Regulation of Receptor Fate by Ubiquitination of Activated β₂-Adrenergic Receptor and β-Arrestin

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CHW1102 cells expressing untagged β₂AR were lysed in lysis buffer [LB; 50 mM Hepes (pH 7.5), 0.5% NP-40, 250 mM NaCl, 2 mM EDTA, 10% (v/v) glycerol, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, leupeptin (10 μg/ml)], mixed with affinity beads (Sepharose 4B–alprenolol), and rotated at 4°C for 4 hours. Nonspecific binding was eliminated by repeated washes with LB, and bound receptor protein was eluted with sample buffer containing SDS. The proteins were transferred to nitrocellulose membrane for Western blotting. Chemiluminiscent detection was performed with SuperSignal West Pico reagent (Pierce). Antibodies Ub P4D1 (SantaCruz) and β₂AR H-20 (SantaCruz) were used to detect ubiquitin and β₂AR, respectively.

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β-Arrestin2 with a COOH-terminal Flag epitope (pcDNA3–β-arr2Flag) was transiently transfected with Lipofectamine (Life Technologies, Rockville, MD) in COS-7 cells, which were then stimulated by isoproterenol. Cells were solubilized in LB supplemented with 5 mM N-ethylmaleimide (NEM). β-Arrestin was immunoprecipitated with Flag affinity beads (Sigma), and the immunoprecipitates were resolved by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoreacted with Ub P4D1 to detect ubiquitinated β-arrestin2. Longer exposure of β-arrestin blots showed broad bands, as in the ubiquitin blots.

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Internalization and degradation assays were done with [¹²⁵I](−)iodocyanopindolol (¹²⁵I-CYP) radioligand binding (18) on whole cells gently resuspended in Dulbecco's modified Eagle's medium (Life Technologies) buffered with 10 mM Hepes (pH 7.5). Binding was performed in triplicates with 400 pM ¹²⁵I-CYP in the presence or absence of the hydrophobic antagonist propranolol (10 μM, to define nonspecific binding) and in the presence or absence of the hydrophilic antagonist CGP12177 (0.3 μM, to assess internalized receptors). Incubation for internalization assays was at 13°C for 3 hours. Binding was terminated by rapid dilution and filtration on Whatman GFC glass fiber filters. To obtain the number of internalized receptors, we determined the percentage of total specific ¹²⁵I-CYP binding sites that could not be displaced by CGP12177. The isoproterenol-stimulated internalization was determined as the difference between the percentage of total receptors internalized after stimulation and the percentage of receptors internalized in untreated cells. For degradation assays, cells were incubated at 25°C for 1 hour, and receptor number (total specific ¹²⁵I-CYP binding sites) was determined after 24 hours of isoproterenol treatment and expressed as the percentage of receptor number assessed in nonstimulated cells. Where necessary, MG132 (20 μM) or lactacystin (20 μM) mixed in serum-free media was added to cells 1 hour before stimulation.

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Flag epitope–tagged WT-β₂AR and β₂AR²⁻, along with β-arrestin2, were transiently transfected in COS-7 cells with Lipofectamine. After agonist treatment, receptors were immmunoprecipitated with Flag affinity beads, and the immunoprecipitates were resolved on SDS-PAGE and probed with antibodies Ub P4D1 and β₂AR H-20.

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Confluent cells on 150-mm dishes were treated or not for 15 min with isoproterenol, after which cells were lysed in LB. Endogenous β₂AR was immunoprecipitated with the antibody β₂AR M-20. In the case of overexpressed β₂AR, cells seeded at 400,000 per 100-mm dish were transfected with pcDNA3-Flag-β₂AR plasmid using Lipofectamine, and receptor was immunoprecipitated using FLAG affinity beads (Sigma). Ubiquitinated species were detected with antibody Ub P4D1.

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For coimmunoprecipitation of endogenous proteins from whole-brain extract, brain tissue was solubilized in LB (10). β-Arrestin was immunoprecipitated from extracts by incubation for 4 hours at 4°C with antibody specific for β-arrestin, covalently cross-linked to Reactigel beads (Pierce). Immunoprecipitated proteins were analyzed by SDS-PAGE and detected by immunoblot analysis. For cellular experiments, COS-7 cells were transfected with COOH-terminal Flag-tagged β-arrestin and untagged Mdm2 plasmids. Flag–β-arrestins were immunoprecipitated with M2 Flag affinity beads (Sigma); the immunoprecipitates were separated by SDS-PAGE, and the presence of Mdm2 was demonstrated by immunoblotting with monoclonal antibodies (mAbs) 2A10 or AB1.

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Recombinant β₂AR reconstituted in vesicles was used as the substrate in a ubiquitination reaction containing 20 mM Hepes (pH 7.5), 5 mM MgCl₂, 2 mM dithiothreitol (DTT), 2 mM adenosine 5′-triphosphate (ATP), 5 μg of ubiquitin, 20 μM MG132, and crude rabbit reticulocyte lysate (RRL, Promega), a source for the ubiquitination machinery (36) (5 μl/reaction). The reactions were supplemented with either His–β-arrestin2 (7 μg) or GST–β-arrestin2 (5 μg), GRK2, (1 μg), or Mdm2 [either GST-Mdm2_1-491 (8 μg) or COS-7 lysate with overexpressed Mdm2] where necessary. Reaction mixtures were incubated for 1 hour at 30°C. Ubiquitinated receptor (vesicles) was isolated by repeated washes with LB and centrifugation at 250,000g, 45 min. After addition of SDS–sample buffer, protein samples were incubated at 37°C for 2 hours before electrophoresis.

A cDNA construct of β-arrestin2 was cloned into pET-29a and was used to isolate recombinant S·TAG-β-arrestin2His6 purified on S-protein agarose beads (Novagen), which was then used as the substrate in the β-arrestin2 in vitro ubiquitination reaction containing 20 mM Hepes (pH 7.5), 5 mM MgCl₂, 2 mM DTT, 2 mM ATP, 5 μg of ubiquitin, 20 μM MG132, and crude RRL, either supplemented or not with 100 μg of COS cell extract (clarified by centrifugation at 21,000g for 15 min) with or without overexpressed Mdm2.

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Mdm2 truncation mutants were prepared by standard polymerase chain reaction protocols and subcloned into pcDNA3. All sequences were confirmed with an automated ABI DNA sequencer (Howard Hughes Nucleic Acid Facility, Duke University).

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We thank D. Addison, M. Holben, and J. Turnbough for secretarial assistance; B. K. Kobilka for 0-Kβ₂AR plasmid; A. J. Levine for the Mdm2 plasmid and mAb 2A10; G. Lozano for providing the Mdm2-p53 null cell line; M. Delahunty for purified rβ₂AR; W. E. Miller for pAS2 constructs and advice in yeast experiments; F. T. Lin for pcDNA3-βarrHis6 constructs; M. Cong for providing pcDNA3-β₂AR²⁻; S. J. Perry and K. L. Pierce for critical reading; H. E. Kendall and G. P. Irons for technical assistance; W. D. Capel for purified GRK2; and G. J. Sabo for assistance. Supported by NIH grant HL16037. R.J.L is an investigator of the Howard Hughes Medical Institute.