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• W2038863418 abstract "Arrestins play a key role in the homologous desensitization of G protein-coupled receptors (GPCRs). These cytosolic proteins selectively bind to the agonist-activated and GPCR kinase-phosphorylated forms of the GPCR, precluding its further interaction with the G protein. Certain mutations in visual arrestin yield “constitutively active” proteins that bind with high affinity to the light-activated form of rhodopsin without requiring phosphorylation. The crystal structure of visual arrestin shows that these activating mutations perturb two groups of intramolecular interactions that keep arrestin in its basal (inactive) state. Here we introduced homologous mutations into arrestin2 and arrestin3 and found that the resulting mutants bind to the β2-adrenoreceptor in vitro in a phosphorylation-independent fashion. The same mutants effectively desensitize both the β2-adrenergic and δ-opioid receptors in the absence of receptor phosphorylation inXenopus oocytes. Moreover, the arrestin mutants also desensitize the truncated δ-opioid receptor from which the C terminus, containing critical phosphorylation sites, has been removed. Conservation of the phosphate-sensitive hot spots in non-visual arrestins suggests that the overall fold is similar to that of visual arrestin and that the mechanisms whereby receptor-attached phosphates drive arrestin transition into the active binding competent state are conserved throughout the arrestin family of proteins. Arrestins play a key role in the homologous desensitization of G protein-coupled receptors (GPCRs). These cytosolic proteins selectively bind to the agonist-activated and GPCR kinase-phosphorylated forms of the GPCR, precluding its further interaction with the G protein. Certain mutations in visual arrestin yield “constitutively active” proteins that bind with high affinity to the light-activated form of rhodopsin without requiring phosphorylation. The crystal structure of visual arrestin shows that these activating mutations perturb two groups of intramolecular interactions that keep arrestin in its basal (inactive) state. Here we introduced homologous mutations into arrestin2 and arrestin3 and found that the resulting mutants bind to the β2-adrenoreceptor in vitro in a phosphorylation-independent fashion. The same mutants effectively desensitize both the β2-adrenergic and δ-opioid receptors in the absence of receptor phosphorylation inXenopus oocytes. Moreover, the arrestin mutants also desensitize the truncated δ-opioid receptor from which the C terminus, containing critical phosphorylation sites, has been removed. Conservation of the phosphate-sensitive hot spots in non-visual arrestins suggests that the overall fold is similar to that of visual arrestin and that the mechanisms whereby receptor-attached phosphates drive arrestin transition into the active binding competent state are conserved throughout the arrestin family of proteins. G protein-coupled receptor GPCR kinase β2-adrenergic receptor phosphorylated β2AR phosphorylated isoproterenol-activated β2AR isoproterenol-activated β2AR antagonist-occupied β2AR antagonist-occupied phosphorylated β2AR triple alanine substitution (F375A,V376A,F377A in the C-tail of arrestin and I286A,V387A,F388A in C-tails of arrestin2 and 3) wild type unphosphorylated dark rhodopsin dark phosphorhodopsin light-activated, unphosphorylated rhodopsin light-activated phosphorhodopsin δ-opioid receptor. Signaling by various members of the superfamily of G protein-coupled receptors (GPCRs)1 is attenuated by a uniform two-step mechanism (1Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar). First, the agonist-activated receptor that catalyzes GDP/GTP exchange on G proteins is specifically phosphorylated by a G protein-coupled receptor kinase (GRK). An arrestin protein then binds to the active phosphoreceptor, which makes further G protein interaction impossible by simple steric exclusion (2Krupnick J.G. Gurevich V.V. Benovic J.L. J. Biol. Chem. 1997; 272: 18125-18131Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Because of the high affinity of non-visual arrestins 2Note that here we use the systematic names of non-visual arrestins. The synonyms of arrestin2 are β-arrestin and β-arrestin1; arrestin3 is also called β-arrestin2. 2Note that here we use the systematic names of non-visual arrestins. The synonyms of arrestin2 are β-arrestin and β-arrestin1; arrestin3 is also called β-arrestin2. for various components of the trafficking machinery (3Laporte S.A. Oakley R.H. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 2000; 275: 23120-23126Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 4Goodman 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 (1153) Google Scholar, 5McDonald P.H. Cote N.L. Lin F.-T. Premont R.T. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1999; 274: 10677-10680Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), the arrestin-receptor complex is then internalized. Arrestin binding also serves to switch GPCR signaling from G proteins to various mitogen-activated protein kinases (6Luttrell 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 (1250) Google Scholar, 7McDonald P.H. Chow C.W. Miller W.E. Laporte S.A. Field M.E. Lin F.T. Davis R.J. Lefkowitz R.J. Science. 2000; 290: 1574-1577Crossref PubMed Google Scholar, 8Lutrell L.M. Roudabush F.L. Choy E.W. Miller W.E. Field M.E. Pierce K.L. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2449-2454Crossref PubMed Scopus (696) Google Scholar). The loss of active receptor conformation in the endosomes (presumably due to ligand dissociation) facilitates arrestin dissociation, rendering the phosphoreceptor accessible to protein phosphatases. The dephosphorylated receptor can then be recycled back to the plasma membrane (9Krueger K.M. Daaka Y. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 5-8Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar).Perfectly timed binding and dissociation of arrestins, which are ensured by their remarkable selectivity for the phosphorylated/activated form of their cognate receptors, are equally important for high fidelity of this quenching mechanism. Destabilization of certain intramolecular interactions that support the basal (inactive) arrestin conformation by the activated phosphoreceptor is the mechanistic basis of arrestin selectivity (10Gurevich V.V. Benovic J.L. J. Biol. Chem. 1995; 270: 6010-6016Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 15Vishnivetsky S.A. Paz C.L. Schubert C. Hirsch J.A. Sigler P.B. Gurevich V.V. J. Biol. Chem. 1999; 274: 11451-11454Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 19Hirsch J.A. Schubert C. Gurevich V.V. Sigler P.B. Cell. 1999; 97: 257-269Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 20Gurevich V.V. Methods Enzymol. 1996; 275: 382-397Crossref PubMed Scopus (58) Google Scholar). The visual arrestin residues involved in the stabilization of its basal state have been identified by extensive mutagenesis (10Gurevich V.V. Benovic J.L. J. Biol. Chem. 1995; 270: 6010-6016Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 11Gurevich V.V. Benovic J.L. Mol. Pharmacol. 1997; 51: 161-169Crossref PubMed Scopus (120) Google Scholar, 12Gray-Keller M.P. Detwiler P.B. Benovic J.L. Gurevich V.V. Biochemistry. 1997; 36: 7058-7063Crossref PubMed Scopus (79) Google Scholar, 13Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, 14Gurevich V.V. J. Biol. Chem. 1998; 273: 15501-15506Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 15Vishnivetsky S.A. Paz C.L. Schubert C. Hirsch J.A. Sigler P.B. Gurevich V.V. J. Biol. Chem. 1999; 274: 11451-11454Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 24Schleicher A. Kuhn H. Hofmann K.P. Biochemistry. 1989; 28: 1770-1775Crossref PubMed Scopus (165) Google Scholar, 26Kovoor A. Nappey V. Kieffer B.L. Chavkin C. J. Biol. Chem. 1997; 272: 27605-27611Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) and crystallography (19Hirsch J.A. Schubert C. Gurevich V.V. Sigler P.B. Cell. 1999; 97: 257-269Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). Here we describe homologous mutations in both non-visual arrestins that also yield proteins with constitutive activity, suggesting that the basal conformation of all arrestins and the activating mechanisms triggering arrestin transition into its high affinity receptor binding state are conserved throughout this family of proteins. The results presented in this study help to define the conserved mechanisms of arrestin activation.RESULTSTwo main groups of intramolecular interactions are largely responsible for the stability of the basal conformation of visual arrestin (14Gurevich V.V. J. Biol. Chem. 1998; 273: 15501-15506Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 15Vishnivetsky S.A. Paz C.L. Schubert C. Hirsch J.A. Sigler P.B. Gurevich V.V. J. Biol. Chem. 1999; 274: 11451-11454Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 19Hirsch J.A. Schubert C. Gurevich V.V. Sigler P.B. Cell. 1999; 97: 257-269Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 25Vishnivetskiy S.A. Schubert C. Climaco G.C. Gurevich Y.V. Velez M.-G. Gurevich V.V. J. Biol. Chem. 2000; 275: 41049-41057Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). One is an unusual network of five buried, solvent-excluded, charged residues in the fulcrum of the two-domain arrestin molecule, which we termed the polar core (15Vishnivetsky S.A. Paz C.L. Schubert C. Hirsch J.A. Sigler P.B. Gurevich V.V. J. Biol. Chem. 1999; 274: 11451-11454Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 19Hirsch J.A. Schubert C. Gurevich V.V. Sigler P.B. Cell. 1999; 97: 257-269Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). The other, termed the three-element interaction, involves bulky hydrophobic residues in β-strand I, α-helix I, and β-strand XX of the C-tail (14Gurevich V.V. J. Biol. Chem. 1998; 273: 15501-15506Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 19Hirsch J.A. Schubert C. Gurevich V.V. Sigler P.B. Cell. 1999; 97: 257-269Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 25Vishnivetskiy S.A. Schubert C. Climaco G.C. Gurevich Y.V. Velez M.-G. Gurevich V.V. J. Biol. Chem. 2000; 275: 41049-41057Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Mutations in both of these hot spots in the molecule change binding selectivity, yielding visual arrestin that binds activated rhodopsin in a phosphorylation-independent fashion (10Gurevich V.V. Benovic J.L. J. Biol. Chem. 1995; 270: 6010-6016Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 11Gurevich V.V. Benovic J.L. Mol. Pharmacol. 1997; 51: 161-169Crossref PubMed Scopus (120) Google Scholar, 12Gray-Keller M.P. Detwiler P.B. Benovic J.L. Gurevich V.V. Biochemistry. 1997; 36: 7058-7063Crossref PubMed Scopus (79) Google Scholar, 13Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, 14Gurevich V.V. J. Biol. Chem. 1998; 273: 15501-15506Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 15Vishnivetsky S.A. Paz C.L. Schubert C. Hirsch J.A. Sigler P.B. Gurevich V.V. J. Biol. Chem. 1999; 274: 11451-11454Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar,23Gurevich V.V. Chen C.-Y. Kim C.M. Benovic J.L. J. Biol. Chem. 1994; 269: 8721-8727Abstract Full Text PDF PubMed Google Scholar, 25Vishnivetskiy S.A. Schubert C. Climaco G.C. Gurevich Y.V. Velez M.-G. Gurevich V.V. J. Biol. Chem. 2000; 275: 41049-41057Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar).The key salt bridge in the polar core of visual arrestin is between Arg-175 and Asp-296 (15Vishnivetsky S.A. Paz C.L. Schubert C. Hirsch J.A. Sigler P.B. Gurevich V.V. J. Biol. Chem. 1999; 274: 11451-11454Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 19Hirsch J.A. Schubert C. Gurevich V.V. Sigler P.B. Cell. 1999; 97: 257-269Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). The homologous residues in arrestin2 are Arg-169 and Asp-290 (19Hirsch J.A. Schubert C. Gurevich V.V. Sigler P.B. Cell. 1999; 97: 257-269Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). Charge reversal mutations of Arg-175 in visual arrestin (10Gurevich V.V. Benovic J.L. J. Biol. Chem. 1995; 270: 6010-6016Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 11Gurevich V.V. Benovic J.L. Mol. Pharmacol. 1997; 51: 161-169Crossref PubMed Scopus (120) Google Scholar, 12Gray-Keller M.P. Detwiler P.B. Benovic J.L. Gurevich V.V. Biochemistry. 1997; 36: 7058-7063Crossref PubMed Scopus (79) Google Scholar, 15Vishnivetsky S.A. Paz C.L. Schubert C. Hirsch J.A. Sigler P.B. Gurevich V.V. J. Biol. Chem. 1999; 274: 11451-11454Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) and Arg-169 in arrestin2 (18Kovoor A. Celver J. Abdryashitov R.I. Chavkin C. Gurevich V.V. J. Biol. Chem. 1999; 274: 6831-6834Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) yield phosphorylation-independent arrestins that bind with high affinity to the activated unphosphorylated forms of their cognate receptors. We hypothesized that these residues participate in the binding of arrestin to receptor-attached phosphates and function as phosphate sensors; phosphate binding neutralizes the charge on Arg, thereby breaking its interaction with its negatively charged partner (10Gurevich V.V. Benovic J.L. J. Biol. Chem. 1995; 270: 6010-6016Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 18Kovoor A. Celver J. Abdryashitov R.I. Chavkin C. Gurevich V.V. J. Biol. Chem. 1999; 274: 6831-6834Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) and destabilizing the polar core. Thus, if the charge is already neutralized or reversed by a mutation, arrestin would not require that the receptor be phosphorylated before it could bind to the agonist-activated receptor. In visual arrestin, alanine substitutions of bulky hydrophobic residues in either participant of the three-element interaction yield mutants with enhanced binding to activated unphosphorylated and phosphorylated inactive rhodopsin (but not to the inactive unphosphorylated form) (25Vishnivetskiy S.A. Schubert C. Climaco G.C. Gurevich Y.V. Velez M.-G. Gurevich V.V. J. Biol. Chem. 2000; 275: 41049-41057Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). The most potent of these is the triple alanine substitution in the C-tail of arrestin (F375A,V376A,F377A, referred to as 3A mutations) (14Gurevich V.V. J. Biol. Chem. 1998; 273: 15501-15506Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 25Vishnivetskiy S.A. Schubert C. Climaco G.C. Gurevich Y.V. Velez M.-G. Gurevich V.V. J. Biol. Chem. 2000; 275: 41049-41057Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Deletions in the C-tail, as well as charge neutralization of another polar core residue localized there, Arg-382, also yield mutants with reduced selectivity (13Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, 14Gurevich V.V. J. Biol. Chem. 1998; 273: 15501-15506Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar).If the overall conformation and the activation mechanism of non-visual arrestins resemble that of visual arrestin, mutations homologous to all known activating mutations in visual arrestin can be expected to yield proteins with similarly reduced selectivity. To test this idea, we introduced three types of mutations that yield robust phosphorylation-independent binding in visual arrestin (11Gurevich V.V. Benovic J.L. Mol. Pharmacol. 1997; 51: 161-169Crossref PubMed Scopus (120) Google Scholar, 14Gurevich V.V. J. Biol. Chem. 1998; 273: 15501-15506Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 15Vishnivetsky S.A. Paz C.L. Schubert C. Hirsch J.A. Sigler P.B. Gurevich V.V. J. Biol. Chem. 1999; 274: 11451-11454Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) into arrestin2 and arrestin3 (Fig. 1). First, we reversed the charge of the putative main phosphate sensor in arrestin3 by the R170E mutation. Second, we introduced triple alanine substitutions (I286A,V387A,F388A) in the C-tail of both arrestin2 and arrestin3. In addition, we introduced mutations in the distal part of the arrestin3 C-tail, neutralized the charge of Arg-393 (homolog of Arg-382 in visual arrestin), and deleted residues 394–409 or 393–409. (The difference between these two deletions is the absence or presence of Arg-393.)The mutants were expressed in tritiated form in a cell-free translation system and tested in a direct binding assay with a purified, reconstituted β2AR (16Gurevich 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). Arrestin2(3A) demonstrated the typical phenotype of a phosphorylation-independent mutant. WT arrestin2 binding to phosphorylated and isoproterenol-activated β2AR (P-β2AR*) was substantially greater than to the unphosphorylated form of the receptor (β2AR*). In contrast, the binding of the arrestin2 (3A) mutant to unphosphorylated β2AR* was high and was not significantly increased by receptor phosphorylation (Fig.2 A). Arrestin3 mutants demonstrate similar binding patterns (Fig. 2 A), supporting the idea that homologous mutations in all arrestins yield similar phenotypes. As predicted, arrestin3-(1–393), which does not have the distant C-tail but has all the key elements of both the polar core and the three-element interaction, shows essentially WT selectivity for P-β2AR*.Figure 2Binding characteristics of mutant arrestins. A, 100 fmol of unphosphorylated (β2AR*) or GRK2-phosphorylated (P-β2AR*) purified β2-adrenergic receptor reconstituted into liposomes was incubated in 50 μl with 50 fmol of the indicated tritiated arrestin (specific activities, 70–110 dpm/fmol) in the presence of 100 μm agonist isoproterenol as described under “Experimental Procedures.” B, 0.3 μg of unphosphorylated (Rh*) or rhodopsin kinase-phosphorylated (P-Rh*) rhodopsin was incubated with the same amount of arrestins under room light. Membrane-bound arrestins were separated by gel filtration on Sepharose 2B and quantified in a liquid scintillation counter. *,p < 0.01; Student's t test, as compared with the binding of a corresponding WT protein to the same functional form of the same receptor. Mean ± S.E. from three experiments, each performed in duplicate, are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Interestingly, the most potent phosphorylation-independent form of visual arrestin (Fig. 2 B), arrestin(3A), that binds with very high affinity to unphosphorylated, light-activated rhodopsin (Rh*) (Fig. 2 B), does not demonstrate enhanced binding to β2AR*, although its binding to P-β2AR* is about 50% higher than that of WT visual arrestin (Fig. 2 A). Similarly, constitutively active forms of both non-visual arrestins demonstrate higher binding to light-activated phosphorhodopsin (P-Rh*) than their parental WT proteins, but none of these mutants shows an enhanced binding to Rh* (Fig. 2 B). Thus, mutational destabilization of either of the two main intramolecular interactions that hold arrestin proteins in the basal state facilitates the binding of these proteins to the phosphorylated active form of a non-preferred receptor but is not sufficient to promote binding to its unphosphorylated active form. The constitutively active forms of arrestin2 and arrestin3 that bind equally well to the phosphorylated and unphosphorylated forms of β2AR* still strongly prefer the phosphorylated form of rhodopsin.To further explore the mechanism underlying constitutive activity of the mutants, we also tested the binding of the most potent phosphorylation-independent forms (3A mutants) to the inactive forms of both receptors. In the case of β2AR, the latter were represented by both empty (P-β2AR and β2AR) and antagonist-occupied (in the presence of 10 μm alprenolol, P-β2ARA and β2ARA) forms (Fig.3 A), whereas in the case of rhodopsin a dark inactive receptor was used (P-Rh and Rh) (Fig.3 B). As we reported earlier (16Gurevich 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 binding of visual arrestin to any form of β2AR is low and appears to depend only on receptor phosphorylation (i.e. the binding to phosphorylated forms is higher than to unphosphorylated forms and it does not depend on receptor activation state). 3A mutation enhances the binding without changing this pattern. Both WT non-visual arrestins demonstrate relatively high binding to inactive forms of both phosphoreceptors. 3A mutations enhance their binding to all phosphorylated forms, further reducing their ability to discriminate between active and inactive phosphoreceptor (Fig. 3 A). However, the binding of these mutations to an unphosphorylated receptor is strictly activation-dependent; both 3A mutants bind to β2AR* with high affinity and do not demonstrate appreciable binding to β2AR or β2ARA (Fig. 3 A). In contrast to the strong preference for activated β2AR*, the binding of constitutively active non-visual arrestins to rhodopsin was not significantly enhanced by receptor activation (compare Rh and Rh* binding on Fig.3 B).Figure 3Comparison of activation and phosphorylation dependence of the binding of WT arrestins and 3A mutants. The binding to indicated functional forms of β2AR (A) and rhodopsin (B) was performed as described in the legend to Fig. 2. Note that the subscript A indicates an antagonist-occupied receptor (in the presence of 10 μm alprenolol), whereas * indicates an active receptor (agonist-occupied β2AR and light-activated rhodopsin). Means ± S.E. from two experiments, each performed in duplicate, are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Next we compared the ability of these arrestins to promote desensitization of β2AR in Xenopus oocytes with and without simultaneous expression of GRK3. The major advantage of this system is that in sharp contrast to cultured cells oocytes do not express detectable amounts of arrestins or GRKs. As a result, the direct effects of mutant arrestins and GRKs on the rate of receptor desensitization in a living cell can be tested without any interference from endogenous proteins. Another major advantage is an easy readout; activation of a Gαi/o or Gαs protein by any GPCR can be monitored in real time via a potassium current through a βγ-gated inwardly rectifying channel (18Kovoor A. Celver J. Abdryashitov R.I. Chavkin C. Gurevich V.V. J. Biol. Chem. 1999; 274: 6831-6834Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 26Kovoor A. Nappey V. Kieffer B.L. Chavkin C. J. Biol. Chem. 1997; 272: 27605-27611Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). As shown in Fig.4, the expression of WT arrestins does not accelerate the rate of β2AR desensitization, whereas the coexpression of GRK3 with either arrestin accelerates it 3–4-fold. In contrast, arrestin2(3A) and several arrestin3 mutants (3A, 1–392, R393Q, and R170E) dramatically accelerate β2AR inactivation even in the absence of GRK3. All phosphorylation-independent mutants fully retain the ability to inactivate GRK3-phosphorylated receptor. This is true even for arrestin3 (R170E) in which the charge of phosphate-binding Arg-170 is reversed, likely precluding direct interaction of this residue with the phosphate. Thus, high affinity arrestin binding to β2AR is possible when its interactions with phosphates is either precluded by their absence on the receptor or impeded by the absence of one of the key phosphate binding arrestin residues. These results suggest that the binding of non-visual arrestins to receptor-attached phosphates per se does not significantly contribute to the overall interaction. This conclusion agrees with the model of sequential multisite binding earlier proposed for visual arrestin-rhodopsin interaction (13Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar). In fact, this is the mechanistic reason why it is possible to construct functionally effective phosphorylation-independent arrestins.Figure 4GRK-independent functional desensitization of β2AR by arrestin2 and 3 mutants. Xenopus oocytes were injected with a mixture of cRNAs for the β2AR (0.05 ng), the G protein-gated inwardly rectifying K+ channel subunits Kir3.1 and Kir3.4 (0.02 ng each), and Gs (0.5 ng) that allows the Gs-coupled β2AR to activate the co-expressed channels as described (26Kovoor A. Nappey V. Kieffer B.L. Chavkin C. J. Biol. Chem. 1997; 272: 27605-27611Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). As indicated, some oocytes were also co-injected with 8 ng of cRNA for the different forms of arrestin either alone or together with 0.5 ng of GRK3 cRNA. All recordings were performed 3–4 days postinjection. Receptor-activated currents were measured in 16 mmK+ buffer (26Kovoor A. Nappey V. Kieffer B.L. Chavkin C. J. Biol. Chem. 1997; 272: 27605-27611Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) at −80 mV holding potential. The agonist-elicited responses were adjusted by baseline subtraction as described (26Kovoor A. Nappey V. Kieffer B.L. Chavkin C. J. Biol. Chem. 1997; 272: 27605-27611Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) and normalized to the peak response. Average peak β2AR responses with different arrestin mutants were not significantly different compared with β2AR responses with the parental WT arrestins. A, representative traces depicting β2AR-activated current responses elicited by 1 μmagonist isoproterenol and reversed by 1 μm of antagonist propranolol. Antagonist perfusion was used to determine the amount of residual receptor response. The short vertical lines through the traces indicate when agonist treatment was discontinued and the antagonist perfusion started. Calibration scales are the same for each trace (2 min). B, the β2AR desensitization rate in each group of oocytes is expressed as a multiple of the desensitization rate in the oocyte group injected with cRNA for the corresponding WT arrestin only. *, p< 0.05; Student's t test, compared with the desensitization rate of the corresponding group expressing the parental WT arrestin. Each bar represents the mean ± S.E. of 8–23 separate oocytes from multiple donors.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Two arrestin3 truncation mutants (1–379 and 1–393) demonstrate essentially WT behavior and inactivate β2AR only in the presence of GRK3 (Fig. 4). The lack of phosphorylation-independent activity of arrestin3-(1–393) agrees with the direct binding results (Fig. 2). However, the behavior of one mutant in this system differs from its in vitro binding pattern; the shortest truncated arrestin3-(1–379) binds to unphosphorylated β2AR* in vitro, but it does not demonstrate phosphorylation-independent activity in oocytes (compare Figs. 2 and 4). In the presence of GRK3, all mutant forms of both non-visual arrestins demonstrate an ability to desensitize β2AR similar to that of corresponding WT proteins (Fig.3), which indicates that expressed arrestin proteins were fully functional in oocytes. There are obvious differences between the two assays, e.g. temperature (25 °C in oocytes, 30 °C during the binding, and 4 °C during the chromatography in direct binding assay) and the presence of competing molecules (G proteins in oocytes and none in the binding assay). Conceivably, the complex of arrestin3-(1–379) with receptor is stable enough to form in the absence of competition and survive a few minutes at 4 °C on minicolumns (see “Experimental Procedures”) but not stable enough to compete out G protein at 25 °C.To ascertain whether phosphorylation-independent forms of non-visual arrestins retain receptor specificity of the parental proteins (1Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar,2Krupnick J.G. Gurevich V.V. Benovic J.L. J. Biol. Chem. 1997; 272: 18125-18131Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 3Laporte S.A. Oakley R.H. Holt J.A. Barak L.S. Caron M.G. J. Biol. Chem. 2000; 275: 23120-23126Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 4Goodman 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 (1153) Go" @default.
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