β-Arrestin1 Knockout Mice Appear Normal but Demonstrate Altered Cardiac Responses to β-Adrenergic Stimulation
Abstract β-Arrestin1 knockout mice were studied to define the physiological role of β-arrestin1 in the regulation of G protein–coupled receptors. β-Arrestin1 is thought to be involved in the desensitization of many G protein–associated cell surface receptors, particularly β-adrenergic receptors. Homozygous knockout mice are overtly normal. Resting cardiovascular parameters modulated by β-adrenergic receptors such as heart rate, blood pressure, and left ventricular ejection fraction are not changed. However, homozygous mutants are more sensitive to β-receptor agonist–stimulated increases in ejection fraction, consistent with a role of β-arrestin1 in β-adrenergic receptor desensitization. We conclude that β-arrestin1 is important for in vivo G protein–coupled receptor desensitization and that this aspect of desensitization represents a mechanism for fine-tuning responses. However, β-arrestin1 does not appear to be required for development or for other essential biological functions.
Arrestins are thought to bind G protein–coupled receptors that have been phosphorylated by G protein–coupled receptor kinases (GRKs). The association of arrestins with phosphorylated receptors blocks receptor interaction with heterotrimeric G proteins, resulting in signal termination. The combined action of these two groups of proteins is thought to mediate rapid desensitization of particular G protein–coupled receptors.1 In vitro GRK and arrestin effects are dependent on each other: neither protein alone can significantly terminate signal transduction.2 In addition to physically blocking receptor coupling with G proteins, β-arrestin1 modulates agonist-promoted β2-adrenergic receptor internalization (sequestration) in HEK 293 cells.3,4 In fact, β-arrestin1 binds directly to clathrin and may act as an adapter to target phosphorylated β2-adrenergic receptors to clathrin-coated vesicles.5 Although these data clearly demonstrate that the β2-adrenergic receptor is regulated by β-arrestin1 in vitro, there is no evidence that β-arrestin1 is a physiological regulator of the receptor. Other reports suggest that β-arrestin1 may regulate β1-adrenergic receptors, M2-muscarinic receptors, and α1B-adrenergic receptors.6–8
β-Arrestin1 is a member of a small protein family that consists of four proteins: arrestin, arrestin-C, β-arrestin1, and β-arrestin2.2,9–11 Arrestin and arrestin-C are expressed primarily in rod and cone cells in the visual system. β-Arrestin1 and β-arrestin2 are widely expressed. β-Arrestin1 is usually more abundant than β-arrestin2.12 The physiological roles of β-arrestin1 and β-arrestin2 are undefined.
Critical questions regarding the precise physiological role of particular arrestins have been hampered by the lack of reagents that specifically block arrestin action. We hypothesized that knockout mice lacking a particular arrestin would provide a valuable tool for studying the role of particular arrestins. Mice lacking β-arrestin1 were created using gene targeting and blastocyst-mediated transgenesis. β-Arrestin1–deficient mice have a normal life expectancy and by a variety of different parameters appear normal. However, the β-arrestin1–deficient mice do demonstrate alterations of in vivo β-adrenergic receptor responses compared with their wild-type littermates.
Materials and Methods
Preparation of Targeting Construct
A fragment of the β-arrestin1 gene was isolated from a custom Lambda Dash II 129/SvJ mouse genomic library (Stratagene Inc) using a bovine β-arrestin1 cDNA probe. An 11-kb EcoRV fragment was inserted into a Bluescript SK II (+)–based plasmid (Stratagene Inc) containing the neomycin resistance gene (neor) under control of the phosphoglycerate kinase promoter.
Cell Culture and Blastocyst Injection
The C1 ES cell line13 was cultured on irradiated mouse embryonic fibroblasts in supplemented DMEM with 500 U/mL ESGRO (GIBCO Laboratories). Construct DNA (1 pmol) was linearized at the unique Kpn I site and transfected by electroporation (125 μF, 400 V: Gene Pulser; Bio-Rad Laboratories) into 2.5×107 C1 cells. Cells surviving G418 (0.2 mg/mL active, GIBCO Laboratories) were screened by Southern blot analysis (Fig 1⇓). Targeted clones were injected into C57BL/6 (Taconic, Germantown, NY) blastocysts (15 cells/embryo). Germ-line transmission by the resultant chimeric males was determined by crossing with Black Swiss females (Taconic). Heterozygous mutants were mated to produce the F2 generation. All experiments were performed with mice in the mixed 129/SvJ and Black Swiss background. Wild-type control animals were age- and sex-matched sibs.
Western Blot Analysis
Cytosolic proteins were separated using nonreducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Western blot analysis was performed using the ECL detection system (Amersham). The monoclonal antibody MabF4C1, which recognizes a conserved arrestin domain, was generously provided by Dr Larry Donoso.12,14
Adenylyl Cyclase Assay
Assays were performed as previously described,15 with some modifications. Briefly, crude membrane fractions were prepared by homogenization of hearts, from 9- to 10-month-old male mice, in 20 mmol/L Tris (pH 7.6) and 5 mmol/L EDTA. Homogenates were centrifuged at 800g for 10 minutes to remove nuclei. The supernatant was centrifuged at 20 000g for 20 minutes. The pellet was resuspended in 75 mmol/L Tris (pH 7.6), 2 mmol/L EDTA, and 12.5 mmol/L MgCl2 at a protein concentration of 1 mg/mL. Reactions in triplicate contained approximately 20 mg of membrane protein, 30 mmol/L Tris (pH 7.6), 0.8 mmol/L EDTA, 5 mmol/L MgCl2 (approximately 4.0 mmol/L free MgCl2), 2.7 μmol/L phosphoenolpyruvate, 50 mmol/L GTP, 100 mmol/L cAMP, 120 mmol/L [α-32P]ATP (0.3 μCi/tube), 20 U/mL myokinase, 4 U/mL pyruvate kinase, and isoproterenol (0 to 30 mmol/L) or NaF (10 mmol/L). Tubes were incubated for 10 minutes at 37°C. Reactions were terminated by the addition of 1 mL of a solution containing 0.3 mmol/L cAMP (with approximately 10 000 cpm of [3H]cAMP), 0.4 mmol/L ATP, and 1% SDS (pH 7.5). cAMP was isolated by sequential chromatography with Dowex (AG 50W-X4) and neutral alumina (WN-3). Results are expressed as a percentage of the NaF response.
In Vivo Determination of Ejection Fraction
All animal care was in accordance with institutional guidelines. Experiments were performed as previously described.16 Briefly, adult male mice (approximately 1 year old and weighing 40 to 60 g) were anesthetized by intraperitoneal injection with ketamine (0.065 mg/g), acepromazine (0.002 mg/g), and xylazine (0.013 mg/g) and allowed to breathe spontaneously. The chest was shaved and the animal was positioned prone on a warmed (37°C) saline bag stand-off suspended between two supports. Echocardiography was performed from below the stand-off using an Interspec Apogee X-200 ultrasonograph (Interspec-ATL) with a 9-MHz annular array transducer. Short axis M-mode measurements of left ventricular internal diameter (LVID) were determined by averaging values from more than 3 beats using the leading edge–to–leading edge convention adopted by the American Society of Echocardiography.17 Left ventricular ejection fraction (LVEF) was calculated by the cubed method as follows: LVEF=[(LVIDd)3−(LVIDs)3]/LVIDd3, where d indicates diastolic and s indicates systolic. Measurements were performed at baseline and during intravenous infusion (via a previously placed jugular catheter) of isoproterenol (0.05, 0.1, and 0.2 μg/kg per minute for 5 minutes each). The total amount of infusate was less than 100 μL. Heart rate was measured continuously from an on-line ECG acquired using subcutaneously placed needle electrodes.
In Vivo Determination of Blood Pressure and Heart Rate
Mean arterial blood pressures were monitored by femoral artery catheterization18,19 in conscious 3-month-old male mice. After anesthesia with avertin (tribromoethyl alcohol and tertiary amyl alcohol), the femoral artery was cannulated with drawn out PE-10 tubing. The cannula was then exteriorized and secured to a swivel device, allowing mobility of the animal. The cannula was attached to a Y-connector, with one arm going to a presssure transducer and the second arm attached to an infusion pump (1 μL/min dextrose/heparin). Animals were allowed to recover for at least 24 hours before studies were performed.
Blood was obtained by cardiac puncture following administration of avertin. Serum and whole blood were submitted to the Tufts Veterinary Diagnostic Laboratory for evaluation.
Spleen cells were prepared from age-matched mice (approximately 3 months old). Cells were stained with fluorescent-labeled antibodies and subjected to flow cytometry in a Cytofluorograf II (Becton Dickinson & Co).
Linkage analysis was performed using Mapmaker 3.0.
Targeted Disruption of the β-Arrestin1 Gene
A bacteriophage λ clone (designated βarr-1) was isolated from a 129/SvJ mouse genomic bacteriophage λ library that encoded part of the murine β-arrestin1 gene. An internal portion of the murine β-arrestin1 gene was found on an 11-kb EcoRV fragment and was used to make a targeting construct (Fig 1A⇑). Although the exact intron/exon structure of the murine β-arrestin1 gene was not determined, hybridization with a bovine β-arrestin1 cDNA confirmed that the EcoRV fragment lacked the 5′ and 3′ end of the coding region. The targeting construct was linearized at a unique Kpn I site within the region of homology and introduced into C1 ES cells by electroporation. ES cell clones surviving G418 selection were screened for insertions in the endogenous β-arrestin1 gene by Southern blot analyses using an “external” EcoRV fragment (Fig 1A⇑). One of every 12 G418-resistant clones exhibited a novel 16.5-kb Ase I fragment consistent with insertion of the entire vector into the β-arrestin1 locus (Fig 1A⇑). Cells from one of the targeted lines were injected into C57BL/6 blastocysts. Seven germ-line chimeric males were identified by mating with wild-type Black Swiss females. Heterozygous offspring were crossed to generate mice homozygous for the β-arrestin1 mutation (Arrb1−/−). Arrb1−/−, Arrb1−/+, and Arrb1+/+ mice were characterized by Southern blot analysis of DNA from the tail (Fig 1B⇑). Homozygous Arrb1−/− mice were viable.
Localization of the β-Arrestin1 Gene to Chromosome 7
Heterozygous Arrb1−/+ mice were derived from mating between chimeric agouti male mice and Black Swiss females. The Arrb1−/+ mice were also heterozygous at two coat color loci, tyr (tyrosinase) and p (pink-eye dilute), that are closely linked on chromosome 7 (Fig 2⇓). Characterization of F2 mice, the offspring of intercrosses between F1 Arrb1−/+ mice, suggested that these coat color loci were linked to the β-arrestin1 gene. That is, 14 of 15 albino F2 mice (homozygous at the tyr locus) were also homozygous for the β-arrestin1 mutation, demonstrating linkage rather than random association between the tyr locus and the β-arrestin1 gene. The genotypes of 68 F2 mice were assessed at the two coat color loci and at D7MIT40 (Fig 2⇓ and Reference 2020 ). Analysis of these genotypes using Mapmaker 3.0 indicated that the β-arrestin1 gene was more likely (P<.0001) to be located between tyr and D7MIT40 than between tyr and p (Fig 2⇓). Genotype data were consistent with the model that the β-arrestin1 gene is about 1.5 centimorgans from the tyr locus (Fig 2⇓).
Absence of β-Arrestin1 Protein in Homozygous Arrb1−/− Mice
Northern blot and Western blot analyses were performed to determine the effect of the β-arrestin1 gene insertion on RNA and protein expression. Heart and kidney RNAs from wild-type mice contained a 7.5-kb β-arrestin1 mRNA species (Fig 3A⇓). However, no β-arrestin1 mRNAs were detected in any tissues derived from homozygous Arrb1−/− mice (Fig 3A⇓). Equivalent amounts of β-actin mRNA were detected in RNA samples from Arrb1−/− and wild-type mice (Fig 3A⇓, lower panel). The absence of β-arrestin1 protein in homozygous mutants was verified by Western blot analysis using a mouse monoclonal antibody against a conserved domain of arrestin (Fig 3B⇓). The 53-kD β-arrestin1 band is present in brain cytosol from wild-type mice and absent in Arrb1−/− mice. Similar results were obtained with cytosol preparations from the heart and with membrane preparations from these tissues (data not shown). The anti-mouse secondary antibody used to detect the primary antibody also recognized IgG in the protein samples (migrating at greater than 100 kD in the nonreducing gel in Fig 3B⇓), demonstrating that equivalent amounts of protein were loaded in each lane. Thus, the introduced mutation results in the complete ablation of the β-arrestin1 gene product.
Characteristics of Arrb1−/− Mice
Adult Arrb1−/− mice appeared identical to wild-type littermates. Male and female Arrb1−/− mice were fertile and produced normal size litters. Hematoxylin and eosin staining of fixed sections revealed no histological abnormalities in heart, kidney, brain, intestine, spleen, and lung (data not shown). Homozygous mutants kept for over 1.5 years showed no reduction in life expectancy when compared with wild-type littermates.
The blood chemistry values obtained from Arrb1−/− mouse blood were similar to those obtained from wild-type littermates (Table 1⇓). Hemoglobin, hematocrit, white blood cell counts, and red blood cell counts of Arrb1−/− and wild-type littermates were indistinguishable (Table 1⇓).
High levels of β-arrestin1 expression in spleen and peripheral lymphocytes has led to the speculation that the protein may modulate lymphocyte signaling.12,21 FACS analysis of Arrb1−/− mouse lymphocytes was used to screen for gross immune system defects. Spleen cells were isolated from Arrb1−/− and wild-type littermates. The total number of cells was similar for the two groups of animals: Arrb1−/−, 5.5±0.5×107; wild-type, 5.2±0.3×107. A variety of antisera were chosen on the basis of their ability to distinguish major classes of lymphocytes (Table 2⇓). No significant differences were observed. Of particular note, normal numbers of B cells were observed in Arrb1−/− spleens as indicated by staining with anti-B220, anti-kappa light chain, and anti-IgM heavy chain. No changes were observed in the number of T cells as indicated by staining with the anti-thy1.1,1.2 antisera. Normal αβ (H57) and γδ (GL3) T-cell subpopulations were also present. No change in the number of macrophages (mac-1) was observed.
Cardiovascular Characteristics of Arrb1−/− Mice
β-Adrenergic receptor function was investigated because of the proposed role of β-arrestin1 in adrenergic receptor signaling. Coupling of the β-adrenergic receptor to adenylyl cyclase was assessed in heart membrane preparations from control and Arrb1−/− mice. Isoproterenol dose-response curves were not significantly different in wild-type and Arrb1−/− heart membranes; no differences in Vmax or EC50 were observed (data not shown). The resting mean blood pressure (114.3± 3.4 mm Hg, n=3; 117.8±1.1 mm Hg, n=4: mean±SEM) and heart rate (603.3±44.1 bpm, n=3; 662.5±43.1 bpm, n=4: mean±SEM) of conscious wild-type and Arrb1−/− mice were also indistinguishable. Anesthetized mutant and wild-type mice had the same basal heart rates and ejection fractions (Fig 4⇓). There was no difference in the isoproterenol-mediated increase in heart rate between the wild-type and mutant mice except at the highest isoproterenol concentration (Fig 4B⇓). However, isoproterenol produced significantly greater increases in ejection fraction in mutant mice at each isoproterenol concentration than was produced in wild-type mice (Fig 4A⇓).
We demonstrate here that the β-arrestin1 gene is on mouse chromosome 7, closely linked to the murine albino locus, and that β-arrestin1–deficient mice are viable with no overt differences from wild-type littermates. β-Arrestin1–deficient mice and wild-type littermates have indistinguishable life expectancy, fertility, blood chemistry, mean resting blood pressure, resting heart rate, and immune systems. β-Arrestin1 is not essential for mouse development or viability. However, the cardiac response of β-arrestin1–deficient mice to isoproterenol appears to be different from that of wild-type mice.
The absence of a severe phenotype in β-arrestin1–deficient mice was unexpected given the widespread expression of β-arrestin12,11,21 and its presumed modulatory role for many G protein–coupled receptors.1 Knockout mice lacking GRK2 (β-adrenergic receptor kinase 1), another component of the G-protein–signaling pathway, are not viable, demonstrating an essential role for this pathway in mouse physiology.22 There is no β-arrestin1 protein in the tissues of the β-arrestin1–deficient mouse (Fig 3B⇑). Perhaps essential arrestin functions in these mice are provided by β-arrestin2. That is, β-arrestin1 and β-arrestin2 are more than 75% identical,11 so β-arrestin2 might be induced in the β-arrestin1–deficient mouse to compensate for the absence of β-arrestin1. However, the monoclonal antibody MabF4C1 recognizes a highly conserved amino acid sequence, DGVVLVD, present near the amino terminus of rat and human arrestin, arrestin-C, β-arrestin1, and β-arrestin2 and should recognize mouse β-arrestin2.12,14,23 The absence of β-arrestin2 immunoreactivity in wild-type and mutant mice is consistent with reports showing that β-arrestin2 is much less abundant than β-arrestin1 in most tissues12 and suggests that there is no compensatory induction of β-arrestin2 in the β-arrestin1–deficient mice. A small increase of β-arrestin2 might not have been detected by this analysis. However, arrestins are believed to function stoichiometrically. Thus, we suggest that a small increase in β-arrestin2 would not be able to compensate for the complete lack of the more abundant β-arrestin1.
In vitro studies suggest that the β-adrenergic receptor is a likely target of β-arrestin1, although there is no direct in vivo data to support this hypothesis. Basal, isoproterenol-stimulated, and NaF-stimulated adenylyl cyclase activities were not altered in Arrb1−/− heart membrane preparations (data not shown). This observation suggests that there is no compensatory change in β-adrenergic receptor density, Gsα abundance, or coupling efficiency in the homozygous mutant mice. However, we recognize that subtle changes in the signal transduction system due to β-arrestin1 deficiency might not have been detected by this assay.
In vivo differences between wild-type and mutant animals were evident. β-Arrestin1 modifies isoproterenol-induced increases in ejection fraction as assessed by echocardiography in anesthetized animals. Mice lacking β-arrestin1 have a significantly greater response than wild-type mice at each isoproterenol concentration (Fig 4⇑). This difference is not observed for the isoproterenol-induced increase in heart rate, except at the greatest isoproterenol concentration, suggesting that β-arrestin1 is a more important regulator of the inotropic effects of isoproterenol than of its chronotropic effects. Although some isoproterenol-stimulated responses were altered, resting heart rate and blood pressure were not different between the two groups of mice. These results are consistent with a current model of β-arrestin1 action1 in which only agonist-activated receptors are targets for β-arrestin1 regulation during desensitization. Although β-arrestin1 probably functions in other tissues and in physiological processes other than the inotropic response to isoproterenol, it appears to have a negligible role in maintaining basal sympathetic tone.
We have demonstrated an in vivo role for β-arrestin1 in the regulation of G protein–coupled receptors. Although the protein is widely expressed, β-arrestin1 is not required for development or for essential biological functions. β-Arrestin1 knockout mice will provide a useful system for determining the receptor specificity of β-arrestin1 and for identifying its role in other aspects of receptor desensitization such as receptor internalization. These studies also demonstrate the usefulness of β-arrestin1 as a new therapeutic target: inhibition of β-arrestin1 action could potentiate the effects of exogenous or endogenous activators of G protein–coupled receptors without producing gross deleterious effects.
This work was supported by the Howard Hughes Medical Institute. We thank Dr Larry Donoso for providing us with the monoclonal antibody MabF4C1 and Juanita Campos-Torres for her assistance with the FACS analysis.
This manuscript was sent to Laurence H. Kedes, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
- Received June 17, 1997.
- Accepted September 9, 1997.
- © 1997 American Heart Association, Inc.
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