Kinetic Analysis Of Proteinprotein Interactions By Means Of Fret

Receptors are activated by agonists, and this event subsequently alters their interactions with G proteins and other downstream effectors such as arrestins. The possibility of studying these conformational changes by agonist application (or withdrawal) and monitoring receptors switching from one state to another and accordingly altering their interaction pattern makes GPCRs a preferred object for kinetically resolved FRET and BRET studies. In most cases, for ligand-induced alterations in BRET and FRET signals, the absolute RET efficiency is of minor interest, and much less care needs to be taken for controlling basal and maximal FRET levels.

Current approaches in GPCR research measure in many cases ligand-induced changes indirectly by means of assessing downstream signaling events at various points along the signaling pathway. For instance, popular assays detect cAMP level, inositol-phosphates or Ca2+ signals. These assays are typically robust and easy to perform; however, they measure events relatively far downstream of the receptor. That such events occur indirectly, for example, as a result of receptor cross-talk driven through other signaling pathways needs to be considered. Furthermore, when the observed signal is downstream of receptor activation, it has been amplified manifold, and the quantitative correlation is very difficult. Similarly, kinetics of second messenger production do not adequately represent kinetics of upstream events. Receptor-induced binding of radioactive GTPyS to G proteins allows for better quantification of receptor activity; however, fast kinetics are difficult to measure and the assay cannot be performed in intact cells.

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