ELSEVIER PII zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFED SOOU-3205(98)00107-6 Life Sciences, Vol. 62, Nos. 17/B, pp. 1561-1565, 1998 copyright 0 1998 Faevicr science Inc. Printed in the USA. All rights reserved 0024-32mp $19.00 t .oo MOLECULAR MECHANISMS OF G PROTEIN-COUPLED RECEPTOR DESENSITIZATION AND RFSENSITIZATION Stephen S. G. Ferguson*, Jie Zhangt, Larry S. Barakt, and Marc G. Caront *John P. Robarts Research Institute and Departments of Physiology, Pharmacology and Toxicology, University of Western Ontario, London, Ontario, N6A 5K8, tHoward Hughes Medical Institute and Departments of Cell Biology and Medicine, Duke University Medical Center, Durham, North Carolina, 277 10. Summary P-Arrestin proteins play a dual role in regulating G protein-coupled receptor (GPCR) responsiveness by contributing to both receptor desensitization and internalization. Recently, p-arrestins were also shown to be critical determinants for P2-adrenergic receptor @AR) resensitization. This was demonstrated by overexpressing wild-type j3-arrestins to rescue the resensitization-defect of a p&R (Y326A) mutant (gain of function) and overexpressing a dominant-negative p-arrestin inhibitor of j32AR sequestration to impair PzAR dephosphorylation and resensitization (loss of function). Moreover, the ability of the PzAR to resensitize in different cell types was shown to be dependent upon p-arrestin expression levels. To further study the mechanisms underlying p-arrestin function, green fluorescent protein was coupled to p- arrestin (parr2GFP), thus allowing the real-time visualization of the agonist- dependent trafficking of p-arrestin in living cells. @rrzGFP 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. @rr~GFp translocation was GRK-dependent and was demonstrated for 16 different ligand-activated GPCRs. Because p-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, parr2GFP provides a valuable new tool to dissect the biological function and regulation of p-arrestin proteins. Key Words: G-protein-coupled receptor, &mesh, resensitization, endocytosis, green fluorescent protein G protein-coupled receptors (GPCRs) transduce the information provided by a wide variety of extracellular stimuli. By doing so, GPCRs play a pivotal role in regulating biological functions that include phototransduction, olfaction, taste, cardiovascular function, neurotransmission and hormonal responsiveness. Agonist activation of a GPCR not only initiates the receptor-mediated signal transduction cascade, but also triggers the activation of cellular and molecular mechanisms that lead to receptor desensitization and endocytosis. Agonist-mediated GPCR signalling requires the isomerization of the receptor to a high-affinity agonist-binding conformation necessary to mediate the exchange of GDP for GTP on the heterotrimeric guanine nucleotide binding protein (G protein) a-subunit. Freely dissociated a- and py-subunits then