beta-Arrestin 1 and 2 differentially regulate heptahelical receptor signaling and trafficking

Abstract

The two widely coexpressed isoforms of beta-arrestin (termed beta arrestin 1 and 2) are highly similar in amino acid sequence. The beta-arrestins bind phosphorylated heptahelical receptors to desensitize and target them to clathrin-coated pits for endocytosis. To better define differences in the roles of beta-arrestin 1 and 2, we prepared mouse embryonic fibroblasts from knockout mice that lack one of the beta-arrestins (beta arr1-KO and beta arr2-KO) or both (beta arr1/2-KO), as well as their wild-type (WT) littermate controls. These cells were analyzed for their ability to support desensitization and sequestration of the beta(2)-adrenergic receptor (beta(2)-AR) and the angiotensin II type 1A receptor (AT(1A)-R). Both beta arr1-KO and beta arr2-KO cells showed similar impairment in agonist-stimulated beta(2)-AR and AT(1A)-R desensitization, when compared with their WT control cells, and the beta arr1/2-KO cells were even further impaired. Sequestration of the beta(2)-AR in the beta arr2-KO cells was compromised significantly (87% reduction), whereas in the beta arr1-KO cells it was not. Agonist-stimulated internalization of the AT(1A)-R was only slightly reduced in the beta arr1-KO but was unaffected in the beta arr2-KO cells. In the beta arr1/2-KO cells, the sequestration of both receptors was dramatically reduced. Comparison of the ability of the two beta-arrestins to sequester the beta(2)-AR revealed beta-arrestin 2 to be 100-fold more potent than beta-arrestin 1. Down-regulation of the beta(2)-AR was also prevented in the beta arr1/2-KO cells, whereas no change was observed in the single knockout cells. These findings suggest that sequestration of various heptahelical receptors is regulated differently by the two beta-arrestins, whereas both isoforms are capable of supporting receptor desensitization and down-regulation.

Figure 1

Analysis of β-arrestin expression in MEF cell lines. Whole cell lysates were prepared from 11 MEF cell lines and resolved (50–70 μg of protein per lane) by SDS/PAGE. Proteins were transferred to a nitrocellulose sheet and immunoblotted with the polyclonal anti-β-arrestin antibody A1CT. The genotype of each MEF line is described beneath the immunoblot. Lines 1–5 are littermates of a βarr2(+/−) × βarr2(+/−) cross, lines 6–9 are littermates from a βarr1(+/−) × βarr1(+/−) cross, and lines 10 and 11 are littermates from a βarr1(+/−) βarr2(−/−) × βarr1(−/−) βarr2(+/−) cross.

Figure 2

Quantitation of β-arrestin levels in MEF cell lines. (A) Whole cell lysates (50 μg) from WT (line 1), βarr1-KO (line 6), and βarr2-KO (line 2) (Left) and known quantities of β-arrestin1-Flag and β-arrestin2-Flag proteins (Right) were separated by SDS/PAGE, transferred to nitrocellulose, and immunoblotted with the A1CT antibody. (B) Concentrations of β-arrestin 1 and β-arrestin 2 in the above MEF lines were quantitated by densitometric analysis of the immunoblots. The resulting β-arrestin levels are plotted as ng of β-arrestin per mg of cell protein. Data are expressed as the mean ± SEM of four to seven experiments.

Figure 3

Effect of reduced β-arrestin levels on second messenger generation. (A and B) Littermate WT (line 8) and βarr1-KO (line 6) cell lines (A) or littermate WT (line 1) and βarr2-KO (line 2) cell lines (B), as well as the βarr1/2-KO cell line 10, all expressing approximately100 fmol of β₂-AR per mg of protein, were stimulated with 10 μM isoproterenol as indicated. Isoproterenol-induced cAMP accumulation in the MEF lines was determined as the percent conversion of [³H]adenine into [³H]cAMP and then normalized to total forskolin (50 μM)-stimulated cAMP accumulation for each cell line. Data are the mean ± SEM of three to six experiments and were analyzed with GRAPHPAD PRISM software. (C) WT (line 1), βarr1-KO (line 6), βarr2-KO (line 2), and βarr1/2-KO (line 10) MEF cell lines, all expressing AT_1A-R at 200–350 fmol/mg of protein, were stimulated with 100 nM AngII for the indicated times. Accumulation of inositol phosphates was measured as the fold difference over basal accumulation. Data are the mean ± SEM of 10 experiments. Unpaired, two-tailed t tests were performed for total cAMP and total inositol phosphate accumulations between WT and βarr1-KO, βarr2-KO, or βarr1/2-KO lines (*, P < 0.005) and between βarr1/2-KO and βarr1-KO or βarr2-KO lines (†, P < 0.03).

Figure 4

Effect of reduced levels of β-arrestins on heptahelical receptor sequestration. (A) Littermate MEF βarr2-KO lines 1–5, βarr1-KO lines 6–9, and βarr1/2-KO lines 10 and 11 expressing 200–300 fmol of β₂-AR per mg of protein were stimulated with 10 μM isoproterenol (iso) for 20 min at 37°C. Receptor sequestration was subsequently measured with a ligand binding assay. Percent isoproterenol-stimulated sequestration was determined as the difference between the agonist-stimulated internalized β₂-ARs and the nonstimulated basally internalized β₂-ARs. (B) Littermate MEF βarr2-KO lines 1–5, βarr1-KO lines 6–9, and βarr1/2-KO lines 10 and 11 expressing 200–350 fmol of AT_1A-R per mg of protein were stimulated with 0.2 nM ¹²⁵I-labeled AngII for 20 min at 37°C. Percent AngII-stimulated sequestration was determined as acid-resistant cpm divided by the total cpm bound. Data are the mean ± SEM of 5–10 experiments. An unpaired two-tailed t test was used to test statistical significance. *, P < 0.0001 between βarr2-KO (lines 2–4) cell lines and their WT controls (lines 1 and 5); †, P < 0.01 between βarr1-KO (lines 6 and 7) cell lines and their WT control (lines 8 and 9); **, P < 0.0001 between βarr1/2-KO (lines 10 and 11) cell lines and all WT lines (lines 1, 5, 8, and 9).

Figure 5

Reconstitution of agonist-induced β₂-AR sequestration by β-arrestin 1 or 2 in βarr1/2-KO MEFs. (A and B) βarr1/2-KO cells (line 10) were infected with various multiplicities of infection of βarr1-Ad (A) or βarr2-Ad (B) and sufficient β₂-AR Ad to express β₂-AR at approximately 200 fmol/mg. The level of β-arrestin expression in each infection was determined by Western blotting of cell lysates (Upper) followed by comparison to a standard curve of β-arrestin1-Flag and β-arrestin2-Flag proteins. Isoproterenol-induced β₂-AR sequestration for each infection was then determined (Lower). A and B show a representative experiment (n = 5). For comparison, the same protein concentration from WT cell lysates was immunoblotted and its isoproterenol-induced sequestration is represented as a dashed line in the bar graph. (C) Pooled data from all experiments showing effect of β-arrestin expression on the ability of βarr1/2-KO cells to sequester the β₂-AR.

Figure 6

Effect of reduced β-arrestin expression on down-regulation of the β₂-AR. WT (line 1), βarr1-KO (line 6), βarr2-KO (line 2), and βarr1/2-KO (line 10) cell lines expressing β₂-AR at approximately 150 fmol/mg of protein or endogenous β₂-AR (approximately 25–50 fmol/mg of protein) were stimulated with 10 μM isoproterenol as indicated. Experiments with overexpressed β₂-AR (n = 3) and with endogenous β₂-AR (n = 4) showed similar results, and thus data were pooled. Receptor number was determined by ligand binding. An unpaired two-tailed t test was used to determine statistical significance as follows. *, P < 0.01 between WT and βarr1/2-KO cells.