Receptor Parrestin Interaction Detected by RET

Many GPCRs undergo desensitization upon prolonged exposure to agonist. Initially, desensitization mechanisms were worked out for rhodopsin and P2-adrenergic receptors [66, 67]. Subsequently, numerous other GPCRs were demonstrated to exhibit similar patterns and mechanisms of desensitization. However, some receptors utilize different mechanisms for desensitization [68]. The principal mechanism of desensitization can be described briefly: upon agonist binding, receptors are recognized and phosphorylated by kinases such as G protein-coupled receptor kinases (GRKs) and protein kinase A (PKA) on intracellular serine and threonine residues. Subsequently, P - arrestins recognize the phosphorylated receptors and bind to their intracellular surface. This, in turn, blocks interactions between receptors and G proteins due to steric hindrance. Kinetic analysis of arrestin recruitment by receptors has been made possible by means of fluorescence imaging of GFP-tagged arrestins [69]. However, membrane translocation of fluorescent arrestins represents only a semiquantitative approach for studying GPCR-arrestin interactions, particularly because arrestins may interact with other membrane proteins subsequent to receptor binding and therefore exhibit prolonged membrane localization. In other words, arrestins may stay at the membrane even after dissociation from the GPCR. A more direct method to study receptor-arrestin interactions is the use of RET. Both BRET and FRET have been successfully employed to study arrestin binding to receptors [70, 71] . These studies have demonstrated that for Class A receptors such as P2- adrenergic receptors, phosphory-lation of the receptors is not the only prerequisite for GPCR- arrestin interaction. Phosphorylated receptors need to be in an agonist-activated state in order to bind arrestins, and withdrawal of agonist leads to a fast dissociation of arrestins from receptors. These studies uncovered that phosphorylation of the receptors represents a rate-limiting step in receptor-arrestin interactions, and phosphorylated receptors actually exhibit an agonist-dependent interaction with arrestins. The onset of arrestin interaction with phosphorylated receptors is fast (within a few seconds), and is only slightly slower than the interaction of receptors with G proteins [30, 71, 72]. The results from RET-based studies highlight that GPCR-arrestin interactions represent a kind of coincidence detector, requiring both the presence of agonist and phosphoryla-tion of the receptor.

0 0

Post a comment