Interaction with β-Arrestin Determines the Difference in Internalization Behavior between β1- and β2-Adrenergic Receptors

Abstract

The β1-adrenergic receptor (β1AR) shows the resistance to agonist-induced internalization. As β-arrestin is important for internalization, we examine the interaction of β-arrestin with β1AR with three different methods: intracellular trafficking of β-arrestin, binding of in vitro translated β-arrestin to intracellular domains of β1- and β2ARs, and inhibition of βAR-stimulated adenylyl cyclase activities by β-arrestin. The green fluorescent protein-tagged β-arrestin 2 translocates to and stays at the plasma membrane by β2AR stimulation. Although green fluorescent protein-tagged β-arrestin 2 also translocates to the plasma membrane, it returns to the cytoplasm 10–30 min after β1AR stimulation. The binding of in vitro translated β-arrestin 1 and β-arrestin 2 to the third intracellular loop and the carboxyl tail of β1AR is lower than that of β2AR. The fusion protein of β-arrestin 1 with glutathione S-transferase inhibits the β1- and β2AR-stimulated adenylyl cyclase activities, although inhibition of the β1AR-stimulated activity requires a higher concentration of the fusion protein than that of the β2AR-stimulated activity. These results suggest that weak interaction of β1AR with β-arrestins explains the resistance to agonist-induced internalization. This is further supported by the finding that β-arrestin can induce internalization of β1AR when β-arrestin 1 does not dissociate from β1AR by fusing to the carboxyl tail of β1AR. The β1-adrenergic receptor (β1AR) shows the resistance to agonist-induced internalization. As β-arrestin is important for internalization, we examine the interaction of β-arrestin with β1AR with three different methods: intracellular trafficking of β-arrestin, binding of in vitro translated β-arrestin to intracellular domains of β1- and β2ARs, and inhibition of βAR-stimulated adenylyl cyclase activities by β-arrestin. The green fluorescent protein-tagged β-arrestin 2 translocates to and stays at the plasma membrane by β2AR stimulation. Although green fluorescent protein-tagged β-arrestin 2 also translocates to the plasma membrane, it returns to the cytoplasm 10–30 min after β1AR stimulation. The binding of in vitro translated β-arrestin 1 and β-arrestin 2 to the third intracellular loop and the carboxyl tail of β1AR is lower than that of β2AR. The fusion protein of β-arrestin 1 with glutathione S-transferase inhibits the β1- and β2AR-stimulated adenylyl cyclase activities, although inhibition of the β1AR-stimulated activity requires a higher concentration of the fusion protein than that of the β2AR-stimulated activity. These results suggest that weak interaction of β1AR with β-arrestins explains the resistance to agonist-induced internalization. This is further supported by the finding that β-arrestin can induce internalization of β1AR when β-arrestin 1 does not dissociate from β1AR by fusing to the carboxyl tail of β1AR. adrenergic receptor G protein-coupled receptor kinase Dulbecco's modified Eagle medium glutathione S-transferase green fluorescent protein polymerase chain reaction fusion protein of βAR with GFP phosphate-buffered saline fusion protein of β-arrestin 2 with GFP carboxyl tail Gs protein enhanced green fluorescent protein The β2-adrenergic receptor (β2AR)1 is one of most extensively studied model systems among G protein-coupled receptors for desensitization consisting of three steps (i.e. uncoupling, internalization, and down-regulation) (1Böhm S.K. Grady E.F. Bunnett N.W. Biochem. J. 1997; 322: 1-18Crossref PubMed Scopus (465) Google Scholar). Within seconds or minutes upon agonist stimulation, β2AR is phosphorylated by GRKs and cAMP-dependent protein kinase. Subsequently, β-arrestin binds to the GRK-phosphorylated β2AR and inhibits coupling of β2AR with Gs. β2AR is also internalized (sequestered) from the plasma membrane to the intracellular endosome through clathrin-coated pits in a similar period of time to that of uncoupling. β-Arrestin plays a central role not only in uncoupling but also in the internalization of β2AR. The overexpression of β-arrestin 1 and β-arrestin 2 restored agonist-induced internalization of the internalization-defective mutant of β2AR (Y326F-β2AR), and the β-arrestin mutants (V53D-β-arrestin 1 and V54D-β-arrestin 2) inhibit internalization of β2AR (2Ferguson S.S.G. Ménard L. Barak L.S. Koch W.J. Colapietro A.-M. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 3Ferguson S.S.G. Downey III, W.E. Colapietro A-M. Barak L.S. Ménard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (846) Google Scholar). After internalization, vesicular acidification leads to conformational changes of the phosphorylated β2AR and facilitates dephosphorylation by vesicle-associated phosphatase (4Krueger K.M. Daaka Y. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 5-8Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). Then the dephosphorylated β2AR returns to the plasma membrane and responds to the new stimulus. In contrast to β2AR, much less is known about the mechanism of β1AR desensitization. Freedman et al. (5Freedman N.J. Liggett S.B. Drachman D.E. Pei G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17953-17961Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar) reported that in transiently transfected HEK293 cells, the exposure to agonist for a short time leads to phosphorylation of β1AR by cAMP-dependent protein kinase and GRKs, and phosphorylation of β1AR decreases the responses to second isoproterenol stimulation. They also showed that overexpression of β-arrestin 1 or β-arrestin 2 enhances uncoupling of β1AR from Gs. These results indicate that β-arrestins are involved in the uncoupling of β1AR as well as β2AR. However, there is a significant difference in the rate of internalization between β1AR and β2AR. Upon agonist stimulation for a short time, β1AR undergoes only slight internalization, whereas β2AR internalizes rapidly in the same cellular environment (6Suzuki T. Nguyen C.T. Nantel F. Bonin H. Valiquette M. Frielle T. Bouvier M. Mol. Pharmacol. 1992; 41: 542-548PubMed Google Scholar). Green and Liggett (7Green S.A. Liggett S.B. J. Biol. Chem. 1994; 269: 26215-26219Abstract Full Text PDF PubMed Google Scholar) reported that the transfer of the proline-rich region in the third intracellular loop of β1AR to the homologous site of β2AR attenuates internalization, and deletion of the same region from β1AR promotes internalization. They suggested that the in agonist-induced internalization between β1- and are of the proline-rich region in the third intracellular loop of β1AR. to the interaction of β1AR with β-arrestin the resistance to agonist-induced internalization. in the that the weak interaction of β1AR with β-arrestins results in the resistance of β1AR to agonist-induced internalization. receptors of β1- and β2ARs, we also that the third intracellular loop and the carboxyl tail of receptors are the difference in internalization between β1- and and from from for β1AR and β2AR, β-arrestin β-arrestin and fusion of the carboxyl tail and the third intracellular loop of β1AR and β2AR by J. Lefkowitz The for and by J. L. HEK293 from and from The from and from from from The and from from The of the β1- and by et al. Y. H. M. T. J. Pharmacol. 1996; PubMed Scopus Google Scholar). The fusion of β2AR, and β-arrestin 2 with by a of of Barak et al. L.S. S.S.G. J. T. Caron M.G. Mol. Pharmacol. 1997; PubMed Scopus Google Scholar, L.S. S.S.G. J. Caron M.G. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). of the β1- and β2AR fusion with for β1AR and for by at the of the of the the of the receptors and of for β1AR or and of for β2AR. the fusion protein of β-arrestin 2 with for and for by at of the of β-arrestin the region of β-arrestin 2 and of The fusion with as or β-arrestin The β1- and 1 fusion as the region of β-arrestin 1 by and of or The of the of by from Then the region of β-arrestin 1 with the β1- or of by of and for β1AR or and for β2AR. of there are between β-arrestin 1 and of receptors for β1AR and for The fusion protein of β-arrestin 1 with GFP also by of the β-arrestin 1 with However, we not the β-arrestin fusion protein as by and not β-arrestin for the intracellular trafficking of the β-arrestin upon agonist stimulation. receptors of β1- and by The of the in β1- and are as to in β1- and β2AR The of the of by from of or of The fusion with the third intracellular loop of β1AR carboxyl tail of β1AR third intracellular loop of β2AR or carboxyl tail of β2AR for the binding of in vitro translated β-arrestin 1 and β-arrestin of the carboxyl tail of β-arrestin 1 by and for the at the or at the for of After the of the the and of The fusion protein with the carboxyl tail of β-arrestin 1 and for as H. Lefkowitz R.J. Mol. Pharmacol. Google Scholar). The the carboxyl tail of β-arrestin 1 β-arrestin 1 but not β-arrestin 2 not of or the of or of by The similar to of for and of fusion and The region of β-arrestin 1 by the As the of β-arrestin 1 in at the at the β-arrestin 1 the and in and the and of The fusion protein for of the The fusion protein by the of for at H. T. J. Biochem. PubMed Scopus Google Scholar). The fusion protein by the same as that of fusion and by HEK293 in with and The at of 2 for or 2 for the of Then of for or for for with or of to the of by the of The for after HEK293 in that transiently transfected at in with or isoproterenol for the The three by and for at with in modified 2 After the with and the binding by the of a from the intracellular trafficking of β1- or β2AR upon the receptor HEK293 transfected with the β1AR or β2AR as the of the medium with The or fluorescent The with isoproterenol for min at the of β-arrestin in cells, HEK293 transfected with β1- or with β-arrestin The medium with The β-arrestin The with isoproterenol for the time at β1AR or β2AR by et al. Y. H. M. T. J. Pharmacol. 1996; PubMed Scopus Google Scholar) and for the The of of β1AR and β2AR and cyclase activities by the of et al. Y. M. Biochem. PubMed Scopus Google Scholar). of membrane and 1 or at the or and at for min in of the concentration of β1AR or β2AR about or The reaction by the and 1 of the and The by and and the The the of β-arrestin 1 or β-arrestin 2 in and translated in vitro in the of to the fusion with the third intracellular loop or the carboxyl tail of β1- or The with in vitro translated β-arrestin 1 and β-arrestin 2 in consisting of of binding of and of in vitro translated β-arrestin 1 or β-arrestin 2 for at After with binding at by in and by The β-arrestins by agonist-induced internalization by the of β1- or at the after agonist stimulation. transiently β1- or by isoproterenol for The of at the about by isoproterenol stimulation for min In contrast to β2AR, the of not by agonist stimulation. This is with the (6Suzuki T. Nguyen C.T. Nantel F. Bonin H. Valiquette M. Frielle T. Bouvier M. Mol. Pharmacol. 1992; 41: 542-548PubMed Google Scholar, S.A. Liggett S.B. J. Biol. Chem. 1994; 269: 26215-26219Abstract Full Text PDF PubMed Google Scholar). the of to the carboxyl of β1- or The fusion in HEK293 cells, and the with β1- and at the plasma membrane in the in as a of agonist stimulation and in that the of the 2 In contrast to β2AR, β1AR at the plasma membrane after agonist stimulation for min 2 β1AR concentration at of the of the upon agonist stimulation 2 These results that β1AR is to the agonist-induced internalization the cellular environment in β2AR can of β1- or upon agonist stimulation. of of upon receptor transiently in HEK293 and with isoproterenol for of of upon receptor transiently in HEK293 and with isoproterenol for In the in the same reported that overexpression of β-arrestin or β-arrestin 2 the agonist-induced internalization of internalization-defective mutant (2Ferguson S.S.G. Ménard L. Barak L.S. Koch W.J. Colapietro A.-M. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). we the that overexpression of GRKs or β-arrestins the resistance of β1AR to the agonist-induced internalization. β1AR or β1AR with β-arrestin β-arrestin or one of the GRKs in HEK293 cells, and the of at the after agonist stimulation by the binding shows that β1AR internalized by agonist stimulation when β-arrestin β-arrestin or GRKs In contrast to agonist stimulation for or min the of the receptors of β2AR to or β2AR with β-arrestin 1 or β-arrestin a slight in internalization of β2AR upon receptor stimulation for min but not of GRKs and β-arrestin 1 by to of the GRKs or β-arrestin 1 the the carboxyl tail of β-arrestin 1 not β-arrestin the of β-arrestin 2 not by not However, overexpression of β-arrestin 2 enhanced the agonist-induced internalization of β1- and we that overexpression of β-arrestin or β-arrestin 2 is not for β1AR to upon agonist stimulation. the interaction of β1AR with β-arrestin in cells, we β-arrestin to intracellular trafficking of β-arrestin 2 upon of β1- or The β-arrestin as by of agonist-induced internalization of β1- and The of β-arrestin in the the cytoplasm and not enhanced at the plasma membrane This is with the J. Barak L.S. S.A. Caron M.G. S.S.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). β2AR with β-arrestin rapidly to the plasma membrane in a β-Arrestin to at the plasma membrane at and showed with time a at of the However, β-arrestin to the plasma membrane not with β2AR In the of β-arrestin the cytoplasm in the of agonist stimulation and rapidly to the plasma membrane upon β1AR stimulation However, of β1AR not in a of β-arrestin in contrast to of β2AR. β-arrestin to the plasma membrane to the cytoplasm 10–30 min after agonist stimulation β-Arrestin not upon agonist stimulation when the not transfected with β1- or not results at the cellular that β-arrestin 2 transiently with β1AR upon agonist stimulation. region of β1- or is for of agonist-induced internalization and intracellular trafficking of β-arrestin we and of β1- and The of receptors are in HEK293 transiently transfected with one of receptors by isoproterenol for and the of receptors at the by binding shows the internalization of the that the of β1AR with the of β2AR. The (1Böhm S.K. Grady E.F. Bunnett N.W. Biochem. J. 1997; 322: 1-18Crossref PubMed Scopus (465) Google Scholar, S.S.G. Ménard L. Barak L.S. Koch W.J. Colapietro A.-M. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 3Ferguson S.S.G. Downey III, W.E. Colapietro A-M. Barak L.S. Ménard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (846) Google Scholar, K.M. Daaka Y. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 5-8Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, N.J. Liggett S.B. Drachman D.E. Pei G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17953-17961Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, T. Nguyen C.T. Nantel F. Bonin H. Valiquette M. Frielle T. Bouvier M. Mol. Pharmacol. 1992; 41: 542-548PubMed Google Scholar, S.A. Liggett S.B. J. Biol. Chem. 1994; 269: 26215-26219Abstract Full Text PDF PubMed Google the carboxyl tail of β1AR with that of β2AR, internalized upon agonist stimulation However, the of agonist-induced internalization of not the same as that of β2AR. This that region is for internalization of β2AR. The (6Suzuki T. Nguyen C.T. Nantel F. Bonin H. Valiquette M. Frielle T. Bouvier M. Mol. Pharmacol. 1992; 41: 542-548PubMed Google Scholar, S.A. Liggett S.B. J. Biol. Chem. 1994; 269: 26215-26219Abstract Full Text PDF PubMed Google in the third intracellular loop to the carboxyl tail of β1AR with the region of β2AR, to a similar to that of β2AR upon agonist stimulation These results suggest that the third intracellular loop and the carboxyl tail of β2AR are of agonist-induced internalization. The of the carboxyl tail of β2AR with the region of β1AR in the that showed the of agonist-induced internalization However, the of internalization of higher than that of β1AR that the carboxyl tail is not the of internalization. The of the third intracellular loop to the carboxyl tail of β2AR with the of β1AR further the of internalization. These results suggest that the in the third intracellular loop and the carboxyl tail between β1AR and β2AR the of agonist-induced internalization. are for trafficking of β-arrestin upon agonist stimulation in cells, (6Suzuki T. Nguyen C.T. Nantel F. Bonin H. Valiquette M. Frielle T. Bouvier M. Mol. Pharmacol. 1992; 41: 542-548PubMed Google Scholar, S.A. Liggett S.B. J. Biol. Chem. 1994; 269: 26215-26219Abstract Full Text PDF PubMed Google Scholar) and with β-arrestin In the β-arrestin to the plasma membrane by agonist stimulation and at the plasma membrane agonist stimulation This trafficking of β-arrestin is similar to that upon agonist stimulation of β2AR. In β-arrestin to the plasma membrane by agonist stimulation. However, β-arrestin to the cytoplasm the by agonist This trafficking of β-arrestin is similar to that of β1AR upon agonist stimulation. These results suggest that the intracellular third loop and the carboxyl tail the of β-arrestin by agonist of β-arrestin GFP fusion protein upon or in HEK293 and of the of β-arrestin to the plasma membrane upon or β-Arrestin with or and with isoproterenol for and The of β-arrestin from the in the same The three we the of β-arrestin 1 and β-arrestin 2 to to The fusion of with the carboxyl and the third intracellular of β1- or and with β-arrestin 1 or β-arrestin 2 by in After with fusion the extensively and the β-arrestins by and β-Arrestin 1 not to to the carboxyl tail and the intracellular third loop of β1AR In contrast to the binding to and the β-arrestin 1 to the carboxyl tail of β2AR Although β-arrestin 1 also to the third intracellular loop of β2AR the of β-arrestin 1 binding to lower than that binding to The of binding of β-arrestin 1 results with in vitro translated β-arrestin 2 β-Arrestin 2 to and but to β-Arrestin 2 also to the but the of binding lower than that of The of binding of β-arrestin 2 to and higher than of β-arrestin These results indicate that the intracellular domains of β1AR with β-arrestin 1 and β-arrestin 2 with lower than of β2AR. examine the interaction of β-arrestin 1 with β1- or as a inhibition of βAR-stimulated adenylyl cyclase activities by β-arrestin 1 β-arrestin 1 from as fusion The stimulation of β2AR by agonist the adenylyl cyclase and the cyclase activity in a by 1 fusion but not The inhibition is about and the of inhibition is The cyclase activity not by 1 when the cyclase by These results indicate that inhibition is by β-arrestin 1 but not and inhibition by 1 is for the of inhibition by the fusion protein and the concentration of β2AR inhibition at a 1 of about The β1AR-stimulated adenylyl cyclase activity also by 1 fusion but not in a However, a higher of 1 for inhibition of the β1AR-stimulated cyclase activity. the of inhibition and the concentration of β1AR inhibition at a 1 of about These results indicate that β-arrestin 1 with β1AR with lower than β2AR. The results in the the that β1AR is not internalized by agonist stimulation of weak interaction with β-arrestin 1 and β-arrestin it that β-arrestin 1 can internalization of β1AR when β-arrestin 1 binds to β1AR. examine β-arrestin 1 to the carboxyl of β1- or β2ARs, and the fusion transiently in HEK293 and by shows that 1 fusion protein internalized by agonist stimulation, in contrast to β1AR. 1 fusion protein internalized by agonist stimulation, the of higher than that of β2AR. a different of HEK293 for the of agonist-induced internalization of β2AR higher than that in the However, results that β1AR is internalized by agonist stimulation when β-arrestin does not dissociate from β1AR. β2AR is a of the of G protein-coupled and the mechanism of β2AR extensively by in and in vitro as a of G protein-coupled receptors (1Böhm S.K. Grady E.F. Bunnett N.W. Biochem. J. 1997; 322: 1-18Crossref PubMed Scopus (465) Google Scholar). Although the mechanism of β1AR to β2AR there are (6Suzuki T. Nguyen C.T. Nantel F. Bonin H. Valiquette M. Frielle T. Bouvier M. Mol. Pharmacol. 1992; 41: 542-548PubMed Google Scholar, M. 1995; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. J. Pharmacol. PubMed Scopus Google Scholar). β2AR rapidly internalizes as a of agonist stimulation, whereas β1AR does not (6Suzuki T. Nguyen C.T. Nantel F. Bonin H. Valiquette M. Frielle T. Bouvier M. Mol. Pharmacol. 1992; 41: 542-548PubMed Google Scholar, M. 1995; PubMed Scopus Google Scholar). the desensitization of β1AR is different from that of β2AR (6Suzuki T. Nguyen C.T. Nantel F. Bonin H. Valiquette M. Frielle T. Bouvier M. Mol. Pharmacol. 1992; 41: 542-548PubMed Google Scholar, M. 1995; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). The agonist of β2AR in desensitization as with adenylyl cyclase activities, but not the with β1AR. The of the carboxyl tail of β1AR with the homologous region of β2AR in a receptor that shows a similar desensitization to β2AR G. Nantel F. Bouvier M. Mol. Pharmacol. 1996; Google Scholar). the rate of β2AR is than that of β1AR (6Suzuki T. Nguyen C.T. Nantel F. Bonin H. Valiquette M. Frielle T. Bouvier M. Mol. Pharmacol. 1992; 41: 542-548PubMed Google Scholar, M. 1995; PubMed Scopus Google Scholar, M. J. Pharmacol. PubMed Scopus Google Scholar). However, there are the mechanism of β1AR at the The of the is to the resistance of β1AR to agonist-induced internalization, one of the et al. (6Suzuki T. Nguyen C.T. Nantel F. Bonin H. Valiquette M. Frielle T. Bouvier M. Mol. Pharmacol. 1992; 41: 542-548PubMed Google Scholar) reported that β1AR in agonist-induced internalization. with we that β1AR transiently in HEK293 not internalized by agonist stimulation when β-arrestin or β-arrestin 2 The of β-arrestin to agonist-induced internalization that β1AR with β-arrestin with In the we that the interaction of β1AR with β-arrestins is weak with that of β2AR. three different to the interaction of β1AR with β-arrestins as intracellular trafficking of β-arrestin binding vitro translated β-arrestins to the intracellular of and inhibition of adenylyl cyclase activity by 1 fusion β1AR with β-arrestin and by 2 rapidly to the plasma However, β-arrestin to the plasma membrane to the cytoplasm after agonist stimulation. Upon β2AR stimulation, β-arrestin 2 to the plasma membrane and at the plasma membrane J. Barak L.S. S.A. Caron M.G. S.S.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the of receptor and β-arrestin 1 or β-arrestin 2 to the plasma membrane as a of agonist stimulation to the endosome with the receptors H. Grady E.F. Bunnett N.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. H. Grady E.F. Bunnett N.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of β-arrestin 2 by β1AR stimulation is among G protein-coupled The of β-arrestin 2 by β1AR stimulation the weak interaction of β-arrestin 2 with also the interaction of β-arrestin 1 with β1- and in vitro by inhibition of the β1- or β2AR-stimulated adenylyl cyclase activities by 1 fusion The β1AR-stimulated adenylyl cyclase activity at about a higher concentration with the β2AR-stimulated that β-arrestin 1 with β1AR with lower than β2AR. The cyclase activity by the fusion protein at about of 1 fusion and at about of 1 fusion This is higher than a with β-arrestin 1 and β2AR Pitcher J. J. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). the not by agonist the β2AR in not to phosphorylated by higher than that from the with and phosphorylated β2AR. the β1- and not with agonist the membrane of 1 of However, when the receptors are with the receptors to β-arrestin 1 and β-arrestin to the of β-arrestins are to the The of inhibition by 1 fusion protein Although β2AR-stimulated adenylyl cyclase activity by agonist β2AR-stimulated Gs is by β-arrestin in the the of inhibition of the in the membrane is different from that in the The the lower of β1AR for β-arrestins also from the binding of in vitro translated β-arrestins to the carboxyl tail and the third intracellular loop of β1- and The β-arrestin 1 and β-arrestin 2 to the carboxyl tail and the third intracellular loop of β1- and in the carboxyl tail of β2AR third intracellular loop of β2AR carboxyl tail of β1AR third intracellular loop of β1AR The carboxyl tail of β2AR that phosphorylation a site for β-arrestin 1 and β-arrestin 2 it reported that phosphorylation of β2AR by the for β-arrestin 1 G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Caron M.G. Lefkowitz R.J. Science. PubMed Scopus Google the in vitro translated β-arrestins the of the carboxyl tail and the third intracellular loop of β1- and for results suggest that β1AR lower for β-arrestins than β2AR. The binding of in vitro translated β-arrestin to the carboxyl tail of β2AR that is not phosphorylated by GRKs internalization of β2AR. et al. Bouvier M. Caron M.G. Lefkowitz R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar) reported that cAMP-dependent protein kinase phosphorylation are for the internalization of β2AR in et al. Full Text PDF PubMed Scopus Google Scholar) reported that of the carboxyl tail of β2AR, phosphorylation does not the agonist-induced internalization. et al. Caron M.G. Lefkowitz R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar) cells, reported that is by of cAMP-dependent protein kinase and et al. (2Ferguson S.S.G. Ménard L. Barak L.S. Koch W.J. Colapietro A.-M. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar) that phosphorylation the receptor or enhances the interaction of the receptor with cellular as β-arrestin, is for agonist-induced internalization. They that β2AR can in the of phosphorylation when β-arrestin 1 or β-arrestin 2 at a Although the of binding of β-arrestin to lower than that of β-arrestin to the and Mol. Pharmacol. 1997; PubMed Scopus Google Scholar) the binding of to the phosphorylated Although not β-arrestin 1 or β-arrestin that to the phosphorylated in They showed that undergoes conformational and binding for interaction The results of in vitro translated β-arrestin and the fusion indicate that β-arrestins to the carboxyl tail of β2AR in the and with the third intracellular loop in the second the of β1- and β2ARs, we the that involved in agonist-induced internalization of β2AR and to agonist-induced internalization of β1AR. The results suggest that the carboxyl tail and the third intracellular loop of β1- and the difference in internalization between β1- and The of the carboxyl tail of β2AR to agonist-induced internalization is with the role of the carboxyl phosphorylation in internalization (2Ferguson S.S.G. Ménard L. Barak L.S. Koch W.J. Colapietro A.-M. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). However, the of is not for agonist-induced internalization of β2AR. et al. Bouvier M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar) reported that internalization is not in of the receptors the third intracellular loop of β2AR. They also that intracellular domains in agonist-induced internalization in a and that the from among intracellular domains is for agonist-induced internalization. The results with receptors also indicate that domains of β1AR domains for internalization. et al. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar) reported that in are similar to internalizes when the carboxyl tail of the receptor is et al. Caron M.G. Lefkowitz R.J. PubMed Scopus Google Scholar) reported that and of β2AR, are to phosphorylation are not for internalization, but leads to conformational changes with the internalization Green and Liggett (7Green S.A. Liggett S.B. J. Biol. Chem. 1994; 269: 26215-26219Abstract Full Text PDF PubMed Google Scholar) reported that receptor of β1AR agonist-induced internalization. They showed that deletion of the proline-rich region in the third intracellular loop of β1AR internalization and of the the homologous of β2AR inhibits internalization, that the proline-rich region in the third intracellular loop of β1AR the internalization of β1AR (7Green S.A. Liggett S.B. J. Biol. Chem. 1994; 269: 26215-26219Abstract Full Text PDF PubMed Google Scholar). β1AR the receptor but β2AR is for the resistance of β1AR to agonist-induced internalization. protein than β-arrestin involved in internalization, binds to β2AR but not to β1AR. et al. M. Mol. Biol. PubMed Scopus Google Scholar) a of and suggested that important role in trafficking of a of receptors as and receptors among is to that β2AR, than to one of the or for internalization. In we that β1AR to β-arrestin upon agonist stimulation, but interaction weak with that of β2AR. β-arrestins to for internalization of but not of the G protein-coupled the results indicate that the of β1AR for β-arrestin explains the that β1AR does not upon agonist stimulation. we that the carboxyl tail and the third intracellular loop of β1- and the internalization of agonist-induced internalization of β2AR and receptor is involved in of the resistance of β1AR to agonist-induced internalization the rate of β1AR in and in J. Lefkowitz and J. L. for in the for