand to the receptor.
- Aptamer addition to biospecimens: The pair of aptamers identified above is added in equimolar concentrations to a biospecimen and unbound aptamers are removed by washing.
- Aptamer quantification: Bound aptamers are extracted using standard molecular biology procedures and quantified using a standard TaqMan RT-qPCR reaction. The TaqMan amplification utilizes a set of PCR primers that bind to the 5′ and 3′ ends of both aptamers, thus amplifying both aptamers proportionately. TaqMan probes that bind to the individual variable central regions of the aptamers, each labeled with a different color, allow for quantification of each of the aptamers individually and for calculation of their ratio of binding to the samples.
- Use of a standard curve to calculate fractional occupancy: A standard curve is constructed based on the fractional occupancy of receptor by ligand using control samples with known fractional occupancy. Results of the test samples are compared to the standard curve. Using the binding ratio provides an internal control so the specificity of the two aptamers (for ligand–receptor complex and unoccupied receptor, respectively) does not have to be absolute, only relative. Thus, by using two aptamers and a standard curve, the relative binding of the two aptamers precisely reflects the fractional occupancy of the receptor by ligand.
8. Measuring Soluble IL2-IL2Ra Complexes in Cancer Biospecimens Using the LIRECAP Assay
9. Potential of the LIRECAP Assay with Other Ligand Receptor Pairs
10. Future Directions and Conclusions
Author Contributions
Funding
Conflicts of Interest
References
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Primary Application | Technique(s) | Limitations | Citations |
---|---|---|---|
Quantify receptors or ligands | RIA and ELISAs using labeled antibodies | Usually used to measure individual receptors or ligands; ELISA-based assays that combine both anti-receptor and anti-ligand antibodies to detect complexes are designed to quantify complexes and not assess fractional occupancy of a receptor by a ligand | [9] |
Determine the ligand–receptor interaction kinetics | Labeled ligands (e.g., radioactively labeled), Surface Plasmon Resonance (SPR) and Fluorescence Resonance Energy Transfer (FRET) | Limited use in clinical diagnosis due to challenges in handling large number of samples simultaneously | [10,11] |
Identify interacting molecules | Co-immunoprecipitation; mass spectrometry | Data is qualitative to semi-quantitative; high-throughput assay is not feasible | [12,13] |
Determine co-localization in the cellular environment | Confocal microscopy | Data is qualitative | [14] |
Measure biological function | Assessment of signal transduction (e.g., arrays, such as ZeptoMARK), cell proliferation or differentiation induced as a result of ligand–receptor binding | Provides indirect analysis of ligand–receptor interaction | [2,15,16] |
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Veeramani, S.; Weiner, G.J. Quantification of Receptor Occupancy by Ligand—An Understudied Class of Potential Biomarkers. Cancers 2020, 12, 2956. https://doi.org/10.3390/cancers12102956
Veeramani S, Weiner GJ. Quantification of Receptor Occupancy by Ligand—An Understudied Class of Potential Biomarkers. Cancers. 2020; 12(10):2956. https://doi.org/10.3390/cancers12102956
Chicago/Turabian StyleVeeramani, Suresh, and George J. Weiner. 2020. "Quantification of Receptor Occupancy by Ligand—An Understudied Class of Potential Biomarkers" Cancers 12, no. 10: 2956. https://doi.org/10.3390/cancers12102956
APA StyleVeeramani, S., & Weiner, G. J. (2020). Quantification of Receptor Occupancy by Ligand—An Understudied Class of Potential Biomarkers. Cancers, 12(10), 2956. https://doi.org/10.3390/cancers12102956