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Differential Affinities of Visual Arrestin, βArrestin1, and βArrestin2 for G Protein-coupled Receptors Delineate Two Major Classes of Receptors

2000; Elsevier BV; Volume: 275; Issue: 22 Linguagem: Inglês

10.1074/jbc.m910348199

1083-351X

Robert H. Oakley, Stéphane A. Laporte, Jason A. Holt, Marc G. Caron, Larry S. Barak,

Neuroscience and Neuropharmacology Research

Visual arrestin, βarrestin1, and βarrestin2 comprise a family of intracellular proteins that desensitize G protein-coupled receptors (GPCRs). In addition, βarrestin1 and βarrestin2 target desensitized receptors to clathrin-coated pits for endocytosis. Whether arrestins differ in their ability to interact with GPCRs in cells is not known. In this study, we visualize the interaction of arrestin family members with GPCRs in real time and in live cells using green fluorescent protein-tagged arrestins. In the absence of agonist, visual arrestin and βarrestin1 were found in both the cytoplasm and nucleus of HEK-293 cells, whereas βarrestin2 was found only in the cytoplasm. Analysis of agonist-mediated arrestin translocation to multiple GPCRs identified two major classes of receptors. Class A receptors (β2 adrenergic receptor, mu opioid receptor, endothelin type A receptor, dopamine D1A receptor, and α1b adrenergic receptor) bound βarrestin2 with higher affinity than βarrestin1 and did not interact with visual arrestin. In contrast, class B receptors (angiotensin II type 1A receptor, neurotensin receptor 1, vasopressin V2 receptor, thyrotropin-releasing hormone receptor, and substance P receptor) bound both βarrestin isoforms with similar high affinities and also interacted with visual arrestin. Switching the carboxyl-terminal tails of class A and class B receptors completely reversed the affinity of each receptor for the visual and non-visual arrestins. In addition, exchanging the βarrestin1 and βarrestin2 carboxyl termini reversed their extent of binding to class A receptors as well as their subcellular distribution. These results reveal for the first time marked differences in the ability of arrestin family members to bind GPCRs at the plasma membrane. Moreover, they show that visual arrestin can interact in cells with GPCRs other than rhodopsin. These findings suggest that GPCR signaling may be differentially regulated depending on the cellular complement of arrestin isoforms and the ability of arrestins to interact with other cellular proteins. Visual arrestin, βarrestin1, and βarrestin2 comprise a family of intracellular proteins that desensitize G protein-coupled receptors (GPCRs). In addition, βarrestin1 and βarrestin2 target desensitized receptors to clathrin-coated pits for endocytosis. Whether arrestins differ in their ability to interact with GPCRs in cells is not known. In this study, we visualize the interaction of arrestin family members with GPCRs in real time and in live cells using green fluorescent protein-tagged arrestins. In the absence of agonist, visual arrestin and βarrestin1 were found in both the cytoplasm and nucleus of HEK-293 cells, whereas βarrestin2 was found only in the cytoplasm. Analysis of agonist-mediated arrestin translocation to multiple GPCRs identified two major classes of receptors. Class A receptors (β2 adrenergic receptor, mu opioid receptor, endothelin type A receptor, dopamine D1A receptor, and α1b adrenergic receptor) bound βarrestin2 with higher affinity than βarrestin1 and did not interact with visual arrestin. In contrast, class B receptors (angiotensin II type 1A receptor, neurotensin receptor 1, vasopressin V2 receptor, thyrotropin-releasing hormone receptor, and substance P receptor) bound both βarrestin isoforms with similar high affinities and also interacted with visual arrestin. Switching the carboxyl-terminal tails of class A and class B receptors completely reversed the affinity of each receptor for the visual and non-visual arrestins. In addition, exchanging the βarrestin1 and βarrestin2 carboxyl termini reversed their extent of binding to class A receptors as well as their subcellular distribution. These results reveal for the first time marked differences in the ability of arrestin family members to bind GPCRs at the plasma membrane. Moreover, they show that visual arrestin can interact in cells with GPCRs other than rhodopsin. These findings suggest that GPCR signaling may be differentially regulated depending on the cellular complement of arrestin isoforms and the ability of arrestins to interact with other cellular proteins. G protein-coupled receptor G protein-coupled receptor kinase β2-adrenergic receptor m2 muscarinic acetylcholine receptor angiotensin II type 1A receptor mu opioid receptor endothelin type A receptor dopamine D1A receptor α1b-adrenergic receptor neurotensin 1 receptor vasopressin V2 receptor thyrotropin-releasing hormone receptor substance P receptor green fluorescent protein yellow fluorescent protein minimal essential medium G protein-coupled receptors (GPCRs)1 comprise a large gene family of more than 1000 members that mediate distinct physiological functions as diverse as phototransduction, olfaction, vascular tone, cardiac output, digestion, and pain. GPCR signaling is regulated by a small family of intracellular arrestin proteins that includes visual arrestin (S-antigen), βarrestin1 (arrestin2), and βarrestin2 (arrestin3) (1.Ferguson S.S. Barak L.S. Zhang J. Caron M.G. Can. J. Physiol. Pharmacol. 1996; 74: 1095-1110Crossref PubMed Scopus (318) Google Scholar, 2.Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (855) Google Scholar). Visual arrestin is 60 and 65% identical in amino acid composition to βarrestin1 and βarrestin2, respectively, and predominantly localized in rod photoreceptor cells of the retina but can also be found in other tissues (3.Breitman M.L. Tsuda M. Usukura J. Kikuchi T. Zucconi A. Khoo W. Shinohara T. J. Biol. Chem. 1991; 266: 15505-15510Abstract Full Text PDF PubMed Google Scholar, 4.Smith W.C. Milam A.H. Dugger D. Arendt A. Hargrave P.A. Palczewski K. J. Biol. Chem. 1994; 269: 15407-15410Abstract Full Text PDF PubMed Google Scholar). The βarrestins are 78% identical in amino acid composition and widely expressed in tissues, but their expression level varies in a cell type-specific fashion (5.Attramadal H. Arriza J.L. Aoki C. Dawson T.M. Codina J. Kwatra M.M. Snyder S.H. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 17882-17890Abstract Full Text PDF PubMed Google Scholar, 6.Sterne-Marr R. Gurevich V.V. Goldsmith P. Bodine R.C. Sanders C. Donoso L.A. Benovic J.L. J. Biol. Chem. 1993; 268: 15640-15648Abstract Full Text PDF PubMed Google Scholar). The variations in arrestin homology, localization, and expression level suggest that arrestin family members may differ in their abilities to regulate GPCR signaling.Arrestins bind to GPCRs that are phosphorylated by G protein-coupled receptor kinases (GRKs) (7.Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (898) Google Scholar, 8.Lohse M.J. Andexinger S. Pitcher J. Trukawinski S. Codina J. Faure J.P. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 8558-8564Abstract Full Text PDF PubMed Google Scholar, 9.Gurevich V.V. Benovic J.L. J. Biol. Chem. 1992; 267: 21919-21923Abstract Full Text PDF PubMed Google Scholar). The binding of a single arrestin to a phosphorylated receptor competitively blocks agonist-mediated signal transduction by uncoupling the receptor from heterotrimeric G proteins (5.Attramadal H. Arriza J.L. Aoki C. Dawson T.M. Codina J. Kwatra M.M. Snyder S.H. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 17882-17890Abstract Full Text PDF PubMed Google Scholar, 7.Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (898) Google Scholar, 8.Lohse M.J. Andexinger S. Pitcher J. Trukawinski S. Codina J. Faure J.P. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 8558-8564Abstract Full Text PDF PubMed Google Scholar, 10.Wilden U. Hall S.W. Kuhn H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1174-1178Crossref PubMed Scopus (570) Google Scholar, 11.Pippig S. Andexinger S. Daniel K. Puzicha M. Caron M.G. Lefkowitz R.J. Lohse M.J. J. Biol. Chem. 1993; 268: 3201-3208Abstract Full Text PDF PubMed Google Scholar). This process is termed desensitization. In contrast to visual arrestins that primarily desensitize light-activated rhodopsin (10.Wilden U. Hall S.W. Kuhn H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1174-1178Crossref PubMed Scopus (570) Google Scholar), the nonvisual arrestins βarrestin1 and βarrestin2 also participate in processes controlling re-establishment of receptor responsiveness (12.Ferguson S.S. Downey III, W.E. Colapietro A.M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (839) Google Scholar, 13.Zhang J. Barak L.S. Winkler K.E. Caron M.G. Ferguson S.S. J. Biol. Chem. 1997; 272: 27005-27014Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 14.Yu S.S. Lefkowitz R.J. Hausdorff W.P. J. Biol. Chem. 1993; 268: 337-341Abstract Full Text PDF PubMed Google Scholar, 15.Pippig S. Andexinger S. Lohse M.J. Mol. Pharmacol. 1995; 47: 666-676PubMed Google Scholar). βArrestins target desensitized receptors for endocytosis and resensitization by functioning as docking proteins that link receptors to components of the endocytic machinery such as AP-2 and clathrin (16.Laporte S.A. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Crossref PubMed Scopus (520) Google Scholar, 17.Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1154) Google Scholar). They also regulate the rate at which endosomal receptors are dephosphorylated and recycled back to the plasma membrane through interactions with specific clusters of GRK-phosphorylated residues in the GPCR carboxyl terminus (18.Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 1999; 274: 32248-32257Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar).Arrestins bind numerous GPCRs at the plasma membrane (19.Barak L.S. Ferguson S.S. Zhang J. Caron M.G. J. Biol. Chem. 1997; 272: 27497-27500Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar), and this finding suggests that receptors contain common arrestin recognition motifs. In vitro studies using purified proteins have shown that specificity exists between arrestin family members and GPCRs. For example, visual arrestin binds rhodopsin in preference to the β2-adrenergic receptor (β2AR) and m2 muscarinic acetylcholine receptor (m2mAChR), whereas βarrestin1 and βarrestin2 bind the β2AR and m2mAChR in preference to rhodopsin (5.Attramadal H. Arriza J.L. Aoki C. Dawson T.M. Codina J. Kwatra M.M. Snyder S.H. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 17882-17890Abstract Full Text PDF PubMed Google Scholar, 8.Lohse M.J. Andexinger S. Pitcher J. Trukawinski S. Codina J. Faure J.P. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 8558-8564Abstract Full Text PDF PubMed Google Scholar, 20.Gurevich V.V. Richardson R.M. Kim C.M. Hosey M.M. Benovic J.L. J. Biol. Chem. 1993; 268: 16879-16882Abstract Full Text PDF PubMed Google Scholar, 21.Gurevich V.V. Dion S.B. Onorato J.J. Ptasienski J. Kim C.M. Sterne-Marr R. Hosey M.M. Benovic J.L. J. Biol. Chem. 1995; 270: 720-731Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). Furthermore, βarrestin1 binds the β2AR with a 2.5-fold greater affinity than βarrestin2, and βarrestin2 binds the m2mAChR with a 1.5-fold greater affinity than βarrestin1 (21.Gurevich V.V. Dion S.B. Onorato J.J. Ptasienski J. Kim C.M. Sterne-Marr R. Hosey M.M. Benovic J.L. J. Biol. Chem. 1995; 270: 720-731Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). However, whether specificity exists between arrestin family members and GPCRs in cells has not been explored.In the following study we investigate the dynamic interactions between arrestin family members and GPCRs in live cells by assessing the redistribution of fluorescent arrestins from the cytoplasm to agonist-activated receptors at the plasma membrane. Two classes of GPCRs, designated A and B, are identified that differ in their affinities for the arrestin isoforms. Class A receptors, such as the β2AR, do not interact with visual arrestin and bind βarrestin1 with less affinity than βarrestin2. Class B receptors, such as the angiotensin II type 1A receptor (AT1AR), interact with visual arrestin and bind both βarrestin1 and βarrestin2 with similar high affinities. The molecular determinants underlying this classification appear to reside in specific serine residues located in the receptor carboxyl-terminal tail. These findings reveal a potential role for visual arrestin in the regulation of GPCRs outside the visual system. Moreover, they suggest that the particular cellular complement of arrestin isoforms and their distinct interactions with intracellular proteins will play a critical role regulating the pattern of GPCR desensitization, sequestration, and resensitization.DISCUSSIONIn this study we identify two major classes of GPCRs that demonstrate marked differences in their interactions with visual arrestin, βarrestin1, and βarrestin2. Class A receptors, which bind both biogenic amines and peptide ligands, demonstrate little to no affinity for visual arrestin and have a higher affinity for βarrestin2 than βarrestin1. Class B receptors, which bind peptide ligands, bind visual arrestin and have a similar high affinity for both βarrestin1 and βarrestin2. Residues in the receptor carboxyl-terminal tail appear entirely responsible for determining class A or B characteristics rather than the nature of the ligand. This classification paradigm provides a foundation for predicting the manner in which the cellular complement of arrestin isoforms will regulate the desensitization, sequestration, and resensitization of a particular GPCR.For many GPCRs, homologous desensitization involves both GRK phosphorylation and arrestin binding (1.Ferguson S.S. Barak L.S. Zhang J. Caron M.G. Can. J. Physiol. Pharmacol. 1996; 74: 1095-1110Crossref PubMed Scopus (318) Google Scholar, 2.Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (855) Google Scholar). Our findings fit a model in which the kinetics of GRK phosphorylation are rate-limiting and facilitate the interaction of arrestin with receptors (Fig.11 A). In support of this interpretation, GRK phosphorylation of rhodopsin and the β2AR is necessary for high affinity binding of arrestins both in vitro and in cells (7.Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (898) Google Scholar, 8.Lohse M.J. Andexinger S. Pitcher J. Trukawinski S. Codina J. Faure J.P. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 8558-8564Abstract Full Text PDF PubMed Google Scholar, 9.Gurevich V.V. Benovic J.L. J. Biol. Chem. 1992; 267: 21919-21923Abstract Full Text PDF PubMed Google Scholar, 19.Barak L.S. Ferguson S.S. Zhang J. Caron M.G. J. Biol. Chem. 1997; 272: 27497-27500Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, 21.Gurevich V.V. Dion S.B. Onorato J.J. Ptasienski J. Kim C.M. Sterne-Marr R. Hosey M.M. Benovic J.L. J. Biol. Chem. 1995; 270: 720-731Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). Moreover, GFP analogues of GRK2 translocate to the SPR at the plasma membrane prior to the binding of βarrestin (28.Barak L.S. Warabi K. Feng X. Caron M.G. Kwatra M.M. J. Biol. Chem. 1999; 274: 7565-7569Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Some βarrestin binding may also occur, however, with agonist-activated receptors in the absence of GRK phosphorylation. For example, in vitro studies have shown that βarrestin binds with low affinity to the unphosphorylated β2AR and m2mAChR (21.Gurevich V.V. Dion S.B. Onorato J.J. Ptasienski J. Kim C.M. Sterne-Marr R. Hosey M.M. Benovic J.L. J. Biol. Chem. 1995; 270: 720-731Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). In addition, in experiments with the SPR under conditions that should significantly reduce GRK-mediated phosphorylation in response to agonist (60 min agonist treatment at 4 °C), both transfected and endogenous βarrestins were found to associate with the receptor at the plasma membrane (31.McConalogue K. Dery O. Lovett M. Wong H. Walsh J.H. Grady E.F. Bunnett N.W. J. Biol. Chem. 1999; 274: 16257-16268Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). βArrestin binding to GPCRs is thought to involve the engagement of two regions of the βarrestin molecule with two corresponding regions of the receptor (2.Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (855) Google Scholar, 21.Gurevich V.V. Dion S.B. Onorato J.J. Ptasienski J. Kim C.M. Sterne-Marr R. Hosey M.M. Benovic J.L. J. Biol. Chem. 1995; 270: 720-731Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). A large region within the amino-terminal half of βarrestin (termed the activation-recognition domain) appears to bind the third loop of agonist-activated GPCRs (32.Wu G. Krupnick J.G. Benovic J.L. Lanier S.M. J. Biol. Chem. 1997; 272: 17836-17842Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). A positively charged smaller domain (termed the phosphorylation-recognition domain) appears to interact with GRK-phosphorylated residues in the GPCR carboxyl terminus (33.Kieselbach T. Irrgang K.D. Ruppel H. Eur. J. Biochem. 1994; 226: 87-97Crossref PubMed Scopus (29) Google Scholar). Thus, although βarrestins may bind intracellular loops of agonist-activated receptors that are not phosphorylated, the affinity of this interaction is presumably weak. GRK phosphorylation and the engagement of both βarrestin recognition domains with the receptor appear to be necessary for a high affinity interaction to take place.From the data presented in Fig. 2, we observed that translocation of βarrestin1 to the β2AR was delayed in onset and slower in rate compared with βarrestin2 translocation. Re-analysis of this data using the model described under “Experimental Procedures” indicates that these qualitative differences correspond to an approximately 10-fold higher affinity of βarrestin2 for the β2AR relative to βarrestin1 (Fig. 11 B). In contrast to these findings in cells, in vitro studies have reported that βarrestin1 has a 2.5-fold greater affinity for the β2AR than βarrestin2 (21.Gurevich V.V. Dion S.B. Onorato J.J. Ptasienski J. Kim C.M. Sterne-Marr R. Hosey M.M. Benovic J.L. J. Biol. Chem. 1995; 270: 720-731Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). The discrepancy may arise from differences in the manner in which βarrestin1 and βarrestin2 interact intracellularly with the GRK-phosphorylated β2AR carboxyl-terminal tail. The β2AR tail contains 13 putative phosphate acceptor sites, and βarrestin1 binding in vivo may require that either a greater number of sites or different residues be phosphorylated. Moreover, proteins other than GRKs apparently influence the interaction between βarrestins and GPCRs. The βarrestin carboxyl-terminal domain contains AP-2 and clathrin-binding sites (16.Laporte S.A. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Crossref PubMed Scopus (520) Google Scholar, 30.Krupnick J.G. Goodman Jr., O.B. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 15011-15016Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). A stronger interaction between βarrestin2 and these endocytic proteins may explain why swapping the βarrestin1 and βarrestin2 carboxyl-terminal domains increases the fraction of the βarrestin1 chimera and decreases the fraction of the βarrestin2 chimera that translocate to the β2AR at the plasma membrane.The difference in the ability βarrestin1 and βarrestin2 to interact in cells with the β2AR is typical of class A receptors. However, receptors belonging to class B, such as the AT1AR, appear to bind βarrestin1 and βarrestin2 equally well (Figs. 6 Cand 11 C). Two lines of evidence suggest that their affinity for βarrestins is greater than that possessed by class A receptors. First, AT1ARs and βarrestins form stable endocytic complexes, whereas the β2AR·βarrestin complexes dissociate at or near the plasma membrane. The ability of βarrestins to internalize with the AT1AR into endocytic vesicles may indicate that this receptor internalizes in a βarrestin-dependent pathway. Consistent with this possibility, overexpression of βarrestin1 potentiates AT1AR sequestration in HEK-293 cells (24.Zhang J. Ferguson S.S.G. Barak L.S. Menard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). Second, visual arrestin translocates to class B but not class A receptors. The ability of class B receptors, such as the AT1AR and V2R, to bind arrestin family members with such high affinity appears to be mediated entirely by clusters of phosphorylated residues in the receptor carboxyl-terminal tail (18.Oakley R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 1999; 274: 32248-32257Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar,22.Zhang J. Barak L.S. Anborgh P.H. Laporte S.A. Caron M.G. Ferguson S.S. J. Biol. Chem. 1999; 274: 10999-11006Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Similar clusters are noticeably absent from the tails of class A receptors. Thus, it is not unexpected that switching tails between class A and class B receptors completely reverses their affinity for both the visual and nonvisual arrestins.Differences in βarrestin affinities for class A receptors suggest that the tissue complement of βarrestin1 and βarrestin2 may provide an additional degree of receptor regulation. βArrestin1 is more abundant than βarrestin2 in many bovine tissues including the heart, lung, and spleen and is the predominant isoform in rat respiratory epithelial cells (6.Sterne-Marr R. Gurevich V.V. Goldsmith P. Bodine R.C. Sanders C. Donoso L.A. Benovic J.L. J. Biol. Chem. 1993; 268: 15640-15648Abstract Full Text PDF PubMed Google Scholar, 34.Dawson T.M. Arriza J.L. Jaworsky D.E. Borisy F.F. Attramadal H. Lefkowitz R.J. Ronnett G.V. Science. 1993; 259: 825-829Crossref PubMed Scopus (165) Google Scholar). Conversely, βarrestin2 is more abundant than βarrestin1 in the rat central nervous system, olfactory epithelial cells, and spermatozoa (5.Attramadal H. Arriza J.L. Aoki C. Dawson T.M. Codina J. Kwatra M.M. Snyder S.H. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 17882-17890Abstract Full Text PDF PubMed Google Scholar, 34.Dawson T.M. Arriza J.L. Jaworsky D.E. Borisy F.F. Attramadal H. Lefkowitz R.J. Ronnett G.V. Science. 1993; 259: 825-829Crossref PubMed Scopus (165) Google Scholar). βArrestin2 was also found to be 3–5-fold more abundant than βarrestin1 in a variety of transformed cell lines (35.Menard L. Ferguson S.S. Zhang J. Lin F.T. Lefkowitz R.J. Caron M.G. Barak L.S. Mol. Pharmacol. 1997; 51: 800-808Crossref PubMed Scopus (213) Google Scholar). Since βarrestin1 and βarrestin2 bind class A receptors with different affinities, signaling of class A receptors will be more affected by relative changes in the ratio of βarrestin1 and βarrestin2 than class B receptors. An important challenge for future research will be to determine the relationship between GPCR behavior and the absolute and relative levels of βarrestin1 and βarrestin2 in normal cells and in diseased tissues.The ability of visual arrestin to interact with class B receptors suggests that it may play an unappreciated role regulating the signaling of GPCRs other than rhodopsin. Visual arrestin is expressed primarily in rod photoreceptor cells of the retina, but it is also found in other brain regions such as the pineal gland and in peripheral tissues including the heart, kidney, and lung (3.Breitman M.L. Tsuda M. Usukura J. Kikuchi T. Zucconi A. Khoo W. Shinohara T. J. Biol. Chem. 1991; 266: 15505-15510Abstract Full Text PDF PubMed Google Scholar, 4.Smith W.C. Milam A.H. Dugger D. Arendt A. Hargrave P.A. Palczewski K. J. Biol. Chem. 1994; 269: 15407-15410Abstract Full Text PDF PubMed Google Scholar). In contrast to βarrestin1 and βarrestin2, visual arrestin is unlikely to promote receptor sequestration as it does not bind clathrin and interacts very poorly with AP-2 2S. A. Laporte, personal communication. (17.Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1154) Google Scholar). Moreover, we observe that visual arrestin does not appear to target class B receptors to clathrin-coated pits. Therefore, visual arrestin by desensitizing class B receptors without promoting internalization may provide a more permanent scaffold for membrane signaling events and an endocytic independent mechanism to resensitize receptors more rapidly than either βarrestin1 or βarrestin2 (36.Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar, 37.Luttrell L.M. Ferguson S.S. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Science. 1999; 283: 655-661Crossref PubMed Scopus (1253) Google Scholar, 38.Palczewski K. McDowell J.H. Jakes S. Ingebritsen T.S. Hargrave P.A. J. Biol. Chem. 1989; 264: 15770-15773Abstract Full Text PDF PubMed Google Scholar, 39.Byk T. Bar-Yaacov M. Doza Y.N. Minke B. Selinger Z. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1907-1911Crossref PubMed Scopus (132) Google Scholar).An unanticipated finding of these studies is that the agonist-independent subcellular distributions of the arrestin isoforms differ. Visual arrestin and βarrestin1 are distributed in both the cell cytoplasm and nucleus, whereas βarrestin2 is found in the cytoplasm but is excluded from the nucleus. The amino acid residues mediating arrestin compartmentalization appear to reside in the arrestin carboxyl terminus. Switching the βarrestin1 and βarrestin2 carboxyl-terminal domains completely reverses their subcellular distributions. Notably, reports have shown that functional prostaglandin E2 receptors reside on the nuclear envelope and other GPCRs, including muscarinic and angiotensin receptors, may be there as well (40.Bhattacharya M. Peri K.G. Almazan G. Ribeiro-da-Silva A. Shichi H. Durocher Y. Abramovitz M. Hou X. Varma D.R. Chemtob S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15792-15797Crossref PubMed Scopus (215) Google Scholar, 41.Bhattacharya M. Peri K. Ribeiro-da-Silva A. Almazan G. Shichi H. Hou X. Varma D.R. Chemtob S. J. Biol. Chem. 1999; 274: 15719-15724Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 42.Lind G.J. Cavanagh H.D. Invest. Ophthalmol. & Visual Sci. 1993; 34: 2943-2952PubMed Google Scholar, 43.Booz G.W. Conrad K.M. Hess A.L. Singer H.A. Baker K.M. Endocrinology. 1992; 130: 3641-3649Crossref PubMed Scopus (123) Google Scholar). Thus, the nuclear pools of visual arrestin and βarrestin1 may specifically regulate nuclear GPCR signaling.In summary, we have shown in real time and in live cells that specificity exists between arrestin family members and GPCRs. Therefore, these findings provide a basis for predicting the arrestin-mediated pattern of GPCR desensitization, sequestration, and resensitization. Moreover, they explain why the regulation of certain GPCRs will be more susceptible to variations in the cellular complement of arrestin isoforms. G protein-coupled receptors (GPCRs)1 comprise a large gene family of more than 1000 members that mediate distinct physiological functions as diverse as phototransduction, olfaction, vascular tone, cardiac output, digestion, and pain. GPCR signaling is regulated by a small family of intracellular arrestin proteins that includes visual arrestin (S-antigen), βarrestin1 (arrestin2), and βarrestin2 (arrestin3) (1.Ferguson S.S. Barak L.S. Zhang J. Caron M.G. Can. J. Physiol. 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