Abstract
βarrestin1 and -2 (also known as arrestin2 and -3, respectively) are G protein-coupled receptor (GPCR) adapter proteins, performing three major functions in the cell: functional desensitization, i.e., G protein uncoupling from the receptor, GPCR internalization via clathrin-coated pits, and formation of signalosomes. The βarrestins elicit a large part of the G protein-independent signaling emanating from GPCRs. Several methodologies have been developed over the past 15 years or so to quantify the GPCR-arrestin interaction/binding, especially since the latter's roles in signal transduction were discovered. One of the simplest and most traditional of these methodologies is the assay of co-immunoprecipitation (co-IP), followed by western blotting. This assay is also one of the most reliable ones, since it does not require any chemical modification of either component in the complex (i.e., neither of the receptor nor of the arrestin). Therefore, it is the only assay that can detect and semi-quantify interactions between native GPCRs and native arrestins. The caveat of this assay is of course that its reliability depends on the quality (specificity and sensitivity) of the utilized antibodies. Here, we describe a simple protocol for performing this co-IP assay to get a measurement of the steady-state levels of agonist-elicited GPCR-arrestin interaction in cells.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.