Francesca Santini

We recently demonstrated that nonvisual arrestins interact via a C-terminal binding domain with clathrin and function as adaptor proteins to promote β2-adrenergic receptor (β2AR) internalization. Here, we investigated the potential utility of a mini-gene expressing the clathrin-binding domain of β-arrestin (β-arrestin (319–418)) to function as a dominant-negative with respect to β2AR internalization and compared its properties with those of β-arrestin and β-arrestin-V53D, a previously reported dominant-negative mutant.In vitro studies demonstrated that β-arrestin-V53D bound better to clathrin than β-arrestin but was significantly impaired in its interaction with phosphorylated G protein-coupled receptors. In contrast, whereas β-arrestin (319–418) also bound well to clathrin it completely lacked receptor binding activity. When coexpressed with the β2AR in HEK293 cells, β-arrestin (319–418) effectively inhibited agonist-promoted receptor internalization, whereas β-arrestin-V53D was only modestly effective. However, both constructs significantly inhibited the stimulation of β2AR internalization by β-arrestin in COS-1 cells. Interestingly, immunofluorescence microscopy analysis reveals that both β-arrestin (319–418) and β-arrestin-V53D are constitutively localized in clathrin-coated pits in COS-1 cells. These results indicate the potential usefulness of β-arrestin (319–418) to effectively block arrestin-clathrin interaction in cells and suggest that this construct may prove useful in further defining the mechanisms involved in G protein-coupled receptor trafficking. We recently demonstrated that nonvisual arrestins interact via a C-terminal binding domain with clathrin and function as adaptor proteins to promote β2-adrenergic receptor (β2AR) internalization. Here, we investigated the potential utility of a mini-gene expressing the clathrin-binding domain of β-arrestin (β-arrestin (319–418)) to function as a dominant-negative with respect to β2AR internalization and compared its properties with those of β-arrestin and β-arrestin-V53D, a previously reported dominant-negative mutant.In vitro studies demonstrated that β-arrestin-V53D bound better to clathrin than β-arrestin but was significantly impaired in its interaction with phosphorylated G protein-coupled receptors. In contrast, whereas β-arrestin (319–418) also bound well to clathrin it completely lacked receptor binding activity. When coexpressed with the β2AR in HEK293 cells, β-arrestin (319–418) effectively inhibited agonist-promoted receptor internalization, whereas β-arrestin-V53D was only modestly effective. However, both constructs significantly inhibited the stimulation of β2AR internalization by β-arrestin in COS-1 cells. Interestingly, immunofluorescence microscopy analysis reveals that both β-arrestin (319–418) and β-arrestin-V53D are constitutively localized in clathrin-coated pits in COS-1 cells. These results indicate the potential usefulness of β-arrestin (319–418) to effectively block arrestin-clathrin interaction in cells and suggest that this construct may prove useful in further defining the mechanisms involved in G protein-coupled receptor trafficking. Signaling by G protein-coupled receptors (GPRs) 1The abbreviations used are: GPR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; m2AChR, m2 muscarinic acetylcholine receptor; β2AR, β2-adrenergic receptor; MES, 4-morpholineethanesulfonic acid; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline. is rapidly attenuated within minutes of agonist exposure, a process often referred to as desensitization. This process, which uncouples GPR-G protein interactions, often involves phosphorylation of the GPR by a G protein-coupled receptor kinase (GRK), ultimately leading to high affinity arrestin binding (1Sterne-Marr R. Benovic J.L. Vitam. Horm. 1995; 51: 193-235Crossref PubMed Scopus (113) Google Scholar). It is the latter step that sterically inhibits G protein binding to the agonist-activated GPR (2Kuhn H. Hall S.W. Wilden U. FEBS Lett. 1984; 176: 473-476Crossref PubMed Scopus (291) Google Scholar, 3Krupnick J.G. Gurevich V.V. Benovic J.L. J. Biol. Chem. 1997; 272: 18125-18131Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). There are currently four known mammalian arrestins: two visual arrestins, arrestin and cone arrestin (4Shinohara T. Dietzschold B. Craft C.M. Wistow G. Early J.J. Donoso L.A. Horwitz J. Tao R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6975-6979Crossref PubMed Scopus (198) Google Scholar, 5Murakami A. Yajima T. Sakuma H. McLaren M.J. Inani G. FEBS Lett. 1993; 334: 203-209Crossref PubMed Scopus (93) Google Scholar), and two nonvisual arrestins, β-arrestin and arrestin3 (6Lohse M.J. Benovic J.L. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (931) Google Scholar, 7Rapoport B. Kaufman K.D. Chazenbalk G.D. Mol. Cell. Endocrinol. 1992; 84: R39-R43Crossref PubMed Scopus (46) Google Scholar). By virtue of their restricted localization predominantly to retinal tissue (4Shinohara T. Dietzschold B. Craft C.M. Wistow G. Early J.J. Donoso L.A. Horwitz J. Tao R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6975-6979Crossref PubMed Scopus (198) Google Scholar, 5Murakami A. Yajima T. Sakuma H. McLaren M.J. Inani G. FEBS Lett. 1993; 334: 203-209Crossref PubMed Scopus (93) Google Scholar, 6Lohse M.J. Benovic J.L. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (931) Google Scholar) and supported by in vitro binding studies (8Gurevich V.V. Richardson R.M. Kim C.M. Hosey M.M. Benovic J.L. J. Biol. Chem. 1993; 268: 16879-16882Abstract Full Text PDF PubMed Google Scholar, 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 (341) Google Scholar), it appears that the visual arrestins specifically regulate photoreceptor signaling. In contrast, the ubiquitous expression of β-arrestin and arrestin3 (6Lohse M.J. Benovic J.L. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (931) Google Scholar, 10Sterne-Marr R. Gurevich V.V. Goldsmith P. Bodine R.C. Sanders C. Donoso L.A. Benovic J.L. J. Biol. Chem. 1993; 268: 15640-15648Abstract Full Text PDF PubMed Google Scholar), as well as in vitro binding studies (8Gurevich V.V. Richardson R.M. Kim C.M. Hosey M.M. Benovic J.L. J. Biol. Chem. 1993; 268: 16879-16882Abstract Full Text PDF PubMed Google Scholar, 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 (341) Google Scholar), strongly suggest that these arrestins regulate a wide variety of GPRs. Indeed, intact cell studies have demonstrated a role for the nonvisual arrestins in regulation of the β2-adrenergic (11Pippig S. Andexinger S. Daniel K. Puzicha M. Caron M.G. Lefkowitz R.J. Lohse M.J. J. Biol. Chem. 1993; 268: 3201-3208Abstract Full Text PDF PubMed Google Scholar), β1-adrenergic (12Freedman 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 (310) Google Scholar), α1B-adrenergic (13Diviani D. Lattion A.-L. Larbi N. Kunapuli P. Pronin A. Benovic J.L. Cotecchia S. J. Biol. Chem. 1996; 271: 5049-5058Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), and odorant (14Dawson T.M. Arriza J.L. Jaworsky D.E. Borisy F.F. Attramadal H. Lefkowitz R.J. Ronnett G.V. Science. 1993; 259: 825-829Crossref PubMed Scopus (170) Google Scholar) receptors. Another level of regulation of GPRs is their internalization or sequestration following agonist exposure. Agonist-induced internalization of GPRs occurs subsequent to the rapid agonist-induced desensitization mediated by GRKs and arrestins. Recent studies have directly demonstrated a role for GRKs in mediating internalization of GPRs. Coexpression of GRK2 with either the m2 muscarinic acetylcholine receptor (m2AChR) or a sequestration-defective β2AR (β2AR-Y326A) resulted in enhanced internalization of these receptors, whereas coexpression with dominant-negative GRK2 attenuated receptor internalization (15Tsuga H. Kameyama K. Haga T. Kurose H. Nagao T. J. Biol. Chem. 1994; 269: 32522-32527Abstract Full Text PDF PubMed Google Scholar, 16Ferguson 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). It is evident that GRKs mediate the enhanced internalization of GPRs through arrestins. Coexpression of β-arrestin or arrestin3 with β2AR-Y326A rescued the sequestration-defective phenotype of this mutant (17Ferguson S.S.G. Downey III, W.E. Colapietro A.-M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-365Crossref PubMed Scopus (856) Google Scholar). Moreover, coexpression of mutant forms of β-arrestin or arrestin3 (containing a single Val to Asp mutation in the far N terminus) with the β2AR inhibited internalization, whereas coexpression of these mutants with GRK2 and β2AR-Y326A prevented GRK2-promoted receptor sequestration (17Ferguson S.S.G. Downey III, W.E. Colapietro A.-M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-365Crossref PubMed Scopus (856) Google Scholar). We recently described a potential molecular mechanism of nonvisual arrestin-promoted GPR internalization (18Goodman 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 (1190) Google Scholar). We observed that β-arrestin and arrestin3 but not visual arrestin interact specifically, stoichiometrically, and with high affinity with clathrin, the major protein component of coated pits. Importantly, the in vitro interaction between β-arrestin and clathrin was substantiated by the immunofluorescence colocalization of β-arrestin, clathrin, and surface β2AR following agonist activation in intact cells. Thus, by virtue of its ability to bind to both the receptor and clathrin, β-arrestin uncouples the β2AR from G protein and then targets it for internalization through clathrin-coated pits. We further demonstrated that the predominant clathrin-binding domain in nonvisual arrestins is localized to a domain (amino acids 367–385 in arrestin3) that contains several hydrophobic and acidic residues critical for clathrin binding (19Krupnick J.G. Goodman Jr., O.B. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 15011-15016Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar) and that the clathrin terminal domain contains a complementary sequence that is involved in arrestin binding (20Goodman Jr., O.B. Krupnick J.G. Gurevich V.V. Benovic J.L. Keen J.H. J. Biol. Chem. 1997; 272: 15017-15022Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). In light of these findings it is interesting that nonvisual arrestins containing a single mutation in their N terminus apparently function as dominant-negative proteins with respect to β2AR sequestration (17Ferguson S.S.G. Downey III, W.E. Colapietro A.-M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-365Crossref PubMed Scopus (856) Google Scholar). In this report, we biochemically characterize the Val to Asp mutant of β-arrestin (β-arrestin-V53D) as well as a β-arrestin clathrin-binding domain construct (β-arrestin (319–418)) and assess the potential utility of these proteins to disrupt β2AR internalization. [3H]Leucine and [γ-32P]ATP were purchased from NEN Life Science Products. All restriction enzymes were purchased from Boehringer Mannheim. Sepharose 2B and all other chemicals were purchased from Sigma. Rabbit reticulocyte lysate was purchased from Promega. SP6 polymerase was prepared as described previously (21Jackson R.J. Hunt T. Methods Enz. 1983; 96: 50-75Crossref PubMed Scopus (438) Google Scholar). Reagents were generously provided by Drs. S. Schmid and H. Damke (dynamin-K44A), V. Gurevich (purified bovine β-arrestin-V53D), M. Hosey (purified m2AChR), S. Ferguson and M. Caron (rat β-arrestin-V53D), B. Kobilka (Flag-tagged β2AR), P. Goldsmith and A. Spiegel (β-arrestin antibodies), and R. K. Crouch (11-cis-retinal). β-Arrestin-V53D was generated by amplifying a ∼206-base pair fragment of bovine β-arrestin by polymerase chain reaction using a sense primer containing a single mutation (underlined) (5′-cttgtggatccggagtatctcaaggagaggagagactatg-3′) and an antisense primer (5′-ggtaggcatgctcgcccagcttat-3′). The resulting product was digested with BamHI and SphI and then ligated into BamHI/SphI digested SP6-modified pBluescript KS containing β-arrestin. β-Arrestin and β-arrestin-V53D cDNAs were then excised with NotI and HindIII, blunted with Klenow, and ligated into pcDNA3 (Invitrogen) that was digested with EcoRV and treated with phosphatase. Orientation was confirmed by restriction analysis. β-Arrestin (319–418) was generated by polymerase chain reaction amplification of bovine β-arrestin using sense (5′-caataagcttaccatggtttcctacaaagtgaaagtg-3′) and antisense (5′-agatggatccctatctgtcgttgagccg-3′) primers. The sense primer contained a Kozak consensus sequence for initiation (underlined) preceding residue 319 in β-arrestin. The resulting product was digested with HindIII and BamHI, purified, and then ligated into HindIII/BamHI-digested pcDNA3. In all of these studies we used the 418-amino acid-long variant of β-arrestin (10Sterne-Marr R. Gurevich V.V. Goldsmith P. Bodine R.C. Sanders C. Donoso L.A. Benovic J.L. J. Biol. Chem. 1993; 268: 15640-15648Abstract Full Text PDF PubMed Google Scholar). All sequences were confirmed by DNA sequencing (Sidney β2AR in pcDNA3 was generated by with by to the The was excised with HindIII and the fragment was ligated into pcDNA3. was excised by BamHI purified, and then ligated into and pcDNA3. cDNAs were with HindIII or EcoRV (β-arrestin (319–418)) and a In vitro and were as described previously V.V. Benovic J.L. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). The and of in vitro proteins were to and The of clathrin and clathrin J.H. Cell. Full Text PDF PubMed Scopus Google Scholar) and the binding (18Goodman 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 (1190) Google Scholar) have previously in arrestins were with or clathrin for in a of in and of and were a in and for in a were in of for and were with enhanced with in and was by analysis binding was by the of in the to the of the and of for β-arrestin, β-arrestin-V53D, and β-arrestin was in the of clathrin and from all were phosphorylated with GRK2 to a of and with as described V.V. Benovic J.L. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). binding to phosphorylated agonist-activated receptors was as described 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 (341) Google Scholar). in vitro arrestins were for with phosphorylated or in a of in were then and arrestins were from arrestins by Sepharose 2B binding was in the of receptor and for β-arrestin, β-arrestin-V53D, and β-arrestin HEK293 and COS-1 cells were in in a containing in Dulbecco's modified Eagle's with bovine and The cells were to and then with the constructs using to the were with or of in of for of was then with the and to a of cells. the was with cells were by with and in of containing were with or for with of PBS, and in of surface receptors were directly by with for with binding in the of were a cell assess expression of the β-arrestin of the cells were by in of and then by and protein was a to and then using a of a as described previously (19Krupnick J.G. Goodman Jr., O.B. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 15011-15016Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). The were with and then by using were by using bovine as a were for immunofluorescence analysis as described previously F. Keen J.H. J. Biol. 1996; PubMed Scopus Google Scholar). stimulation was by the cells in containing for and clathrin was using either J.H. Biol. Scholar) or and β-arrestin was using a a C-terminal (10Sterne-Marr R. Gurevich V.V. Goldsmith P. Bodine R.C. Sanders C. Donoso L.A. Benovic J.L. J. Biol. Chem. 1993; 268: 15640-15648Abstract Full Text PDF PubMed Google Scholar). were with either or or to microscopy was a to a using a expressing the of proteins but those of in the were for of were in the using single Recent that nonvisual arrestins function as clathrin adaptor proteins (18Goodman 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 (1190) Google Scholar), a function that their ability to promote GPR internalization (17Ferguson S.S.G. Downey III, W.E. Colapietro A.-M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-365Crossref PubMed Scopus (856) Google Scholar, 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 (1190) Google Scholar). that nonvisual arrestins are directly involved in GPR internalization from the of mutant forms of these proteins and These mutants were reported to significantly the sequestration of coexpressed β2AR and to block the ability of GRK2 to the sequestration-defective phenotype of a mutant β2AR containing a mutation (17Ferguson S.S.G. Downey III, W.E. Colapietro A.-M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-365Crossref PubMed Scopus (856) Google Scholar). Thus, these mutants were referred to as proteins with respect to GPR internalization. light the ability of β-arrestin-V53D to function as a dominant-negative we its binding to both clathrin and GPRs interaction with either component to its In vitro β-arrestin-V53D and β-arrestin were with of clathrin and following bound arrestins were by and analysis. demonstrated in β-arrestin-V53D is not in clathrin binding in appears to bind better to clathrin than β-arrestin. Moreover, in which β-arrestin significantly from clathrin, we β-arrestin-V53D from clathrin not β-arrestin-V53D in to clathrin with high affinity not The of a clathrin binding is with the mutation in β-arrestin-V53D in a not directly involved in clathrin binding (19Krupnick J.G. Goodman Jr., O.B. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 15011-15016Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). The ability of β-arrestin-V53D to interact better with clathrin than β-arrestin may in light of the role of the far N terminus of arrestins in with the far terminus to regulate the of arrestins 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 (341) Google Scholar, V.V. Benovic J.L. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, V.V. Kim C.M. Benovic J.L. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). It is that this mutation this the far terminus containing the clathrin-binding domain in β-arrestin-V53D, is to of β-arrestin and β-arrestin-V53D were not We the GPR binding properties of of in vitro β-arrestin and β-arrestin-V53D with phosphorylated that β-arrestin-V53D is impaired in with phosphorylated Moreover, β-arrestin-V53D binding to phosphorylated agonist-activated was also significantly impaired not The in GPR binding of β-arrestin-V53D is with the of the mutation to the in arrestins (8Gurevich V.V. Richardson R.M. Kim C.M. Hosey M.M. Benovic J.L. J. Biol. Chem. 1993; 268: 16879-16882Abstract Full Text PDF PubMed Google Scholar, 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 (341) Google Scholar, V.V. Benovic J.L. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, V.V. Benovic J.L. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). Thus, it that this mutation either directly or with the in β-arrestin. The ability of β-arrestin-V53D to interact well with clathrin with its ability to interact with phosphorylated agonist-activated GPRs suggest that this mutant in GPR internalization and may function as a dominant-negative protein as previously reported (17Ferguson S.S.G. Downey III, W.E. Colapietro A.-M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-365Crossref PubMed Scopus (856) Google Scholar). However, we that a construct that only the clathrin-binding of β-arrestin function as a dominant-negative for arrestin-promoted receptor internalization. localization of the clathrin-binding domain in nonvisual arrestins to the far terminus (18Goodman 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 (1190) Google Scholar, J.G. Goodman Jr., O.B. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 15011-15016Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar) and localization of the to the (8Gurevich V.V. Richardson R.M. Kim C.M. Hosey M.M. Benovic J.L. J. Biol. Chem. 1993; 268: 16879-16882Abstract Full Text PDF PubMed Google Scholar, 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 (341) Google Scholar, V.V. Benovic J.L. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, V.V. Benovic J.L. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar), we a construct expressing the C-terminal acids of β-arrestin (β-arrestin In vitro β-arrestin (319–418) specifically to clathrin its of binding appears modestly However, the of binding of β-arrestin (319–418) to clathrin compared with β-arrestin is to in the of the in vitro proteins a protein containing the C-terminal acids of β-arrestin bound to clathrin with a affinity and as β-arrestin not Importantly, β-arrestin (319–418) binding to phosphorylated Moreover, (319–418) and a construct of arrestin3 which was to interact well with clathrin (19Krupnick J.G. Goodman Jr., O.B. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 15011-15016Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar), also not interact with phosphorylated not We the ability of β-arrestin-V53D and β-arrestin (319–418) to agonist-induced GPR internalization. HEK293 cells were a high level of agonist-induced β2AR internalization mediated by arrestins (17Ferguson S.S.G. Downey III, W.E. Colapietro A.-M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-365Crossref PubMed Scopus (856) Google Scholar). HEK293 cells were with β2AR or with β-arrestin constructs or with a dominant-negative mutant that is an of Damke H. Schmid J. Biol. 1993; PubMed Scopus Google Scholar). β-arrestin-V53D, and β-arrestin (319–418) were all nonvisual arrestin We the of these proteins cell surface receptor by of with β-arrestin-V53D a β2AR internalization, β-arrestin (319–418) inhibits β2AR internalization as well as In β-arrestin-V53D and bovine were also dominant-negative proteins in HEK293 cells not These findings that β-arrestin (319–418) is a of β2AR internalization than Interestingly, immunofluorescence of HEK293 cells demonstrated that β-arrestin (319–418) is localized in clathrin-coated pits in the of whereas β-arrestin-V53D is not that the ability of β-arrestin (319–418) to β2AR internalization may in its in clathrin-binding Moreover, the of β-arrestin to β2AR internalization in HEK293 cells and S.S.G. Downey III, W.E. Colapietro A.-M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-365Crossref PubMed Scopus (856) Google Scholar), is with the of β-arrestin to with clathrin-coated pits in an in HEK293 cells not in to the colocalization of β-arrestin with clathrin-coated pits observed in COS-1 cells (18Goodman 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 (1190) Google Scholar). We investigated the ability of β-arrestin-V53D and β-arrestin (319–418) to receptor internalization in COS-1 cells. COS-1 cells a level of agonist-induced GPR internalization in the of arrestin or coexpression than HEK293 L. Ferguson S.S.G. Barak L.S. L. Colapietro A.-M. Lefkowitz R.J. Caron M.G. 1996; PubMed Scopus Google Scholar). Thus, we the of β-arrestin-V53D and β-arrestin (319–418) expression β2AR internalization in these cells. Coexpression of the β2AR with β-arrestin results in a of β2AR internalization, whereas coexpression with β-arrestin (319–418) the agonist-induced internalization Interestingly, coexpression of either β-arrestin-V53D or β-arrestin (319–418) significantly inhibited the of β-arrestin. Importantly, β-arrestin (319–418) and β-arrestin-V53D not have internalization of the receptor not that expression of either of these constructs not all both β-arrestin-V53D and β-arrestin (319–418) the ability of β-arrestin to promote β2AR internalization in COS-1 cells, we immunofluorescence in these cells. demonstrated in we localization of both β-arrestin-V53D and β-arrestin (319–418) in clathrin-coated pits in the of receptor This is in to the localization of β-arrestin in clathrin-coated pits only in the of the agonist-activated receptor and 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 (1190) Google Scholar). Thus, the ability of β-arrestin-V53D and β-arrestin (319–418) to arrestin-promoted β2AR internalization in COS-1 cells may in their ability to block clathrin-binding The localization of β-arrestin-V53D in coated pits is with the clathrin binding by this mutant and that the clathrin-binding domain is in β-arrestin-V53D than in β-arrestin. Moreover, β-arrestin is only with coated pits in the of the agonist-activated β2AR, it is to that binding to the phosphorylated agonist-activated GPR, β-arrestin a that the clathrin-binding and that this is by the β-arrestin-V53D The localization of β-arrestin (319–418) in coated pits in both COS-1 and HEK293 cells and not is with the clathrin binding properties of β-arrestin (319–418) (319–418) not and (19Krupnick J.G. Goodman Jr., O.B. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 15011-15016Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). Moreover, the localization of β-arrestin (319–418) in coated pits also that its clathrin-binding domain is than in β-arrestin. the in the localization of β-arrestin-V53D in COS-1 HEK293 cells and not as well as the in of β-arrestin in COS-1 HEK293 cells 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 (1190) Google and not suggest that mechanisms may involved in agonist-induced of GPRs and that may in cells. into these are currently In we have biochemically a C-terminal construct of β-arrestin and an Val to Asp mutant of β-arrestin with respect to their ability to interact with both receptors and clathrin and to disrupt agonist-induced GPR internalization in intact cells. We observed that β-arrestin-V53D well to clathrin but to phosphorylated agonist-activated GPRs. Moreover, β-arrestin-V53D inhibited the agonist-induced β2AR internalization by β-arrestin in COS-1 cells, it was to only modestly in HEK293 cells. In contrast, β-arrestin a construct that well to clathrin and completely GPR binding effectively inhibited agonist-induced β2AR internalization in both COS-1 and HEK293 cells. immunofluorescence demonstrated that β-arrestin (319–418) is constitutively localized in clathrin-coated pits in both COS-1 and HEK293 cells, β-arrestin-V53D is constitutively localized in coated pits only in COS-1 cells. These results that β-arrestin (319–418) effectively the arrestin-clathrin interaction in cells and indicate the potential usefulness of this construct as a to the agonist-induced of other GPRs.

Synaptic degeneration, including impairment of synaptic plasticity and loss of synapses, is an important feature of Alzheimer disease pathogenesis. Increasing evidence suggests that these degenerative synaptic changes are associated with an accumulation of soluble oligomeric assemblies of amyloid beta (Abeta) known as ADDLs. In primary hippocampal cultures ADDLs bind to a subpopulation of neurons. However the molecular basis of this cell type-selective interaction is not understood. Here, using siRNA screening technology, we identified alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits and calcineurin as candidate genes potentially involved in ADDL-neuron interactions. Immunocolocalization experiments confirmed that ADDL binding occurs in dendritic spines that express surface AMPA receptors, particularly the calcium-impermeable type II AMPA receptor subunit (GluR2). Pharmacological removal of the surface AMPA receptors or inhibition of AMPA receptors with antagonists reduces ADDL binding. Furthermore, using co-immunoprecipitation and photoreactive amino acid cross-linking, we found that ADDLs interact preferentially with GluR2-containing complexes. We demonstrate that calcineurin mediates an endocytotic process that is responsible for the rapid internalization of bound ADDLs along with surface AMPA receptor subunits, which then both colocalize with cpg2, a molecule localized specifically at the postsynaptic endocytic zone of excitatory synapses that plays an important role in activity-dependent glutamate receptor endocytosis. Both AMPA receptor and calcineurin inhibitors prevent oligomer-induced surface AMPAR and spine loss. These results support a model of disease pathogenesis in which Abeta oligomers interact selectively with neurotransmission pathways at excitatory synapses, resulting in synaptic loss via facilitated endocytosis. Validation of this model in human disease would identify therapeutic targets for Alzheimer disease.