The Nature of the Arrestin·Receptor Complex Determines the Ultimate Fate of the Internalized Receptor

The vast majority of G protein-coupled receptors are desensitized by a uniform two-step mechanism: phosphorylation of an active receptor followed by arrestin binding. The arrestin·receptor complex is then internalized. Internalized receptor can be recycled back to the plasma membrane (resensitization) or targeted to lysosomes for degradation (down-regulation). The intracellular compartment where this choice is made and the molecular mechanisms involved are largely unknown. Here we used two arrestin2 mutants that bind with high affinity to phosphorylated and unphosphorylated agonist-activated β2-adrenergic receptor to manipulate the receptor-arrestin interface. We found that mutants support rapid internalization of β2-adrenergic receptor similar to wild type arrestin2. At the same time, phosphorylation-independent arrestin2 mutants facilitate receptor recycling and sharply reduce the rate of receptor loss, effectively protecting β2-adrenergic receptor from down-regulation even after very long (up to 24 h) agonist exposure. Phosphorylation-independent arrestin2 mutants dramatically reduce receptor phosphorylation in response to an agonist both in vitro and in cells. Interestingly, co-expression of high levels of β-adrenergic receptor kinase restores receptor down-regulation in the presence of mutants to the levels observed with wild type arrestin2. Our data suggest that unphosphorylated receptor internalized in complex with mutant arrestins recycles faster than phosphoreceptor and is less likely to get degraded. Thus, targeted manipulation of the characteristics of an arrestin protein that binds to a G protein-coupled receptors can dramatically change receptor trafficking and its ultimate fate in a cell. The vast majority of G protein-coupled receptors are desensitized by a uniform two-step mechanism: phosphorylation of an active receptor followed by arrestin binding. The arrestin·receptor complex is then internalized. Internalized receptor can be recycled back to the plasma membrane (resensitization) or targeted to lysosomes for degradation (down-regulation). The intracellular compartment where this choice is made and the molecular mechanisms involved are largely unknown. Here we used two arrestin2 mutants that bind with high affinity to phosphorylated and unphosphorylated agonist-activated β2-adrenergic receptor to manipulate the receptor-arrestin interface. We found that mutants support rapid internalization of β2-adrenergic receptor similar to wild type arrestin2. At the same time, phosphorylation-independent arrestin2 mutants facilitate receptor recycling and sharply reduce the rate of receptor loss, effectively protecting β2-adrenergic receptor from down-regulation even after very long (up to 24 h) agonist exposure. Phosphorylation-independent arrestin2 mutants dramatically reduce receptor phosphorylation in response to an agonist both in vitro and in cells. Interestingly, co-expression of high levels of β-adrenergic receptor kinase restores receptor down-regulation in the presence of mutants to the levels observed with wild type arrestin2. Our data suggest that unphosphorylated receptor internalized in complex with mutant arrestins recycles faster than phosphoreceptor and is less likely to get degraded. Thus, targeted manipulation of the characteristics of an arrestin protein that binds to a G protein-coupled receptors can dramatically change receptor trafficking and its ultimate fate in a cell. G protein-coupled receptors GPCR-kinase wild type β2-adrenergic receptor human embryonic kidney Dulbecco's modified Eagle's medium green fluorescence protein hemagglutinin phosphate-buffered saline PBS supplemented with 1 mm CaCl2 and 0.5 mm MgCl2 phosphorylated G protein-coupled receptors (GPCRs)1 are the largest known group of sensor proteins. There are over 1,000 members of this family that respond to a wide variety of stimuli: light, odorants, hormones, neurotransmitters, peptides, extracellular calcium, etc. (1Claing A. Laporte S.A. Caron M.G. Lefkowitz R.J. Progr. Neurobiol. 2002; 66: 61-79Google Scholar). Activated GPCRs catalyze GDP/GTP exchange on heterotrimeric G proteins, whereupon the GTP-liganded G protein α-subunit and the free βγ-dimer modulate the activity of various effectors, including adenylyl cyclase, phospholipase C, cGMP phosphodiesterase, ion channels, etc. (1Claing A. Laporte S.A. Caron M.G. Lefkowitz R.J. Progr. Neurobiol. 2002; 66: 61-79Google Scholar). The same active receptor conformation that interacts with G proteins is phosphorylated by GPCR-kinases (GRKs) (1Claing A. Laporte S.A. Caron M.G. Lefkowitz R.J. Progr. Neurobiol. 2002; 66: 61-79Google Scholar). Arrestins then bind to the active phosphorylated state of the receptor (2Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Google Scholar). Arrestin binding prevents further G protein interaction (apparently, by simple steric exclusion (3Krupnick J.G. Gurevich V.V. Benovic J.L. J. Biol. Chem. 1997; 272: 18125-18131Google Scholar)), often targeting receptors to the coated pits due to the high affinity of non-visual arrestins for various components of the internalization machinery: clathrin (4Goodman 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: 447-450Google Scholar), clathrin adaptor AP2 (5Laporte S.A. Oakley R.H. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 2000; 275: 23120-23126Google Scholar), and N-ethylmaleimide-sensitive fusion protein (NSF) (6McDonald J.A. Lefkowitz R.J. J. Biol. Chem. Scholar). Arrestins GPCRs to G (1Claing A. Laporte S.A. Caron M.G. Lefkowitz R.J. Progr. Neurobiol. 2002; 66: 61-79Google Scholar), of F. Caron M.G. Lefkowitz R.J. Scholar), kinase Laporte S.A. R.J. Lefkowitz R.J. 2000; Scholar), Lefkowitz R.J. A. Scholar), and extracellular kinase F. Caron M.G. Lefkowitz R.J. Lefkowitz R.J. A. Scholar). to the internalized arrestin·receptor complex likely due to in the (1Claing A. Laporte S.A. Caron M.G. Lefkowitz R.J. Progr. Neurobiol. 2002; 66: 61-79Google Scholar). The of active receptor conformation arrestin whereupon the receptor can be and recycled to the plasma The of by the of the arrestin·receptor is the in this non-visual arrestins bind to the phosphorylated agonist-activated of GPCRs V.V. Benovic J.L. J. Biol. Chem. 1997; 272: A. J. Gurevich V.V. J. Biol. Chem. V.V. J. Benovic J.L. J. Biol. Chem. J. S.A. Gurevich V.V. J. Biol. Chem. 2002; Scholar). binding to the phosphorylated is high A. J. Gurevich V.V. J. Biol. Chem. V.V. J. Benovic J.L. J. Biol. Chem. J. S.A. Gurevich V.V. J. Biol. Chem. 2002; Scholar), that arrestin from internalized phosphoreceptor be we mutants of both non-visual arrestins that bind to receptors in a phosphorylation-independent V.V. Benovic J.L. J. Biol. Chem. 1997; 272: A. J. Gurevich V.V. J. Biol. Chem. J. S.A. Gurevich V.V. J. Biol. Chem. 2002; Scholar). to wild type arrestin2 binding to the binding of the and phosphorylation-independent mutants to unphosphorylated receptor is We that in mutants bind to the unphosphorylated receptor and that internalization the mutant arrestin·receptor complex faster than the complex of arrestin2 with Here we used phosphorylation-independent mutants to this change in the of the arrestin·receptor complex receptor trafficking in cells. that in this we the of arrestin proteins. The of arrestin2 are and is with β2-adrenergic receptor and in V.V. J. Benovic J.L. J. Biol. Chem. Scholar). the receptor phosphorylated with in the presence of of and to the arrestins in mm 0.5 mm with phosphorylated or unphosphorylated in the presence of or in a of The then for on and with mm and mm The arrestin with the in the 0.5 and binding in the presence of of the binding and of the arrestin in the vitro receptor phosphorylation by of with of in the presence of of in of mm mm and activity with or the of arrestin2 or mutant V.V. Benovic J.L. J. Biol. Chem. 1997; 272: A. J. Gurevich V.V. J. Biol. Chem. for both arrestin and to the the by the of and by the of of The to the and the in a a of human embryonic kidney the The in Dulbecco's modified Eagle's medium supplemented with in a At in the with of A.W. Benovic J.L. J. Biol. Chem. and of or its or the in and the with of A.W. Benovic J.L. J. Biol. Chem. Scholar). the in the with of the arrestin2 with of of hemagglutinin F. Caron M.G. Lefkowitz R.J. in used to the for phosphorylation-independent mutant down-regulation to and arrestin2 or mutant with or of with for the and with The in receptor binding and is the receptor due to agonist of in arrestin and levels and are with mutant rapid by the of and with arrestin2 or mutant with then with of and of internalization are of group is in the with by The then and in in PBS supplemented with 1 mm 1 and with or At the of the the with the in and after the and receptor with and A.W. Benovic J.L. J. Biol. Chem. Scholar). binding in the presence of and Internalized receptor the and 1 of protein The of arrestins and in by proteins A. J. Gurevich V.V. J. Biol. Chem. Scholar). and used the and used the The with and A. J. Gurevich V.V. J. Biol. Chem. Benovic J.L. Gurevich V.V. 1997; Scholar). on an The and on and on Arrestins with followed by with the with by its data and the data by of arrestin and for in we and the on the intracellular in the presence of arrestin2 or mutant of green and with and and The of the and to the of the after to the of fluorescence to the receptor on the and the of fluorescence and intracellular fluorescence for cell. intracellular we the receptor with the membrane by that the membrane The for a of the fluorescence the cell. The data by of with protein and the of intracellular on time, of for arrestin protein with the and to used where receptor 2000; J. Biol. Chem. Scholar). with a with and arrestin2 and 24 the in and The with PBS supplemented with 1 mm CaCl2 and 0.5 mm MgCl2 on with of in for The with with 1 and for in supplemented with 1 mm in the presence or of with or J. Scholar). At the of the on and with internalized by two of with mm in mm followed by for with in the rate of receptor the after agonist and further in with for and and then and The in in of mm mm 1 mm mm 1 mm supplemented with The to an with 0.5 of with and for on by for The 0.5 to an of and with used to arrestin by the with with and with The then in of The to and and The with and A. J. Gurevich V.V. J. Biol. Chem. Benovic J.L. Gurevich V.V. 1997; Scholar), on after for the of and after for the of The of in The receptor in used a for the of internalized the data from the where the of high and the of the J. Biol. Chem. 2002; Scholar). with to receptor of protein and or or mutants of with of in for and with the same medium with receptor and arrestin by binding and the mm mm mm mm 1 and by the for in 1 of supplemented with mm of with or with receptor the on with and in 0.5 of mm mm mm mm mm 1 for reduce by with of protein for the of the to a and for 1 with 1 of the of of protein h) for with 1 of with high and mm and with the same then in and on receptor on The of the with further by of with of the arrestin is for receptor binding (2Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Google A. S.A. J.A. Gurevich V.V. J. Biol. Chem. J.A. Gurevich V.V. Gurevich V.V. S.A. Scholar). The conformation of arrestin is in by the an of in the of the arrestin S.A. J.A. Gurevich V.V. J. Biol. Chem. J.A. Gurevich V.V. Gurevich V.V. S.A. V.V. Benovic J.L. J. Biol. Chem. S.A. Gurevich Gurevich V.V. J. Biol. Chem. 2000; 275: Scholar). the and its S.A. J.A. Gurevich V.V. J. Biol. Chem. J.A. Gurevich V.V. Gurevich V.V. S.A. V.V. Benovic J.L. J. Biol. Chem. S.A. Gurevich Gurevich V.V. J. Biol. Chem. 2000; 275: Scholar). of the sensor in the in arrestin Benovic J.L. Gurevich V.V. 1997; S.A. J.A. Gurevich V.V. J. Biol. Chem. V.V. Benovic J.L. J. Biol. Chem. Scholar), in arrestin2 V.V. Benovic J.L. J. Biol. Chem. 1997; 272: A. J. Gurevich V.V. J. Biol. Chem. Scholar), and in J. S.A. Gurevich V.V. J. Biol. Chem. 2002; Scholar), the to the mutant to bind with high affinity to of the phosphorylated or V.V. Benovic J.L. J. Biol. Chem. 1997; 272: A. J. Gurevich V.V. J. Biol. Chem. J. S.A. Gurevich V.V. J. Biol. Chem. 2002; Benovic J.L. Gurevich V.V. 1997; S.A. J.A. Gurevich V.V. J. Biol. Chem. J.A. Gurevich V.V. Gurevich V.V. S.A. V.V. Benovic J.L. J. Biol. Chem. S.A. Gurevich Gurevich V.V. J. Biol. Chem. 2000; 275: Scholar). the binding of arrestin2 to phosphorylated is by receptor its binding to the presence of an is 1 The phosphorylation-independent mutant similar high binding to active and its binding to unphosphorylated is 1 of binding to unphosphorylated can be to with receptor in to mutant effectively phosphorylation by in vitro 1 we used of in the human are in the of both proteins for the same agonist-activated a of mutant is for of receptor phosphorylation characteristics of suggest that its in the phosphorylation of by be and that unphosphorylated with the mutant are likely to receptor due to agonist in faster than the of arrestin2 with phosphorylated the change in arrestin2 characteristics due to the the trafficking of we with fusion is a A.W. Benovic J.L. J. Biol. Chem. with arrestin2 and arrestins are to the plasma membrane agonist and can be with the receptor in intracellular the membrane that the mutant receptor in we found a of in the in the presence of the mutant than with arrestin2. the of the receptor is on the membrane or in to and of the mutant in the a green of the plasma membrane is after of agonist in arrestin2 of and of arrestin2 in intracellular and a of is found or the plasma that the green is and often is and in of we the binding of the and the (4Goodman 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: 447-450Google A.W. Benovic J.L. J. Biol. Chem. Scholar). with a of the agonist in the presence of an of the receptor is found the with with of the receptor is internalized in the presence of the mutant the of internalized receptor with the rate of receptor in with a A.W. Benovic J.L. J. Biol. Chem. Scholar). the presence of the mutant is very of even after 24 of with the agonist of the receptor is with arrestin2 after 24 Interestingly, the of the mutant on receptor down-regulation with its in the of of protein over arrestin2 in by The its the of the mutant than of protein over the of arrestin2 in the same down-regulation data support the that receptor trafficking in the presence of arrestin2 and its phosphorylation-independent mutant is and that a of the mutant is to arrestin2 likely receptor 1 a of with arrestin2 and mutant we found that receptor down-regulation with its to lysosomes in with A.W. Benovic J.L. J. Biol. Chem. Scholar). The very of internalized with receptor to lysosomes and down-regulation in the presence of be by an of the mutant to support receptor rapid by the of the mutant with on the membrane and in the in with an recycling back to the plasma membrane for is the we of the of the interaction to We that a of an to the extracellular of the the receptor in the of its presence even after the receptor of this this we receptor and with arrestin2 and then with The free and the with to receptor The and the by its fluorescence or after with a in of the on the plasma membrane in in in arrestin2 and The of the in the of an agonist change with the we the internalization of the agonist and this and levels by green and in The internalized the of and to an agonist rapid internalization of the in the presence of both arrestin2 and mutant of the found the At this the mutant even than arrestin2 in internalization of the the the of the internalized in the mutant than in the arrestin2. the mutant the of internalized a after of and change in the of internalized with to the the in on in the presence of is to lysosomes and We further the for this is and for the of receptor down-regulation in the presence of the data that the receptor is effectively internalized with both arrestin2 and Interestingly, in phosphorylation-independent of the receptor is on the plasma membrane even after 1 agonist in a of the green receptor with in data support the that the of internalization with both of arrestin2 are Thus, the in the rapid recycling of the receptor internalized in complex with the that this is the a we 2000; J. Biol. Chem. Scholar). this proteins including are with a an the and the internalization is then by agonist for with the this from that internalized the agonist is and the are further with a of the for to the is from that recycled back to the plasma that a of the receptor to the rate of The are then and is to and with is then and by of is internalized after of with of arrestin2 the of internalized receptor from to with of mutant with and in with binding data and Interestingly, and receptor recycling by mm J. Scholar), the same of internalized in the that and mutant of arrestin2 support receptor internalization and that the of found the in the presence of is due to its the of recycling after the same with to receptor we the agonist and and the to internalized receptor for in the presence of to the of that the of the receptor the can be Thus, the the of internalized receptor after agonist with and recycled we the of the recycled receptor the of this to the of the receptor internalized. We found that the same of internalized receptor is recycled in in and in even the of internalized receptor in the of the of receptor internalized in the presence of is recycled in is the same in and arrestin2. over of the receptor internalized in complex with mutant even in that to of the receptor by that the rate of recycling on the of the arrestin is internalized with and that phosphorylation-independent arrestin2 mutant rapid recycling of the of suggest that internalization effectively wild type arrestin2. We observed rapid of mutant to the receptor in response to agonist rapid of in in the presence of high receptor internalization in the presence of mutant recycling is by data are with in vitro that interacts with clathrin and AP2 with even affinity than arrestin2 Benovic J.L. J. Biol. Chem. 2002; Scholar). Thus, very recycling of internalized in complex with mutant is the for the levels of internalized in agonist The arrestin2 and the mutant is that the binds to unphosphorylated We that the observed in receptor down-regulation is the of the of an arrestin complex with unphosphorylated is to receptor the binding of mutant to and agonist-activated on 1 that is then receptor phosphorylation the the binding of both arrestin2 and mutant to and agonist-activated on this we with and mutant the with and of protein by the of the kinase in to or than that of arrestin to that the mutant effectively with the kinase for we observed a receptor with arrestin2 and very down-regulation with in the presence of is a with both of arrestin2 down-regulation in both The is after 24 of with in the where receptor from to in the presence of is in the of receptor in the presence of the two of arrestin2 after 24 is that internalized in complex with can be targeted to lysosomes and degraded. in the of the two of arrestin2 to with the internalization that receptor that phosphorylation-independent binding of to the receptor than of this mutant is the of the observed change in receptor we arrestin2 mutant with a similar J. S.A. Gurevich V.V. J. Biol. Chem. 2002; we to to the the and a interaction arrestin in its the interaction and of the J. S.A. Gurevich V.V. J. Biol. Chem. 2002; J.A. Gurevich V.V. Gurevich V.V. S.A. S.A. Gurevich Gurevich V.V. J. Biol. Chem. 2000; 275: V.V. J. Biol. Chem. Scholar). to its arrestin J. S.A. Gurevich V.V. J. Biol. Chem. 2002; V.V. J. Biol. Chem. and the mutant the to bind to unphosphorylated receptor in a The of in the same in the in a in down-regulation agonist with arrestin2 of with this mutant levels to or than that of receptor down-regulation Thus, similar of the two phosphorylation-independent mutants 1 and of on receptor trafficking in and that high levels of and mutants unphosphorylated we in phosphorylation of in with arrestin and in protein of or phosphorylation-independent of arrestin2 we used a of the agonist to the of receptor phosphorylation by protein kinase (1Claing A. Laporte S.A. Caron M.G. Lefkowitz R.J. Progr. Neurobiol. 2002; 66: 61-79Google Scholar). The with for two to internalization and the with agonist in the presence of to that the majority of the receptor is internalized that the phosphorylation by this that of the internalized We found that arrestin2 with and of arrestin2 The in receptor phosphorylation the same receptor recycling is by mm in phosphorylation observed in of the phosphorylation-independent the presence of or mutants the of receptor phosphorylation even in the presence of and that in the internalization of over of We found in phosphorylation receptor and receptor that phosphorylation on the of the arrestin on its in receptor phosphorylation found and that to bind to an active receptor in a phosphorylation-independent is for the of receptor phosphorylation in cells. data that in the presence of high levels of phosphorylation-independent mutant of is internalized in the unphosphorylated in its recycling and down-regulation and data suggest that the of the and mutants to bind agonist-activated unphosphorylated is for the dramatically fate of the internalized receptor observed in The binding of mutants to is on receptor the binding of both mutants to and agonist-activated on 1 and We that the of and to receptor in with the complex 1 and is likely to an in the change in receptor in is of the in two the can receptors to recycling A. Nature. A. F. J. J. 2000; J. Biol. Chem. 2000; 275: A. Lefkowitz R.J. J. Biol. Chem. R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. Scholar). to be interaction of receptors with components of the in A. Laporte S.A. Caron M.G. Lefkowitz R.J. Progr. Neurobiol. 2002; 66: 61-79Google Scholar). change the trafficking and ultimate fate of the same receptor Here we are to this and phosphorylation-independent arrestin2 mutants and that effectively this receptor A. J. Gurevich V.V. J. Biol. Chem. J. S.A. Gurevich V.V. J. Biol. Chem. 2002; and change the of the arrestin·receptor complex 1 and of that agonist-activated is internalized the coated pits in complex with arrestin (1Claing A. Laporte S.A. Caron M.G. Lefkowitz R.J. Progr. Neurobiol. 2002; 66: 61-79Google 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: 447-450Google S.A. Oakley R.H. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 2000; 275: 23120-23126Google F. Caron M.G. Lefkowitz R.J. A.W. Benovic J.L. J. Biol. Chem. A. F. J. J. 2000; R.H. J. R.B. 1996; R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. is a complex with high agonist affinity V.V. Benovic J.L. J. Biol. Chem. 1997; 272: A.W. Benovic J.L. J. Biol. Chem. Scholar), in arrestin to R.H. J.L. J. Scholar). in the likely agonist receptor the state arrestin whereupon to protein of protein receptor recycling J. R.H. J. R.B. 1996; Scholar), that receptor is back to the plasma is a the receptor and recycling in the presence of an agonist R.H. J. R.B. 1996; R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. Scholar). agonist to receptor in a of the receptor due to its degradation in the lysosomes and in the of intracellular observed after of agonist R.H. Laporte S.A. Holt J.A. Caron M.G. Barak L.S. J. Biol. Chem. 2000; 275: Scholar). is mechanisms are on by or the receptor from the recycling to the is a of the same mechanisms for a long GPCRs to internalized for a long J. Biol. Chem. 2000; 275: R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. J. Biol. Chem. Scholar). The presence of of and phosphorylated by in the of receptors to long arrestin in the complex with the receptor and the receptor R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. Scholar). receptors for the two non-visual arrestins V.V. J. Benovic J.L. J. Biol. Chem. J. Biol. Chem. Benovic J.L. 2000; Lefkowitz R.J. A. Scholar), in trafficking even of the same receptor R.H. J.L. J. J. Biol. Chem. Benovic J.L. 2000; Lefkowitz R.J. A. Scholar). in or an exchange of GPCRs often arrestin trafficking J. Biol. Chem. 2000; 275: R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. Benovic J.L. 2000; Lefkowitz R.J. A. J. Biol. Chem. 2000; 275: Scholar). used to the mechanisms of receptor trafficking and of of GPCRs in the same A.W. Benovic J.L. J. Biol. Chem. R.H. Laporte S.A. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. J. Biol. Chem. Benovic J.L. 2000; Lefkowitz R.J. A. J. Biol. Chem. 2000; 275: Scholar). that the two non-visual arrestins the of the receptors with in A. Laporte S.A. Caron M.G. Lefkowitz R.J. Progr. Neurobiol. 2002; 66: 61-79Google and A. Lefkowitz R.J. J. Biol. Chem. is are likely to be by the for the of mechanisms that change the fate of the same receptor in the same Phosphorylation-independent mutants of non-visual arrestins a for manipulation of the in the to the of and (1Claing A. Laporte S.A. Caron M.G. Lefkowitz R.J. Progr. Neurobiol. 2002; 66: 61-79Google A. Lefkowitz R.J. J. Biol. Chem. Scholar), internalized receptor in free and and receptor arrestin binding receptor (2Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Google S.A. Gurevich Gurevich V.V. J. Biol. Chem. 2000; 275: A. A. J. J. 2000; 2002; Scholar), free phosphoreceptor is the of the in its phosphorylation and its that receptor is recycled J. R.H. J. R.B. 1996; Scholar), There are two of mechanisms internalized receptor to the phosphoreceptor complex be and of that receptor degradation Lefkowitz R.J. is a of a of this be a of proteins from to the of the of recycling of is trafficking of phosphoreceptor arrestin·receptor complex to the lysosomes by of the of the and its to the majority of GPCRs and mechanisms are by an of mechanisms of by the variety of trafficking observed in various cells. The of a phosphorylation-independent arrestin mutant can be to change the of in the of the majority of in complex with mutant arrestin be unphosphorylated for two its binding on receptor phosphorylation and and with we found that of phosphorylation-independent of arrestin2 or in phosphorylation in even where the majority of the receptor is internalized the of the complex is likely to the of active receptor conformation dramatically the affinity of the mutant for unphosphorylated 1 of phosphorylated a on arrestin2 binding 1 arrestin of the receptor in unphosphorylated Thus, in the of internalized the of receptor in complex with arrestin and that of free phosphoreceptor be sharply the of unphosphorylated receptor to be to the plasma membrane dramatically The of arrestin complex with unphosphorylated receptor is likely to in receptor Lefkowitz R.J. Scholar). a in the of of the internalized receptor can the of the receptor in the facilitate its and its degradation less of the of receptor targeting to lysosomes that the of receptor the complex of arrestin with receptor and and and receptor receptor in and reduce the of receptor down-regulation in the presence of both and mutant observed and Thus, phosphorylation-independent arrestin2 mutants bind to and the internalization of unphosphorylated and and the of to internalized receptor the state intracellular receptor time, by the of its internalization and of internalized receptor and unphosphorylated receptor internalized in complex with recycles faster than phosphoreceptor internalized with arrestin2 of this is receptor even after very long agonist and that receptor and down-regulation to the of R.J. 2000; and L.S. Oakley R.H. Laporte S.A. Caron M.G. A. Scholar). The of phosphorylation-independent mutants of non-visual arrestins to receptors from and down-regulation in with in A. J. Gurevich V.V. J. Biol. Chem. J. S.A. Gurevich V.V. J. Biol. Chem. 2002; arrestins for in of this in non-visual arrestins are involved in binding to GPCRs and a can be by (1Claing A. Laporte S.A. Caron M.G. Lefkowitz R.J. Progr. Neurobiol. 2002; 66: 61-79Google V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Google 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: 447-450Google S.A. Oakley R.H. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 2000; 275: 23120-23126Google J.A. Lefkowitz R.J. J. Biol. Chem. F. Caron M.G. Lefkowitz R.J. Laporte S.A. R.J. Lefkowitz R.J. 2000; Lefkowitz R.J. A. V.V. Benovic J.L. J. Biol. Chem. 1997; 272: A. J. Gurevich V.V. J. Biol. Chem. V.V. J. Benovic J.L. J. Biol. Chem. J. S.A. Gurevich V.V. J. Biol. Chem. 2002; Scholar). We that mutant arrestins with characteristics a of for of various of and for of various with in We J. Benovic for and J. J. for A. for and Caron for We for on A. for and Gurevich for the in