Molecular mechanisms of G protein-coupled receptor desensitization and resensitization

beta-Arrestin proteins play a dual role in regulating G protein-coupled receptor (GPCR) responsiveness by contributing to both receptor desensitization and internalization. Recently, beta-arrestins were also shown to be critical determinants for beta(2)-adrenergic receptor (beta(2)AR) resensitization. This was demonstrated by overexpressing wild-type beta-arrestins to rescue the resensitization-defect of a beta(2)AR (Y326A) mutant (gain of function) and overexpressing a dominant-negative beta-arrestin inhibitor of beta(2)AR sequestration to impair beta(2)AR dephosphorylation and resensitization (loss of function). Moreover, the ability of the beta(2)AR to resensitize in different cell types was shown to be dependent upon beta-arrestin expression levels. To further study the mechanisms underlying p-arrestin function, green fluorescent protein was coupled to beta-arrestin2 (beta arr(2)GFP), thus allowing the real-time visualization of the agonist-dependent trafficking of beta-arrestin in living cells. beta arr(2)GFP translocation from the cytoplasm to the plasma membrane proceeded with a time course, sensitivity and specificity that was indistinguishable from the most sensitive second messenger readout systems. beta arr(2)GFP translocation was GRK-dependent and was demonstrated for 16 different ligand-activated GPCRs. Because beta-arrestin binding is a common divergent step in GPCR signalling, this assay represents a universal methodology for screening orphan receptors, GRK inhibitors and novel GPCR ligands. Moreover, beta arr(2)GFP provides a valuable new tool to dissect the biological function and regulation of beta-arrestin proteins.