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Author's personal copy Life Sciences 85 (2009) 339–344
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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Mutagenesis of important amino acid reveals unconventional homologous internalization of β1-adrenergic receptor Murad Hossain a, Mamunur Rashid a, Mohiuddin Ahmed Bhuiyan a, Takashi Nakamura a, Masanobu Ozaki b, Takafumi Nagatomo a,⁎ a b
Department of Pharmacology, Niigata University of Pharmacy and Applied Life Sciences, Niigata 956-8603, Japan Department of Fundamental Pharmacology and Therapeutics, Niigata University of Pharmacy and Applied Life Sciences, Niigata 956-8603, Japan
a r t i c l e
i n f o
Article history: Received 1 April 2009 Accepted 26 June 2009 Keywords: β1-adrenergic receptor Mutation Internalization β-arrestin1
a b s t r a c t Aims: The study was designed to examine the internalization of Asp104Lys mutant of β1-adrenergic receptor (β1-AR) and compared to other mutant (Asp104Ala) and wild type receptors. Moreover, this study needs to perform the role of GRK2 (βARK1) and β-arrestin1 on this internalization of Asp104Lys mutant of β1-AR. Main methods: Binding affinity, functional potency of agonist and agonist-induced internalization were determined for wild type and both mutants of β1-ARs stably expressed in HEK 293 cells as assessed by [3H] CGP12177 radioligand. We have performed GRK2 and β-arrestin1 expression levels by western blot analysis and also performed internalization of this mutant receptor after over expression and deletion of β-arrestin1 gene. Key findings: In the present study, the binding affinity of (−)-isoproterenol for both mutants were significantly decreased compared to wild type. Though the mutant Asp104Ala showed agonist-induced receptor activation, interestingly this mutant was not internalized. However, the mutant Asp104Lys, which showed uncoupling with G protein, was internalized 31.77±3.13% from cell surface. Asp104Lys mutant produced the same level of GRK2 expression in (–)-isoproterenol induced stimulation of wild type receptor and addition of (–)-isoproterenol further increased GRK2 expression in mutant receptors. In addition, overexpression of β-arrestin1 in mutant Asp104Lys promoted (39.75±2.19%) and knockdown of β-arrestin1 by siRNA decreased (3.55±1.75%) internalization compared to Asp104Lys mutant of β1-ARs. Significance: The present studies suggest that Asp104Lys mutant β1-ARs triggers unconventional homologous internalization induced by G protein independent signals, where GRK2 and β-arrestin1 play an important role for β1AR internalization. © 2009 Elsevier Inc. All rights reserved.
Introduction The mammalian β1-adrenergic receptor (β1-AR) (Brodde 1993) is coupled through Gs to the activation of adenylyl cyclase which promotes the receptor internalization and down regulation (Brodde 1993; Bristow 2000). It is important in the regulation of heart rate and contractility (Brodde 1993), lipolysis by adipose tissue (Arner et al. 1991), and blood pressure homeostasis (Kopp and DiBona 1983), among other vital functions. During chronic heart failure (HF) increase of catecholamine causes β1-AR desensitization and down regulation. The resulting partial loss of β1-AR function is believed to be an adaptive mechanism to counteract the cardiotoxicity of chronic adrenergic signaling (Bristow 2000).
⁎ Corresponding author. Department of Pharmacology, Niigata University of Pharmacy and Applied Life Sciences, 265-1, Higashijima, Akihaku, Niigata-shi 9568603, Niigata, Japan. Tel.: +81 250 25 5234; fax: +81 250 25 5135. E-mail address:
[email protected] (T. Nagatomo). 0024-3205/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2009.06.015
Agonist binding to a G protein coupled receptors (GPCRs) induces conformational changes in the receptor, leading to activation of G protein. Upon activation it leads to activate G protein regulated kinases (GRKs) that in turn phosphorylate the specific receptor for desensitization. Consequently, β-arrestins bind to the GRK-phosphorylated motifs of the receptor and induce the receptor internalization. This homologous GPCR desensitization/internalization is agonist-specific and GRK-dependent. This type of feedback regulation is conventional, since it requires activation of classic G proteins (Ferguson 2001; Kohout and Lefkowitz 2003; Lodowski et al. 2003). Initially, internalization of the GPCR was viewed as a means to uncouple the receptor from its signaling components, thereby dampening the overall response (Hertel et al. 1985; Waldo et al. 1983; Gagnon et al. 1998; Tsao et al. 2001). The results of many studies indicated that the itinerary of the internalized GPCR was receptor- and cell-specific (Zhang et al. 1997). At least four pathways of agonistinduced internalization of GPCRs exist (Claing et al. 2000, 2002), and they may be cell type specific. The classical GPCR internalization pathway involves GPCR kinases (GRKs), β-arrestin, clathrin-coated pits, and the GTPase dynamin and is exemplified by the β2-adrenergic
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receptor (Claing et al. 2000, 2002; Ferguson et al. 1996; Ferguson 2001; Krupnick and Benovic 1998; Lefkowitz 1998; Pitcher et al. 1998). We have previously demonstrated that Aspartic acid at position 104 is a highly conserved residue in transmembrane helix II (TMH II) through molecular modeling (Ahmed et al. 2006a,b) and mutation at this position by Alanine (Asp104Ala) showed constitutive activity (Ahmed et al. 2006a,b); however, mutation at position 104 by lysine showed uncoupling to G protein and made the receptor functionally inactive (Hossain et al. 2008). In the present study, we demonstrated that agonist-induced receptor internalization through uncoupling of the receptor from G protein was due to the mutation of Asp104 to Lys (Asp104Lys) at the highly conserved residue in TMH II of the β1-AR. Interestingly, although mutation of Asp104 to Ala (Asp104Ala) showed coupling with GPCRs receptor, mutant receptor was not internalized. Therefore, the present study may provide important information that might have an implication for the elucidation of the mechanism of the internalization of β1-ARs in unconventional pathway.
Ligand binding Crude membranes were prepared from cells expressing the β1-AR and its mutant. Radioligand binding studies were carried out in assay buffer containing 75 mM Tris–HCl (pH 7.4), 12.5 mM MgCl2 and 2 mM EDTA at 37 °C for 60 min using 5–10 μg of membrane protein. The total reaction volume was 250 μl. For saturation isotherms, membranes were incubated with varying concentrations (10 pM–2000 pM) of [3H]-CGP12177 in the absence (total binding) or presence (nonspecific binding) of 1 μM propranolol. Competition binding studies were carried out using 100 pM [3H]-CGP12177 for wild type β1-AR and 300 pM for Asp104Lys mutant of β1-AR. The reactions were stopped by rapid filtration using Brandel cell harvester over Whatman GF/C glass fiber filters that had been treated with 0.1% polyethylenimine followed by washing with 4 ml cold wash buffer containing 25 mM Tris–HCl (pH 7.5) and 1 mM MgCl2. The radioactivity remaining on the filter was counted by liquid scintillation counter. cAMP accumulation assay
Lipofectamine ™2000 reagent was obtained from Invitrogen Life Technology, CA, USA, Inc. Fetal bovine serum was purchased from JRH Biosciences (Lenexa, KS). HEK293 cells were obtained from TAKARA Shuzo (Kyoto, Japan). (–)-Isoproterenol·hydrochloride was from Sigma, St. Louis, USA. [3H]CGP-12177 and [3H]cAMP assay kit were from GE Healthcare Biosciences, Piscataway, USA. The 100-mm dishes, glass-bottomed 35-mm dishes, and collagen-coated 6-well plates were purchased from IWAKI GLASS (Chiba, Japan). Isobutyl-methylxanthine was from Sigma, St. Louis, USA.
cAMP accumulation assay was performed as described previously (Ahmed et al. 2006a,b). In brief, adherent cells in 12-well plates (5 × 105 cells/well) were incubated for 30 min at 37 ºC in 500 μl/well of Hank's balance salt solution (HBSS) buffered with 25 mM HEPES, 1 mM ascorbic acid and 1 mM isobutyl-methylxanthine, in the presence of different concentrations (1 nM to 100 μM) of agonist. The reaction was stopped by washing twice with 1 ml ice-cold (–) phosphate buffered saline and immediate addition of 250 μl 1 N NaOH. After 20 min at 37 ºC the samples were neutralized with 1 N acetic acid and the dissolved cells were centrifuged at 3000 ×g for 10 min at 4 ºC. The amount of cAMP produced was determined on 50 μl of supernatant using Amersham [3H]cAMP assay kit according to the manufacturer's protocol.
Mutagenesis of cDNA and transfection
Internalization assays
Asp104Ala and Asp104Lys mutants were prepared by QuikChange® site-directed mutagenesis kit (Stratagene, CA, USA), according to the manufacturer's protocol as described previously (Ahmed et al. 2006a,b). HEK293 cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Stably expressing cells lines were constructed in HEK293 cells by transfecting with Lipofectamine ™2000 reagent and selected with 0.5 mg/ml G418containing growth medium as described by the manufacturer's protocol. Asp104Lys mutants were also cotransfected together with β-arrestin1 siRNA (Santa Cruz Inc, California, USA ) and plasmid DNA encoding rat β-arrestin1 (kindly gifted from Dr. R. J. Lefkowitz in Duke University Medical Center, Durham) by using Hiperfect (Qiagen, USA ) and Polyfectamine (Qiagen, USA ) transaction reagent respectively, as described by the manufacturer's protocol.
Internalization of the β1-ARs was defined as the fraction of bound [3H]CGP121-77 in whole cell binding assays (Shiina et al. 2000). Internalization studies were performed in duplicate at 37 °C. Each well was aspirated and rinsed with serum-free DMEM containing 20 mM HEPES pH 7.4 in addition with or without 10 μM (–)-isoproterenol for the indicated time. After incubation of indicated time at 37 °C, binding was terminated by rapid removal of the incubation medium and was gently rinsed three times with ice-cold PBS and incubated for 3 h at 4 °C with 6 nM [3H]CGP-12177 in modified KRH buffer (136 mM NaCl, 4.7 mM KCl, 1.25 mM MgSO4, 1.25 mM CaCl2, 20 mM HEPES, pH 7.4, 2 mg/ml bovine serum albumin). After incubation, the cells were washed with ice-cold PBS, and then the cell-associated radio activities were counted by using 1 N NaOH. Finally cell solution was neutralized by adding 1 N HCl and then the cell-associated radioactivities were counted. Nonspecific binding was determined by the addition of 10 µM propranolol. Internalized receptors are expressed as a percentage loss of cell surface binding compared with cell not exposed to (–)isoproterenol. The percentage of internalized receptors was plotted against time and analyzed as a one-phase exponential association using Graph-Pad Prism software. The t1/2 (in min) to reach a maximal level of internalization (Ymax, in %) was determined for each association curve.
Materials and methods Materials
Membrane preparation from HEK293 cells The stably transfected HEK293 cells were rinsed twice with 3 ml of ice-cold homogenize buffer containing 50 mM Tris–HCl (pH 7.4), 100 mM NaCl, and 2 mM EDTA and mechanically detached by cell scraper. The cells were homogenized using Polytron homogenizer for 10 s (Ika Labortechnik, Germany) and after removal of nuclei by centrifugation at 3000 ×g for 5 min, the membrane fractions were centrifuged at 45,000 ×g for 30 min at 4 °C. The resultant pellets were resuspended by Teflon homogenizer in assay buffer containing 75 mM Tris–HCl (pH 7.4), 12.5 mM MgCl2, 2 mM EDTA and kept frozen at −80 °C until used. Protein concentrations of the membrane fractions were measured by the method of Lowry et al. (1951) using bovine serum albumin as the standard.
Western blot analysis Stably transfected HEK 293 cells expressing β1-AR wild type and mutant receptors were constructed according to the methods described above. Equal amounts of protein samples were resolved by SDS-polyacrylamide gel electrophoresis (7.5 % gradient gels) and transferred onto Hybond ECL nitrocellulose membranes (Amersham
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Biosciences, USA) using a semi dry system in immunotransfer buffer. The membranes were blocked in blocking buffer (TBS with 5% nonfat dried milk) and incubated for 1 h at room temperature. After blocking membranes were exposed to donky polyclonal anti-goat β-arrestin1 and anti-rabbit β-ARK1(GRK2) polyclonal antibody (Santa Cruz Inc, California, USA) at 1:1000 dilutions in blocking buffer with 1% milk for 1 h and kept at 4 °C for whole night in rotating shaker. The membranes were washed three times with tris-buffer saline (TBS) containing 0.1% Tween-20 and incubated with HRP-conjugated anti-rabbit IgG (Promega, USA) and anti-goad IgG for (Santa Cruz Inc, California, USA) for 1 h at room temperature at 1:2000 dilutions in blocking buffer with 1% milk. The membranes were washed and then the blots on the membranes were visualized by adding Amersham ECL western blotting detection reagent (GE Healthcare, UK Limited UK).
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Fig. 1. Determination of the functional potency (pEC50) of (–)-isoproterenol to the mutants and wild type β1-ARs by using [3H]cAMP assay kit.
Data analysis Nonlinear regression analysis of saturation and competition binding assay was performed using Graph-Pad Prism software (San Diego, CA, USA). The results of experiments are expressed as means ± SEM. The inhibition concentrations (IC50) in displacement analysis were determined as the concentrations of ligands that inhibited [3H]CGP-12177 binding by 50%; the values of inhibition constants (Ki) were calculated by the equation of Cheng and Prusoff (1973) and expressed as pKi (−log Ki). Student's unpaired t-test was performed to assess the significance of the difference. A P value of less than 0.05 was taken as significant. Results Determination of functional potency for wild type and mutant receptor assessed by cAMP assay
wild type receptor and the Asp104Lys mutant were calculated from the maximal specific binding of [3H]CGP-12177 as 1475.8 ± 30.2 and 1206.7 ± 33.0 fmol/mg of protein, respectively. On the other hand, mutant Asp104Ala showed maximal specific binding of [3H]CGP-1277 as 10,233 ± 70.4 fmol/mg of protein (Table 2). The result demonstrated that receptor expression for the mutant Asp104Ala was increased 10 fold higher compared to wild type receptors. In competition binding experiments, the binding affinity (pKi) of isoproterenol to both mutants Asp 104Ala and Asp104Lys receptors were markedly decreased compared to wild type receptor. The binding affinity of (–)-isoproterenol for both the mutants Asp104Ala and Asp104Lys were decreased 10 folds and 50 folds respectively compared to wild type β1-AR (P b 0.05) (Table 3 and Fig. 2). Internalization of Asp104Ala and Asp104Lys of mutant receptor
As cAMP accumulation is changed with the β1-ARs activation or inactivation, we performed to investigate the constitutive activity of Asp104Ala and Asp104Lys mutants of β1-ARs by measuring its amount of accumulation and compared with that of Wild type receptor. In the present study, we have determined the pEC50 value of (–)-isoproterenol by using [3H]cAMP assay kit. The pEC50 values measured for (–)isoproterenol in wild type and Asp104Ala mutant β1-ARs were 7.40 ± 0.1and 7.2 ± 0.2 respectively (Table 1). The hill slopes of both the wild type and mutant receptor's curve showed unity (Fig. 1). On the other hand, we could not determine the pEC50 value of (–)-isoproterenol in Asp104Lys mutant of β1-AR (Fig. 1), because this mutant did not induce activation by (–)-isoproterenol.
In view of the apparent difference in G protein coupling and activation between mutant Asp104Lys and the wild-type β1-ARs, we examined the capacity of the mutant to undergo homologous internalization. Treatment of the wild type and Asp104Lys mutant with 10 μM (–)-isoproterenol for 20 min induced 51.67 ± 4.69% and 31.77 ± 3.13% receptor internalization, respectively (Table 4 and Fig. 3). The kinetics of the internalization of the mutant Asp104Lys was similar to the wild type β1-AR. In contrast, Asp104Ala mutant showed 2.29 ± 0.49% receptor internalization which was 23% lower (P b 0.05) compared to wild type β1-ARs. (Table 4 and Fig. 3). Western blot analysis
Ligand binding characterization of wild type and mutants (Asp 104Ala and Asp104Lys) of β1AR Wild type and both mutants receptor of β1-AR genes were stably expressed in HEK293 cells. [3H]CGP-12177 radioligand binding assay showed that the wild type receptor and the Asp104Lys were bound as expected, with a dissociation constant (Kd) of 128.4 ± 25.6 pM and 297.4 ± 46.3 pM, respectively. On the other hand, Bmax values for the
To investigate the role of GRK2 and β-arrestin1 in mutant receptor internalization, we have performed western blot analysis for the detection of GRK2 and β-arrestin1 expression. The result showed that expression of GRK2 in Asp104Lys mutant transfected in HEK293 cells was higher compared to wild type receptor without (–)-isoproterenol induction (Fig. 4A). However, expression level of GRK2 in Asp104lys mutant was further increased in the presence of (–)-isoproterenol (Fig. 4B). Moreover, we have confirmed the increased and decreased
Table 1 Analysis of functional potency (pEC50) of (–)-isoproterenol for both wild type and mutants receptors of β1-ARs.
Table 2 Saturation analysis of [3H]-CGP-12177 -labeled wild-type and mutant's β1-AR.
Receptor
(–)-isoproterenol
Receptor
Kd
Bmax (fmol/mg of protein)
pEC50
WT Asp104Ala Asp104Lys
127.4 ± 25.6 228.4 ± 42.5⁎ 298.4 ± 44.3⁎
1465.8 ± 30.9 10,233.0 ± 70.4⁎⁎ 1204.8 ± 33.0
WT Asp104Ala Asp104Lys
7.4 ± 0.1 (4) 7.2 ± 0.4 (4) ND
pEC50 values were determined using [3H]cAMP assay kit in stably transfected HEK293 cell as described in Materials and methods. WT, wild type. ND., not determined. The data are means ± SEM for four independent experiments, each performed in duplicate.
WT = Wild type. The data are means ± SEM for four independent experiments, each performed in duplicate. ⁎ P b 0.05 vs. wild type. ⁎⁎ P b 0.001 vs. wild type.
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Table 3 Studies on binding affinity (pKi) of (–)-isoproterenol for both wild type and mutants receptors of β1-ARs. Receptor
(–)-isoproterenol pKi
WT Asp104Ala Asp104Lys
6.8 ± 0.6 (4) 5.7 ± 0.2⁎ (4) 5.1 ± 0.3⁎ (4)
pKi values were determined using [3H]CGP12177 in cell membrane in stably transfected HEK293 cell as described in Materials and methods. WT, wild type. The data are means± SEM for four independent experiments, each performed in duplicate. ⁎ P b 0.05 vs. wild type β1-ARs.
level of β-arrestin1 by over expression and knockdown of β-arrestin1 gene in mutant receptors respectively by western blot analysis (Fig. 5A). Role of β-arrestin1 in Asp104Lys mutant internalization To examine the exact role of β-arrestin1 signaling in the internalization of Asp104Lys mutant of β1-ARs, we have separately cotransfected Asp104Lys mutant together with DNA plasmid encoding β-arrestin1 (for over expression), siRNA β-arrestin1 (for knock down) and plasmid only (control) in HEK293 cells. The present study showed that over expression of β-Arrestin1 encoding gene and knock down of β-Arrestin1 in Asp104Lys mutant expressed in HEK293 cells were internalized 39.75 ± 2.19% (P b 0.05) and 3.55 ± 1.75 % (P b 0.001) respectively compared to Asp104Lys mutant receptor (control) internalization (Fig. 5B and C). Discussion Internalization plays an important role in receptor internalization and signal transduction. Homologous internalization of a GPCR is an active process that requires the following: 1) specific ligand binding 2) conformational change of receptor, 3) GRK-mediate phosphorylation of the receptor and 4) signal transductions initiated by the activated receptor (Ferguson 2001). It is generally believed that conventional homologous GPCRs depend on the activation of G protein. Earlier, we reported that Asp104Lys mutant was unable to activate due to uncouple with the G protein in an agonist dependent pathway. Moreover, mutant Asp104Ala showed higher cAMP production without agonist-induced stimulation compared to wild type receptor. The present study using Asp104Lys mutated β1-AR shows that there is no correlation between internalization and signal transduction through G protein dependent pathway. Although the abnormalities of both the mutant receptors were at the same position 104, the present studies demonstrated that Asp104Lys mutant receptor showed homologous unconventional internalization.
Fig. 2. Evaluation of binding affinity (pKi) of (–)-isoproterenol to the mutants and wild type β1-ARs as assessed by [3H]CGP12177 radioligand.
Table 4 Agonist-induced internalization of wild type, Asp104Ala and Asp104Lys mutants of β1-ARs. Receptor
Ren (% of surface receptors internalized)
Ken (fraction/min)
t1/2 (min)
WT Asp104Ala Asp104Lys
51.7 ± 4.7 2.4 ± 0.5⁎ 31.8 ± 3.1⁎
0.2 ± 0.1 0.1 ± 0.1 0.1 ± 0.3
4.4 11.1 5.9
The kinetic parameters are derived from experiments shown in Fig. 2 by Prism 4 software as described in Materials and methods. The parameters for internalization are as follows: Ren, percent of surface receptors internalized; ken, first order rate constant of internalization with its t1/2. The data are means ± SEM for four independent experiments, each performed in duplicate. ⁎ P b 0.05 vs. wild type β1-ARs.
In saturation binding experiment, the data represented the differential level of cell expression of mutant receptors compared to wild type β1-ARs. The mutant Asp104Ala showed 10 fold higher Bmax value assessed by radioligand [3H]CGP12177 binding compared to wild type β1-ARs. It was reported that thromboxane receptor density was increased in pulmonary hypertensive patient compared to a nondisease patient assessed by [125I]-BOP radioligand (Katugampola and Davenport 2001). This result leads us to investigate further to find out the actual reason behind this abnormality of receptor expression. The competition binding experiment demonstrated that the binding affinity of (–)-isoproterenol was significantly decreased for both the mutant receptors compared to wild type β1-ARs. The binding affinity of (–)-isoproterenol was decreased due to the mutation of Asp104 to Ala and Lys, which identified that the aspartic acid at position 104 is a very important site for agonist binding (Table 3 and Fig. 2). The present study also investigated that the agonist potency of mutant Asp104Ala was similar to wild type receptor; however, the functional potency of agonist could not be determined for the mutant Asp104Lys (Table 1 and Fig. 1). The result demonstrated that mutation of negatively charged aspartic acid at position 104 to positively charged amino acid of lysine functionally inactivated the receptor by uncoupling with G protein. Earlier, other similar types of investigation has been reported in β2-adrenergic (Chung et al. 1988), α2A adrenergic (Wang et al. 1991) and AT1 (Feng et al. 2005) receptor due to mutation at conserved aspartic acid. On the other hand, the mutant Asp104Ala showed the higher basal level of cAMP production without agonistinduced stimulation, which might be another possible reason for demonstrating this abnormal signaling characteristic of receptor. To determine the possible reason for receptor abnormal behavior in signaling transduction, the present study had to investigate the receptor internalization for cell localization of receptor protein. The mutant Asp104Lys showed the almost similar internalization behavior compared to wild type receptor; however, it was very interesting that Asp104Lys had not been internalized by G protein dependent pathway, because Asp104Lys was functionally inactive due to uncoupling with G protein. Therefore, the present study has proposed
Fig. 3. Internalization kinetics of the wild type, Asp104Ala and Asp104Lys mutants of β1-ARs expressed into HEK 293 cells. The kinetic parameters are derived from experiments by Prism 4 software as described in Materials and methods. The data are means ± SEM of four independent experiments, each performed in duplicate.
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charged aspartic acid to neutral charged alanine which is unable to bind with adaptor protein that may cause the mutant receptor not to be internalized and thus increase the receptor protein in cell surface. This in turn increases higher expression as well as higher cAMP production without agonist stimulation. Thus, our present study first suggests that higher receptor expression of mutant and their signal transduction may be due to lack of conventional internalization of receptor from cell surface. The majority of point mutation in TMH of the β1-ARs and other GPCR may increase or decrease receptor expression on the cell surface, possibly due to impairment of receptor internalization or folding defects (Myburgh et al. 1998). Thus, for
Fig. 4. Effect of (–)-isoproterenol on GRK2 activities in HEK293 cells stably transfected with wild type and mutant Asp104Lys β1-AR. HEK293 cells with both wild and mutant β1-ARs were incubated with the presence and absence of (–)-isoproterenol (10 μM) for 20 min. (A) Western blot analysis showing the band of GRK2 for both wild type and mutant. (B) A quantitative analysis was performed to show the activities of GRK2 in both wild type and mutant receptors in the presence or absence of (–)-isoproterenol. βactin was used for internal standard. Results were expressed as means ± SEM. n = 3–4 for each experiments performed in duplicate.
that the mutant Asp104Lys is internalized in an agonist independent pathway. Ying-Hong Feng et al. (2005) reported the similar abnormality of receptor internalization and receptor activation of AT1 receptor due to mutation. The data showed that Asp125Ala/ Arg126Lue mutant was unable to couple with G protein, however mutant receptor showed similar internalization compared to wild type AT1 receptor. This report also suggested that mutant Asp125/ Arg126Lue was internalized in G protein independent pathway by activating the GRK pathway mechanism. We may therefore suggest that internalization of Asp104Lys mutant in agonist independent pathway may be due to GRK activation or activation of other protein kinases which may be the possible reason for internalization. We next investigate whether GRK and β-arrestin1 signaling pathways are involved in the internalization process of Asp104Lys mutant in agonist independent pathway. The results showed that GRK2 (β-ARK1) expression was higher in absence of (–)-isoproterenol induction than that of wild type receptor and its expression was further increased after induction of (–)-isoproterenol. It had been reported that increased activity of GRK2 caused phosphorylation of β1-ARs and activate β-arrestin1 which leads to internalization (Freedman et al. 1995). Thus, the present studies suggest that GRK2 may play an important role for the internalization of this Asp104Lys mutant receptor through phosphorylation of it. Moreover, the present data clearly showed that internalization of Asp104lys were increased significantly due to over expression of β-arrestin1 in mutant receptor. On the other hand, knock down of β-arrestin1 by siRNA β-arrestin1 in Asp104Lys mutant decreased the internalization significantly. Therefore, taken together the present findings may suggest that GRK2 and β-arrestin1 play an important role for this Asp104Lys mutant receptor internalization through G protein independent signaling. In our previous study, Asp104Ala mutant showed higher basal cAMP production without agonist-induced stimulation which might act like constitutive active mutant receptor (Ahmed et al. 2006a,b). More interestingly, the present study showed that this mutant (Asp104Ala) receptor was unable to be internalized from cell surface. On the other hand, this mutant showed the similar binding characteristic of (–)-isoproterenol with wild type. The reason of these differential findings may be due to the mutation of negatively
Fig. 5. Effect of β-arrestin1 and knock down β-arrestin1 on agonist mediated internalization. Cells were transiently cotransfected with β-arrestin1 encoding plasmid and siRNA of β-arrestin1 by using polyfectamine and hyperfect described in Materials and methods. After 24–48 h, the cells were incubated for 20 min with or without 10 M isoproterenol and assayed for surface receptor as described as Materials and methods, (A) western blot analysis of Asp104Lys mutant expressed HEK293 cell for detection of β-arrestin1 expression level in β-arrestin1 overexpression and knock down effect, (B) Rate of internalization of Asp104Lys mutant receptor with overexpression of βarrestin1 and knock down of β-arrestin1 effect on internalization process. (C) Summary of quantification at internalization of Asp104Lys mutant receptor with overexpression of β-arrestin1 and knock down of β-arrestin1 by [3H]CGP-12177 binding. Values are expressed as percentage of control and represent the means ± SEM. n = 3, each experiments performed in duplicate.
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receptor internalization it is unusual to find a mutation in the β1-ARs which results in an increased receptor expression. Conclusion In conclusion, the present study reveals that receptor over expression and internalization are correlated with each other and the β1-AR internalization may occur in an unconventional pathway through G protein independent signaling which may be very important for understanding the mechanism of GPCRs trafficking in mammalian cell. Therefore, the present study may imply the important clue to find out the mechanism of internalization and receptor activation which may have preferred implication in pathophysiology of cardiovascular disease. Acknowledgement We thank Dr. R. J. Lefkowitz (Duke University Medical Center, Durham) for the human recombinant β1-ARs plasmid and rat encoding β-arrestin1 plasmid. References Ahmed M, Ishiguro M, Nagatomo T. Molecular modeling of SWR-0342SA, a β3-selective agonist, with β1- and β3-adrenoceptor. Life Sciences 78 (17), 2019–2023, 2006a. Ahmed M, Muntasir HA, Hossain M, Ishiguro M, Komiyama T, Muramatsu I, Kurose H, Nagatomo T. Beta-blockers show inverse agonism to a novel constitutively active mutant of β1-adrenoceptor. Journal of Pharmacological Sciences 102 (2), 167–172, 2006b. Arner P, Kriegholm E, Engfeldt P. In vivo interactions between β1 and β2 adrenoceptors regulate catecholamine tachyphylaxia in human adipose tissue. Journal of Pharmacological Experimental Therapeutics 259, 317–322, 1991. Bristow MR. β-adrenergic receptor blockade in chronic heart failure. Circulation 101 (5), 558–569, 2000. Brodde OE. Beta-adrenoceptors in cardiac disease. Pharmacology and Therapeutics 60, 405–430, 1993. Cheng Y-C, Prusoff WH. Relationship between the inhibition constant (ki) and the concentration of inhibition which causes 50% inhibition (I50) of an enzymatic reaction. Biochemical Pharmacology 22, 3099–3108, 1973. Claing A, Laporte SA, Caron MG, Lefkowitz RJ. Endocytosis of G protein-coupled receptors: Roles of G protein-coupled receptor kinases and β-arrestin proteins. Progress in Neurobiology 66, 61–79, 2002. Claing A, Perry SJ, Achiriloaie M, Walker JK, Albanesi JP, Lefkowitz RJ, Premont RT. Multiple endocytic pathways of G protein-coupled receptors delineated by GIT1 sensitivity. The Proceedings of the National Academy of Sciences Online (US) 97, 1119–1124, 2000. Chung FZ, Wang CD, Potter PC, Venter JC, Fraser CM. Site-directed mutagenesis and continuous expression of human beta-adrenergic receptors. Identification of a conserved aspartate residue involved in agonist binding and receptor activation. Journal of Biological Chemistry 263 (9), 4052–4055, 1988. Feng YH, Ding Y, Ren S, Zhou L, Xu C, Karnik SS. Unconventional homologous internalization of the AT1 receptor induced by G-protein-independent signals. Hypertension 46 (2), 419–425, 2005.
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