Role of β-arrestin in mediating agonist-promoted G protein-coupled receptor internalization

Science

Volumn 271, Issue 5247, 1996, Pages 363-366

  Ferguson, Stephen S G ^a   Downey III, William E ^a   Colapietro, Anne Marie ^a   Barak, Larry S ^a   Ménard, Luc ^a   Caron, Marc G ^a  

^a DUKE UNIVERSITY MEDICAL CENTER   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ANTIGEN; BETA 2 ADRENERGIC RECEPTOR; BETA ADRENERGIC RECEPTOR KINASE; BETA ADRENERGIC RECEPTOR STIMULATING AGENT; BETA ARRESTIN; COMPLEMENTARY DNA; CYCLIC AMP DEPENDENT PROTEIN KINASE; EYE PROTEIN; GUANINE NUCLEOTIDE BINDING PROTEIN; ISOPRENALINE; RETINA S ANTIGEN;

ARTICLE; CELL LINE; GENETIC TRANSFECTION; GENETICS; HUMAN; METABOLISM; MUTATION; PHOSPHORYLATION; PHYSIOLOGY; POINT MUTATION;

ADRENERGIC BETA-AGONISTS; ANTIGENS; ARRESTINS; BETA-ADRENERGIC RECEPTOR KINASE; CELL LINE; CYCLIC AMP-DEPENDENT PROTEIN KINASES; DNA, COMPLEMENTARY; EYE PROTEINS; GTP-BINDING PROTEINS; HUMANS; ISOPROTERENOL; MUTATION; PHOSPHORYLATION; POINT MUTATION; RECEPTORS, ADRENERGIC, BETA-2; TRANSFECTION;

EID: 0030593035     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.271.5247.363     Document Type: Article

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25. 54 → D) was generated by PCR to mutate codon GTG (valine) to GAT (aspartic acid). Positive clones were isolated for each mutant, and the integrity of the coding sequences as well as the mutation was confirmed by dideoxy DNA sequencing.
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31. βARK1 protein was overexpressed greater than 20-fold above endogenous levels (7), and ß-arrestin-1 and -2 proteins were overexpressed greater than 30-fold above endogenous levels as measured by densitometric analysis of immunoblots (S S. G Ferguson et al., data not shown).
32. 125I-pindolol (1 nM) binding performed at 14°C for 3 hours (3), which because of hydrophobicity can measure both surface and intracellular receptors not competed for by CGP-12177 (a hydrophilic ligand) minus the basal level of sequestered receptors as measured without exposure to agonist. Basally internalized receptors represented 28 5 ± 2% of total cell receptors. Overexpression of βARK1, β-arrestin-1, β-arrestin-2, β-arrestin-1-V53D, and β-arrestin-2-V54D had no effect on the number of basally internalized receptors.
33. 6 cells per 100-mm dish and transiently transfected with a modified calcium phosphate method [B. R. Cullen, Methods Enzymol. 152, 684 (1987)].
34. 2AR expression binding studies were done at 30°C for 30 min for the measurement of receptor expression, and bound ligand was separated on glass fiber filters (Whatman, GF/C) by vacuum filtration. The filters were washed four times with 4 ml of cold wash buffer [50 mM Tris and 120 mM NaCl (pH 7.2)] and counted in a gamma-counter. Protein concentrations were determined with a Bio-Rad assay kit with bovine serum albumin as the standard.
35. 32P]orthophosphate (100 μCi/ml) per well in serum- and phosphate-free Dulbecco's modified Eagle's medium (DMEM), then treated with serum- and phosphate-free DMEM containing 100 μM ascorbate with or without ISO (10 μM final concentration) and incubated at 37°C for 15 min. The cells were then assayed for whole-cell phosphorylation as descnbed (7). The extent of receptor phosphorylation was quantitated with a Molecular Dynamics phosphonmaging system and ImageQuant software.
36. 2AR cDNA constructs Supported in part by a grant from the National Institutes of Health (NS 19575) and a Bristol Myers Squibb unrestricted grant award to M.G.C S.S.G.F. is a fellow of the Medical Research Council of Canada, W.E.D. III is a recipient of a Howard Hughes Medical Institute research training fellowship for medical students, and L.S.B. is a recipient of a Howard Hughes postdoctoral fellowship