Agonist-Receptor-Arrestin, an Alternative Ternary Complex with High Agonist Affinity

The rapid decrease of a response to a persistent stimulus, often termed desensitization, is a widespread biological phenomenon. Signal transduction by numerous G protein-coupled receptors appears to be terminated by a strikingly uniform two-step mechanism, most extensively characterized for the β2-adrenergic receptor (β2AR), m2 muscarinic cholinergic receptor (m2 mAChR), and rhodopsin. The model predicts that activated receptor is initially phosphorylated and then tightly binds an arrestin protein that effectively blocks further G protein interaction. Here we report that complexes of β2AR-arrestin and m2 mAChR-arrestin have a higher affinity for agonists (but not antagonists) than do receptors not complexed with arrestin. The percentage of phosphorylated β2AR in this high affinity state in the presence of full agonists varied with different arrestins and was enhanced by selective mutations in arrestins. The percentage of high affinity sites also was proportional to the intrinsic activity of an agonist, and the coefficient of proportionality varies for different arrestin proteins. Certain mutant arrestins can form these high affinity complexes with unphosphorylated receptors. Mutations that enhance formation of the agonist-receptor-arrestin complexes should provide useful tools for manipulating both the efficiency of signaling and rate and specificity of receptor internalization. The rapid decrease of a response to a persistent stimulus, often termed desensitization, is a widespread biological phenomenon. Signal transduction by numerous G protein-coupled receptors appears to be terminated by a strikingly uniform two-step mechanism, most extensively characterized for the β2-adrenergic receptor (β2AR), m2 muscarinic cholinergic receptor (m2 mAChR), and rhodopsin. The model predicts that activated receptor is initially phosphorylated and then tightly binds an arrestin protein that effectively blocks further G protein interaction. Here we report that complexes of β2AR-arrestin and m2 mAChR-arrestin have a higher affinity for agonists (but not antagonists) than do receptors not complexed with arrestin. The percentage of phosphorylated β2AR in this high affinity state in the presence of full agonists varied with different arrestins and was enhanced by selective mutations in arrestins. The percentage of high affinity sites also was proportional to the intrinsic activity of an agonist, and the coefficient of proportionality varies for different arrestin proteins. Certain mutant arrestins can form these high affinity complexes with unphosphorylated receptors. Mutations that enhance formation of the agonist-receptor-arrestin complexes should provide useful tools for manipulating both the efficiency of signaling and rate and specificity of receptor internalization. Agonist binding activates G protein 1The abbreviations used are: G protein, guanyl nucleotide binding protein; β2AR, unphosphorylated β2-adrenergic receptor; P-β2AR, phosphorylated β2-adrenergic receptor; m2 mAChR, unphosphorylated m2 muscarinic cholinergic receptor; P-m2 mAChR, phosphorylated m2 muscarinic cholinergic receptor; BSA, bovine serum albumin; ISO, isoproterenol.-coupled receptors and initiates two intimately intertwined cascades of events, resulting in signal transduction and signal termination (desensitization). The receptor-agonist complex initially interacts with G protein(s) to form a transient agonist-receptor-G protein ternary complex that is the first intermediate in transmembrane signaling (1Lefkowitz R.J. Caron M.G. Michel T. Stadel J.M. Fed. Proc. 1982; 41: 2664-2670PubMed Google Scholar, 2Limbird L.E. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 228-232Crossref PubMed Scopus (86) Google Scholar). This ternary complex has a higher affinity for agonists than receptor alone (1Lefkowitz R.J. Caron M.G. Michel T. Stadel J.M. Fed. Proc. 1982; 41: 2664-2670PubMed Google Scholar, 2Limbird L.E. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 228-232Crossref PubMed Scopus (86) Google Scholar). Formation of this complex promotes GDP release from the G protein, which is followed by rapid GTP binding and dissociation of the active Gα·GTP and Gβγ subunits. The agonist-occupied receptors are then phosphorylated by G protein-coupled receptor kinases, resulting in arrestin binding and consequent disruption of receptor-G protein interaction (3Sterne-Marr R. Benovic J.L. Vitam. Horm. 1995; 51: 193-234Crossref PubMed Scopus (113) Google Scholar). Recent studies suggest that arrestin binding also targets the receptors for internalization (4Ferguson S.S.G. Downey III, W.E. Colapietro A.-M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (846) Google Scholar, 5Ferguson S.S.G. Menard 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), apparently by virtue of the ability of non-visual arrestins to interact with clathrin (6Sohlemann P. Hekman M. Puzicha M. Buchen C. Lohse M.J. Eur. J. Biochem. 1995; 232: 464-472Crossref PubMed Scopus (41) Google Scholar), a process that appears to be a prerequisite for resensitization (3Sterne-Marr R. Benovic J.L. Vitam. Horm. 1995; 51: 193-234Crossref PubMed Scopus (113) Google Scholar). Thus, the formation of the arrestin-receptor complex is not only the final step of signal termination but also an initial step of subsequent resensitization, representing a critical juncture in the signaling process. Because of this the arrestin-receptor complex appears to be a tempting target for a more detailed characterization. Bovine arrestin cDNAs were subcloned using theNcoI and HindIII sites of pTrcB (Invitrogen). BL-21 cells transformed with the pTrcB-arrestin constructs were grown at 30 °C in LB containing 0.1 mg/ml ampicillin to anA 600 of 0.2–0.4, induced with 30 μm isopropyl-β-d-thiogalactopyranoside, and grown for an additional 4–6 h. Cells were harvested by centrifugation and lysed, and arrestins were purified by sequential heparin-Sepharose (6Sohlemann P. Hekman M. Puzicha M. Buchen C. Lohse M.J. Eur. J. Biochem. 1995; 232: 464-472Crossref PubMed Scopus (41) Google Scholar) and Q-Sepharose chromatography as described (6Sohlemann P. Hekman M. Puzicha M. Buchen C. Lohse M.J. Eur. J. Biochem. 1995; 232: 464-472Crossref PubMed Scopus (41) Google Scholar, 7Goodman 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 (1172) Google Scholar, 8Gray-Keller M.P. Detwiler P.B. Benovic J.L. Gurevich V.V. Biochemistry. 1997; 36: 7058-7063Crossref PubMed Scopus (80) Google Scholar), adjusting salt gradients for elution of different arrestin proteins. Hamster β2-adrenergic receptor (β2AR) and human m2 muscarinic cholinergic receptor (m2 mAChR) were expressed in Sf9 cells and purified by affinity chromatography as described (9Gurevich 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 (339) Google Scholar). Purified β2AR (90–120 pmol) was mixed with 0.6–0.8 mg of sonicated soybean phosphatidylcholine in 0.4 ml of 10 mm Tris-HCl, pH 7.4, 100 mm NaCl (buffer A) containing 3 mg/ml BSA and incubated on ice for 7 min. Samples were loaded onto Extracti-Gel columns (Pierce) at 4 °C, previously equilibrated with 3 ml of buffer A containing 2 mg/ml BSA and 2 ml of buffer A containing 20 mm MgCl2, and then eluted with 1.7 ml of the latter buffer. 50% polyethylene glycol 8000 (0.6 ml) was added to the eluant, mixed, and incubated for 7 min at 22 °C. Samples were diluted with 30 ml of ice-cold buffer A, and liposomes with reconstituted receptor were pelleted by centrifugation at 35,000 × g for 90 min. Pellets were resuspended in 0.4 ml of 20 mm Tris-HCl, pH 7.4, 2 mm EDTA. The m2 mAChR was reconstituted and phosphorylated, as described (10Richardson R.M. Kim C. Benovic J.L. Hosey M.M. J. Biol. Chem. 1993; 268: 13650-13656Abstract Full Text PDF PubMed Google Scholar). Receptors were phosphorylated in the presence of respective agonists by purified β-adrenergic receptor kinase to a stoichiometry of 2.7 ± 0.2 mol/mol (P-β2AR) or 8 ± 0.2 mol/mol (P-m2 mAChR), as described (7Goodman 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 (1172) Google Scholar, 9Gurevich 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 (339) Google Scholar, 10Richardson R.M. Kim C. Benovic J.L. Hosey M.M. J. Biol. Chem. 1993; 268: 13650-13656Abstract Full Text PDF PubMed Google Scholar). To remove the agonist, P-β2AR was washed with 20 mm Tris-HCl, pH 7.4, 2 mmEDTA three times by centrifugation as above, whereas P-m2 mAChR was gel-filtered on a 2-ml Sephadex G-50 column. Control receptors were similarly prepared but in the absence of kinase. P-β2AR or β2AR (10–15 fmol/assay) were incubated in 0.25 ml of buffer A containing 0.1 mg/ml BSA in the presence of 65–75 fmol of 125Iiodopindolol (NEN Life Science Products) and the indicated concentrations of arrestins and agonists for 60 min at 22 °C. Samples were then cooled on ice and loaded at 4 °C onto 2-ml Sephadex G-50 columns. Receptor-containing liposomes with bound radioligand were eluted with buffer A (between 0.6 and 1.5 ml), and radioactivity was quantitated in a liquid scintillation counter. P-m2 mAChR and m2 mAChR (10Richardson R.M. Kim C. Benovic J.L. Hosey M.M. J. Biol. Chem. 1993; 268: 13650-13656Abstract Full Text PDF PubMed Google Scholar) (50 fmol/assay) were incubated in 0.5 ml of the same buffer A/BSA in the presence of 250 fmol of 3Hquinuclidinyl benzilate (Amersham Corp.) with the indicated concentrations of arrestins and agonists for 60 min at 22 °C. 150 μl of ice-cold 30% (w/v) polyethylene glycol were subsequently added to each assay tube, and the samples were incubated on ice for 10 min and then filtered through GF/F filters (presoaked for 1 h in 1% polyethyleneimine to reduce nonspecific binding). The radioactivity retained on filters was then quantitated in a liquid scintillation counter. Nonspecific binding was determined in the presence of 10 μm alprenolol (β2AR) or atropine (m2 mAChR). All binding experiments were repeated 2–4 times, and data are presented as means ± S.D. Our initial studies examined ligand binding characteristics of phosphorylated β2AR in the absence or presence of arrestins (Fig. 1). Strikingly, both β-arrestin and arrestin3, but not visual arrestin, induced a pronounced leftward shift of the isoproterenol (ISO) competition curve (Fig. 1 A). We next tested the effects of varying the arrestin concentration on ligand binding to P-β2AR in the presence of either 30 or 100 nm isoproterenol. This yielded EC50 values of 19.4 ± 7.4 and 5.0 ± 1.8 nm for β-arrestin and arrestin3, respectively, values well within the physiologically relevant range (6Sohlemann P. Hekman M. Puzicha M. Buchen C. Lohse M.J. Eur. J. Biochem. 1995; 232: 464-472Crossref PubMed Scopus (41) Google Scholar, 11Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar). Similar curve shifts were observed for the β-agonists epinephrine and norepinephrine (Fig. 1, B and C), whereas affinities for the antagonists alprenolol (IC50,∼3.47 ± 0.14 nm) and propranolol (IC50,∼1.19 ± 0.06 nm) were unchanged by arrestins. As shown in Fig. 1, the agonist competition curves in the presence of β-arrestin or arrestin3 are shallower than control curves performed in the absence of arrestins. Analysis of these curves reveals the presence of two distinct sites that differ in their agonist affinity (Fig. 1). β-Arrestin and arrestin3 induce very similar high and low affinity sites for isoproterenol with IC50 values of 26.7 ± 9.1 and 871 ± 71 nm, respectively, the latter value being very similar to the affinity for the receptor in the absence of arrestin (IC50 of 656 ± 127 nm). β-Arrestin and arrestin3 also promote similar high and low affinity binding sites for epinephrine and norepinephrine with IC50 values of 90.3 ± 21.8 nm and 3.84 ± 0.69 μm for epinephrine and 1.20 ± 0.40 and 43.1 ± 13.4 μm for norepinephrine, respectively (Fig. 1). However, the percentage of high affinity sites is clearly dependent upon the particular arrestin present: 31.5 ± 1.4% with β-arrestin and 56.9 ± 1.0% with arrestin3. Thus, it appears that arrestin3 more efficiently shifts the equilibrium of the receptor between the two distinct functional states than does β-arrestin. Although neither arrestin drives 100% of the receptors into a high affinity state, these results are in agreement with direct binding studies where arrestin3 was found to bind better than β-arrestin to the P-β2AR (9Gurevich 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 (339) Google Scholar). Arrestins bind preferentially to phosphorylated receptors (9Gurevich 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 (339) Google Scholar, 11Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar). To further explore this relationship, we tested the phosphorylation dependence of the high affinity agonist binding to ascertain whether the increased affinity directly reflects arrestin-receptor interaction. β-Arrestin did not increase the affinity of unphosphorylated β2AR for ISO, whereas arrestin3 evoked a marginal shift of the curve (Fig. 1 D), apparently reflecting the stronger propensity of arrestin3 to interact with unphosphorylated receptor (9Gurevich 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 (339) Google Scholar). A more systematic study using β2AR phosphorylated by the β-adrenergic receptor kinase to various stoichiometries (0.6–3.4 mol/mol) revealed no effect of β-arrestin on the ISO competition curve at stoichiometries of 0.6 or 1.4 mol/mol, a marginal effect at 1.9 mol/mol, and significant and effects at data are with that two are and for high affinity arrestin interaction (9Gurevich 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 (339) Google Scholar) and provide additional that it is the arrestin-receptor complex that higher affinity for these data suggest that the receptor complex with high agonist affinity is the same arrestin-receptor complex previously characterized by direct binding studies (9Gurevich 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 (339) Google Scholar). of the agonist-receptor-arrestin ternary complex reveals that it is to concentrations of to μm ± 1 μm or GTP mm ± 1 mm or 100 nm G protein subunits. Although both β-arrestin and arrestin3 bind with high affinity to clathrin (6Sohlemann P. Hekman M. Puzicha M. Buchen C. Lohse M.J. Eur. J. Biochem. 1995; 232: 464-472Crossref PubMed Scopus (41) Google Scholar) clathrin formation Jr., O.B. Krupnick J.G. Gurevich V.V. Benovic J.L. Keen J.H. J. Biol. Chem. 1997; Full Text Full Text PDF Scopus Google Scholar), the of nm purified clathrin no effect on the ISO competition curve shift induced by either 10 or the non-visual arrestins Thus, clathrin interaction with arrestins does not with arrestin binding to P-β2AR, that different of the arrestin are in these two arrestin mutations were previously shown to the propensity of arrestins to form a high affinity complex with receptors (9Gurevich 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 (339) Google Scholar, 11Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar) and to the of the arrestins M.P. Detwiler P.B. Benovic J.L. Gurevich V.V. Biochemistry. 1997; 36: 7058-7063Crossref PubMed Scopus (80) Google Scholar, V.V. Benovic J.L. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, V.V. Benovic J.L. 1997; 51: PubMed Scopus Google Scholar). the of β-arrestin with the of visual arrestin the was found to increase binding to P-β2AR (9Gurevich 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 (339) Google Scholar). this was purified and tested on the ISO competition curves for P-β2AR and β2AR, it induced a more significant in the curve than did ± of P-β2AR into a high affinity state with no effect on β2AR binding (Fig. 2 We previously that a within the of visual arrestin a arrestin an arrestin of high affinity binding to both phosphorylated and unphosphorylated M.P. Detwiler P.B. Benovic J.L. Gurevich V.V. Biochemistry. 1997; 36: 7058-7063Crossref PubMed Scopus (80) Google Scholar, V.V. Benovic J.L. 1997; 51: PubMed Scopus Google Scholar). a similar form of β-arrestin was and for ability to induce high affinity agonist binding to P-β2AR and this mutant not only promotes high affinity agonist binding to β2AR ± high affinity but also a more significant shift in the ISO competition curve of P-β2AR ± high affinity than does β-arrestin ± (Fig. studies that arrestin also receptor binding (9Gurevich 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 (339) Google Scholar, 11Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar), we tested a of arrestin3. Purified promotes significant high affinity agonist binding to both P-β2AR ± and β2AR ± (Fig. the EC50 values of both ± nm) and ± nm) for high affinity agonist binding are with the values previously determined in direct binding (9Gurevich 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 (339) Google Scholar), whereas the for β-arrestin and arrestin3 are be to the complex of arrestin binding to receptor (9Gurevich 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 (339) Google Scholar, 11Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar), which appears to be for arrestin (9Gurevich 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 (339) Google Scholar). All non-visual arrestins tested a effect on agonist affinity and no effect on that the effect with the intrinsic activity of the To this we used two with high intrinsic and with low intrinsic with these we used three arrestin with and high propensity to induce the high agonist affinity state of P-β2AR, arrestin3, and arrestins induced leftward shifts of the competition curves of both with the effect on being than that on full stronger than that on The competition curves in the presence of arrestins were and of these curves revealed the presence of high and low affinity The values observed for the high affinity sites for and were ± and ± nm, respectively, in the presence of three arrestins The affinities of low affinity sites were ± and ± 3 μm for and respectively, to the affinities observed in the absence of arrestins ± 0.2 and ± The percentage of high affinity sites was clearly dependent on the of both the arrestin and the the presence of arrestin3, and the percentage of high affinity sites for was 20 ± ± and ± respectively, whereas in the of the percentage was ± 1, ± and ± As shown in A, the percentage of high affinity sites in the presence of a arrestin appears to be proportional to the intrinsic activity of the the coefficient varies with different arrestin from for β-arrestin through for arrestin3 to for results suggest that various arrestins each a propensity to form an arrestin-receptor complex with high agonist We to this propensity of an arrestin the percentage of high affinity sites in the presence of a full agonist appears to a of of a arrestin 1, To whether the high agonist affinity of the arrestin-receptor complexes is observed with G protein-coupled we performed similar experiments with reconstituted m2 β-arrestin and arrestin3 promote a increase in agonist affinity for phosphorylated m2 mAChR whereas was no significant shift in affinity for the unphosphorylated m2 mAChR (Fig. 4 in to the β2AR, was no significant in the of the that most of the phosphorylated m2 mAChR a high agonist affinity complex with either arrestin. This be to the high stoichiometry of phosphorylation of the P-m2 mAChR mol/mol) or to between the two receptors. the same effect as β-arrestin on the P-m2 mAChR, whereas induced shifts on P-m2 mAChR and significant interaction with mAChR (Fig. and the complex similarly to the previously receptor-G protein complex (1Lefkowitz R.J. Caron M.G. Michel T. Stadel J.M. Fed. Proc. 1982; 41: 2664-2670PubMed Google Scholar, 2Limbird L.E. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 228-232Crossref PubMed Scopus (86) Google Scholar, P. S. T. Lefkowitz R.J. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). complexes promote high affinity agonist binding to receptors. The percentage of receptor in the high affinity state appears to be proportional to the intrinsic activity of the ligand in both The increase in agonist affinity to the formation of either of these complexes is A between complex and receptor-G protein complex in the of the agonist-receptor-G protein ternary complex to which promotes G protein whereas the agonist-receptor-arrestin complex is and to and the agonist affinity of two and the β2AR and m2 mAChR, upon arrestin it is tempting to that this is a G protein-coupled receptors. The ability of visual arrestin to the active of A. Biochemistry. Scopus Google Scholar) this The formation of the complex promotes internalization (7Goodman 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 (1172) Google Scholar), that the bound agonist also be the biological of this is β-arrestin and arrestin3 to be similar in a of in and in cells (4Ferguson S.S.G. Downey III, W.E. Colapietro A.-M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (846) Google Scholar, 5Ferguson S.S.G. Menard 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 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 (1172) Google Scholar, 9Gurevich 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 (339) Google Scholar, J.L. C. J. M. Caron M.G. Lefkowitz R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, R. Gurevich V.V. P. C. Benovic J.L. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar), we observed a significant in their the ability to form a high affinity agonist-receptor-arrestin complex with arrestin3 also has a higher affinity for binding to clathrin (7Goodman 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 (1172) Google Scholar). Thus, whereas β-arrestin and arrestin3 similarly the β2AR differ in their ability to promote receptor internalization and subsequent The characteristics of the various arrestin ability to form complexes with phosphorylated and unphosphorylated receptors more the specificity of P-β2AR and β2AR, and more of these useful tools for of the and of receptor signaling and internalization in The ability of a arrestin to form arrestin-receptor complexes with high agonist affinity appears to be intrinsic which we termed arrestin affinity for a the propensity of an arrestin to a into the active state, or We found no between the EC50 values of different arrestins and their with However, mutations that increased to reduce arrestin 2 and that reflects the with which an arrestin a into active The of the of of different arrestin with the P-β2AR and P-m2 mAChR 1, this has no for G the high affinity state of receptor has not in the presence of more than of G the most to arrestin in receptor-G protein appears to be Our data suggest that mutations can increase arrestin and we that additional with higher can be with ability to promote internalization of a receptor in but this to be We L. A. for the arrestin and J. Keen for purified S. for purified G protein J. for in protein by and J. Ptasienski for the of m2 mAChR, and R. Penn for critical of the