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Co-IP assays for measuring GPCR-arrestin interactions
Shelby L Wertz1, Victoria L Desimine1, Jennifer Maning1
1Laboratory for the Study of Neurohormonal Control of the Circulation, Department of Pharmaceutical Sciences, Nova Southeastern University College of Pharmacy, Ft. Lauderdale, FL, United States.
Methods in Cell Biology
|January 9, 2019
Summary
Co-immunoprecipitation (co-IP) is a reliable method to study G protein-coupled receptor (GPCR) and βarrestin interactions. This protocol quantifies native GPCR-βarrestin binding without chemical modification.
Area of Science:
- Cellular signaling and molecular biology.
- Biochemistry and biophysics.
Background:
- βarrestins (arrestin2 and arrestin3) are key adapter proteins for G protein-coupled receptors (GPCRs).
- They mediate receptor desensitization, internalization, and G protein-independent signaling.
- Quantifying GPCR-βarrestin interactions is crucial for understanding signal transduction.
Purpose of the Study:
- To describe a simple and reliable co-immunoprecipitation (co-IP) protocol.
- To measure steady-state levels of agonist-elicited GPCR-βarrestin interaction in cells.
- To enable detection and semi-quantification of native GPCR-arrestin complexes.
Main Methods:
- Co-immunoprecipitation (co-IP) followed by western blotting.
- Utilizes native receptor and arrestin proteins without chemical modification.
- Assay relies on specific and sensitive antibody detection.
Main Results:
- The co-IP assay provides a reliable measurement of GPCR-βarrestin interactions.
- It allows for the semi-quantification of steady-state binding levels.
- The method is applicable to native, unmodified receptor-arrestin complexes.
Conclusions:
- Co-immunoprecipitation is a valuable and traditional method for studying GPCR-βarrestin interactions.
- This protocol offers a straightforward approach to assess these crucial signaling events.
- The reliability of the assay is dependent on antibody quality.
Keywords:
Adrenal cortexAldosteroneAngiotensin II type 1 receptorCo-immunoprecipitationG protein-coupled receptorHeart failureβarrestin1More Related Videos
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