Interaction between Angiotensin Receptor and β-Adrenergic Receptor Regulates the Production of Amyloid β-Protein

Abstract

Alzheimer’s disease (AD) is characterized by the formation of extracellular amyloid plaques containing the amyloid β-protein (Aβ) within the parenchyma of the brain. Aβ is considered to be the key pathogenic factor of AD. Recently, we showed that Angiotensin II type 1 receptor (AT₁R), which regulates blood pressure, is involved in Aβ production, and that telmisartan (Telm), which is an angiotensin II receptor blocker (ARB), increased Aβ production via AT₁R. However, the precise mechanism underlying how AT₁R is involved in Aβ production is unknown. Interestingly, AT₁R, a G protein-coupled receptor, was strongly suggested to be involved in signal transduction by heterodimerization with β₂-adrenergic receptor (β₂-AR), which is also shown to be involved in Aβ generation. Therefore, in this study, we aimed to clarify whether the interaction between AT₁R and β₂-AR is involved in the regulation of Aβ production. To address this, we analyzed whether the increase in Aβ production by Telm treatment is affected by β-AR antagonist using fibroblasts overexpressing amyloid precursor protein (APP). We found that the increase in Aβ production by Telm treatment was decreased by the treatment of β₂-AR selective antagonist ICI-118551 more strongly than the treatment of β₁-AR selective antagonists. Furthermore, deficiency of AT₁R abolished the effect of β₂-AR antagonist on the stimulation of Aβ production caused by Telm. Taken together, the interaction between AT₁R and β₂-AR is likely to be involved in Aβ production.

INTRODUCTION

Alzheimer’s disease (AD) is a progressive, fatal neurodegenerative disease characterized clinically by progressive loss of memory and cognitive decline. The neuropathological hallmarks of AD are the accumulation of amyloid β-protein (Aβ) in the extracellular plaques, neurofibrillary tangles made from intracellular abnormally phosphorylated tau, and neuronal loss.¹⁾ Aβ is generated from amyloid precursor protein (APP) through sequential cleavages by two proteases called β- and γ-secretases. The two most common isoforms of Aβ are 40 and 42 residues in length, depending on the site of γ-secretase cleavage.²⁾ Although secreted Aβ40 is much more abundant than Aβ42, Aβ42 is considered as the causative molecule for triggering the onset of AD because it is more prone to aggregation and is the major component in senile plaques.²⁾

Previously, we showed that Angiotensin II type 1 receptor (AT₁R), which regulates blood pressure, is involved in Aβ production, and that telmisartan (Telm), an angiotensin II receptor blocker (ARB), increased Aβ production via AT₁R.^3,4) We found that AT₁R-knockout mice exhibited a decrease in Aβ accumulation in the brain, due to a decrease in γ-secretase activity.³⁾ Deficiency of AT₁R was found to incompletely generate presenilin complex, which is responsible for γ-secretase activity.³⁾ Therefore, AT₁R is likely to regulate Aβ generation through changing γ-secretase activity. However, the precise mechanism underlying how AT₁R is involved in Aβ production is not clear. On the other hand, β₂-adrenergic receptor (β₂-AR) has also been shown to modulate γ-secretase activity.^5,6) Very interestingly, AT₁R, one of G protein-coupled receptors (GPCRs), was strongly suggested to be regulated in signal transduction by heterodimerization with other GPCR family members, including β₂-AR.^7–9)

GPCRs comprise the largest family of membrane proteins and are the most common target for therapeutic drugs.¹⁰⁾ Surprisingly, over 90% of GPCRs are expressed in the brain, where they appear to have important roles including cognition, mood, synaptic transmission and so on.¹¹⁾ In addition, GPCRs, classically considered to function as monomers, are actually organized as homodimers and heterodimerize with other GPCR family members.¹²⁾ A growing number of observations demonstrate that GPCR oligomerization may occur in native tissues and may have important consequences in receptor function.¹³⁾ However, the precise mechanism underlying the regulation of the signal transduction by heterodimers or homodimers of GPCR family members remains to be elucidated. It is also noted that the binding of an agonist or an antagonist to a GPCR possibly promotes a conformational change that results in the activation of receptor-associated heterodimeric G protein and consequent downstream signaling.¹²⁾

Regarding Alzheimer’s disease, GPCRs are reported to be associated with multiple stages of APP proteolysis, including modulation of processing of APP by the α-, β-, and γ-secretase¹⁴⁾; however, at present, it is not known how these GPCRs modulate APP processing. Therefore, in this study, in order to know the potential drug targets for the treatment of AD, we aimed to clarify whether the interaction between AT₁R and β₂-AR is required for the regulation of Aβ production. To address this, we analyzed whether the increase in Aβ production induced by Telm treatment is affected by β₂-AR antagonist.

MATERIALS AND METHODS

Cell Lines and Cell Culture

To generate AT₁R-deficient mouse embryonic fibroblasts (MEFs), we isolated MEFs from 13.5-d-old embryos of AT_1aR deficient mice (The Jackson Laboratory, U.S.A.) with the C57BL/6 background, following the procedures as described previously.³⁾ C57BL/6 MEFs and AT₁R-deficient MEFs were infected with human 695-amino acid amyloid precursor protein (hAPP695) cDNA by a retrovirus-mediated method according to published methods.¹⁵⁾ The two kinds of cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, U.S.A.). Cells were maintained at 37°C in an atmosphere of 5% CO₂ in a tissue culture incubator. The hAPP695-infected fibroblasts were passaged with the same cell concentration for each pathway inhibitor administration.

Reagents

ARB, Telm, and β-AR antagonists, propranolol (Prop) were purchased from Sigma-Aldrich dissolved in dimethyl sulfoxide (DMSO). β₁-AR selective antagonists, atenolol (Aten), and bisoprolol (Biso), and β₂-AR selective antagonist, ICI-118551 (ICI) were purchased from Sigma-Aldrich and dissolved in DMSO.

Aβ Enzyme-Linked Immunosorbent Assay (ELISA)

Cells were plated in 10% FBS-DMEM (Wako Pure Chemical Corporation) and Aβ levels in the culture media were determined after 72 h with ELISA kit purchased from Wako. All samples were measured in triplicate.

Preparation of Cell Lysate

Cell lysate from fibroblasts expressing APP was prepared in RIPA buffer (50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and 0.1% sodium lauryl sulfate) containing a protease inhibitor mixture. The protein concentration of the lysate was determined using bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA, U.S.A.).

Statistical Analysis

All data are shown as the means ± standard error of the mean (S.E.M.). We compared group difference by one-way ANOVA followed by the post-hoc Bonferroni–Dunn test for two or more groups against a control group. All data was analyzed by GraphPad Prism 5. p values <0.05 were considered statistically significant.

RESULTS

Effect of β-AR Antagonist, Prop on the Increase in Aβ Generation Caused by Telm Treatment

Previously, we reported that AT₁R is involved in Aβ production, although the precise mechanism remains to be clarified. We also found that Telm, an ARB, increased Aβ production via the AT₁R.^3,4) In order to study how AT₁R regulates Aβ production, we focused on the interaction between AT₁R and β₂-AR, because AT₁R, one of GPCRs, has been strongly suggested to be regulated in signal transduction by heterodimerization with another GPCR, including β-AR,^12,13) and β₂-AR has also been shown to modulate γ-secretase activity.^5,6) Thus, we first asked whether an increase in Aβ production caused by treatment with Telm was affected by Prop of β₁- and β₂-AR non-selective antagonist using fibroblasts overexpressing APP. As shown in Fig. 1, an increase in Aβ production following treatment with Telm was clearly found to be decreased by the addition of Prop in a dose-dependent manner, suggesting that the interaction of AT₁R and β-AR may be involved in the pathway of stimulation of Aβ production caused by Telm.

Fig. 1. Effect of β-AR Antagonist, Prop on the Increase in Aβ Generation Caused by Treatment with Telm

Effect of Prop on the increase in Aβ generation following treatment with Telm. Fibroblasts overexpressing APP were cultured with Prop in the presence or absence of Telm for 72 h. Aβ level in the cultured medium was measured with ELISA. Data were the average of four independent experiments. Error bars are means ± S.E.M. * p < 0.05, *** p < 0.005 by one-way ANOVA followed by a post hoc Bonferroni test. N.S., not significant.

β₂-AR Antagonist Reduced an Increase in Aβ Generation Caused by the Treatment with Telm

So, we next clarified which, β-AR, β₁- or β₂-AR, is involved in the decrease in the stimulation of Aβ production by Telm. For this purpose, we examined the effect of β₁-AR selective antagonists of Aten, and Biso, and the β₂-AR selective antagonist, ICI on an increase in Aβ production by Telm. We found that ICI clearly inhibited Aβ production by Telm in a dose-dependent manner (Fig. 2A), while the treatment of Aten failed to inhibit an increase in Aβ production by Telm and Biso did not exhibit a dose-dependent inhibition of the stimulation of Aβ production by Telm. This result showed that an increase in Aβ production by Telm was selectively reduced by β₂-AR antagonist, suggesting that the interaction between AT₁R and β₂-AR is involved in Telm-stimulation of Aβ production.

Fig. 2. β₂-AR Antagonist Reduced the Increase in Aβ Generation Caused by Treatment with Telm

A, Effect of β₂-AR selective antagonist, ICI on the increase in Aβ production caused by Telm. Fibroblasts overexpressing APP were cultured with β₂-AR selective antagonist, in the presence or absence of Telm for 72 h. Aβ level in the cultured medium was measured with ELISA. Data are the average of four independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.005. N.S., not significant. B, Effect of β₁-AR selective antagonists of Aten, and Biso on an increase in Aβ production caused by Telm. Fibroblasts overexpressing APP were cultured with β₁-AR selective antagonists in the presence or absence of Telm for 72 h. Aβ level in the cultured medium was measured with ELISA. Data were the average of four independent experiments. * p < 0.05. N.S., not significant.

Biso appeared to slightly inhibit Telm-stimulation of Aβ production without a dose dependency. Since a small portion of Biso is known to antagonize β₂-AR,¹⁶⁾ the binding of β₂-AR with Biso could diminish a part of Telm-stimulated Aβ generation by affecting the interaction of β₂-AR and AT₁R. Alternatively, β₁-AR could be somehow involved in the Aβ generation pathway although the precise mechanism is not known.

The Effect of β-AR Antagonist and Telm on Aβ Production in AT₁R-Deficient Cells

To further confirm that the interaction of AT₁R and β₂-AR regulates Aβ production, we next addressed whether β₂-AR antagonist can influence Aβ generation in AT₁R deficient cells. As shown in Fig. 3A, the effect of Telm on Aβ generation in AT₁R deficient cells was not significantly inhibited by 20 µM of ICI or Prop, which is the same concentration with Telm, however, Telm-enhancing Aβ generation in wild type cells was inhibited by the treatment with ICI or Prop (Fig. 3B). Indeed, 10 µM ICI was sufficient to inhibit Telm-enhancing Aβ generation in wild type cells (Fig. 2A). Thus, it appears that a decrease in Aβ generation by the treatment with β₂-AR, ICI or β-AR, Prop requires the presence of AT₁R, strongly suggesting that Telm stimulates Aβ generation through affecting the interaction of AT₁R and β₂-AR. It is also found that Aβ40 generation is not changed by the treatment with Telm, but Aβ42 generation is slightly increased by the treatment with Telm, although the exact reason is not known at present (Fig. 3A). Unlike humans, mice have two AT₁R subtypes, AT_1a and AT_1b, although AT_1a isoform predominates over the AT_1b in all tissues.^17,18) Because the mice we used in this study is AT_1a-deficient mice (see “Materials and Methods”), a low level of AT_1b is likely to be expressed. Since ARB has a high affinity for both AT₁R subtypes,¹⁷⁾ Aβ42 generation can be stimulated by Telm through AT_1b.

Fig. 3. Deficiency of AT₁R Abolished the Effects of β₂-AR Antagonist and Telm on Aβ Production

A, AT₁R-deficient fibroblasts overexpressing APP were cultured with ICI or Prop in the presence or absence of Telm for 72 h. Aβ level in the cultured medium was measured with ELISA. Data were the average of four independent experiments. Agtr^−/−/APP⁺ cells: AT₁R-deficient fibroblast cells overexpressing APP B, wild-type fibroblasts overexpressing APP were cultured with ICI or Prop in the presence or absence of Telm for 72 h. Aβ level in the cultured medium was measured with ELISA. Data were the average of four independent experiments. Agtr^+/+/APP⁺ cells: wild-type fibroblasts expressing human APP. Error bars are means ± S.E.M. ** p < 0.01, *** p < 0.005. N.S., not significant.

DISCUSSION

Accumulating evidence demonstrates that GPCRs, classically considered to function as monomers, are actually organized as homodimers and heterodimerize with other GPCR family members.¹²⁾ AT₁R, one of GPCRs, was also strongly suggested to be regulated in signal transduction by heterodimerization with other GPCR members, including β₂-AR.^7–9) For example, the interaction of AT₁R and β₂-AR was shown by coimmunoprecipitation study.⁷⁾ In addition, selective blockade of β₂-AR in mouse cardiomyocytes inhibited angiotensin-induced AT₁R-mediated contractility, and the administration of the ARB to mice resulted in a significant reduction in the maximal response to catecholamine-induced elevation of heart rate.⁷⁾ This trans-inhibitory effect of β-blocker and ARB appears to be through receptor-G protein uncoupling, because the binding of an antagonist to a GPCR possibly results in the inactivation of receptor-associated heterodimeric G protein and consequent downstream signaling. However, the precise mechanism underlying the regulation of the signal transduction by the heterodimers of GPCR family members remains to be elucidated, and also the GPCR family members forming the heterodimer need to be clarified.

Aβ is thought to be the cause of cognitive impairment of AD, and it is produced by the proteolytic cleavage of APP by β- and γ-secretase. Recently, we reported that AT₁R knockout in mice generates incomplete presenilin complex, resulting in a reduction of Aβ deposits in the brain. Therefore, AT₁R is involved in the formation of presenilin complex, regulating γ-secretase activity.³⁾ Our previous study showed that one of ARBs, Telm, increased Aβ production via AT₁R,⁴⁾ although AT₁R-deficient cells exhibited a reduction in Aβ generation. We found that Telm treatment stimulated the phosphorylation of Akt and increased Aβ generation, which was also found in angiotensin II treated cells, as previously shown.⁴⁾ Because Telm has the strongest binding affinity to AT₁R among the various ARBs, we hypothesize that this strong binding may trigger the similar signal transduction pathway to increase Aβ generation as angiotensin II does.^3,4)

It is also shown that β₂-AR affects γ-secretase activity by regulating the formation of presenilin complex.⁵⁾ However, it is not known whether the heterodimerization of AT₁R and β₂-AR is necessary for the regulation of γ-secretase activity. Therefore, in this study, we aimed to elucidate whether the heterodimerization or crosstalk of AT₁R with β₂-AR is crucial for γ-secretase activity. We found that the stimulation of Aβ production by Telm was inhibited by β₂-AR antagonist, while β₁-AR antagonists did not exhibit a significant inhibition. In addition, β₂-AR antagonist did not inhibit Aβ production in AT₁R-deficient cells. These results strongly suggested that the inhibitory effect of β₂-AR antagonist on Telm stimulation of Aβ production is dependent on the presence of AT₁R. Therefore, the stimulatory effect of Telm on Aβ production appears to result from the promotion of the interaction of AT₁R and β₂-AR. Taken together, the heterodimerization or crosstalk of AT₁R with β₂-AR is likely to increase Aβ production. β-arrestin, which is known as one of the adaptor proteins, interacts with β₂-AR¹⁹⁾ and the interaction of β₂-AR and AT₁R is suggested to promote the interaction of β-arrestin with β₂-AR.^20,21) Because β-arrestin 2 is likely to regulate γ-secretase activity,²²⁾ the binding of Telm on AT₁R causes the conformational change of AT₁R that could promote the interaction of β-arrestin with β₂-AR, resulting in an increase in γ-secretase activity.

Since AT₁R and β₂-AR exist in the brain,^23,24) our present study raised the following possibilities: (i) Aβ accumulation in the brain could be caused by the promotion of the heterodimerization or crosstalk of AT₁R and β₂-AR with aging, and (ii) some medicines targeting GPCR family members may affect the stimulation or inhibition of Aβ accumulation. Since the oxidation or glycation of membrane proteins or lipids is known to progress with aging,^25,26) these changes, directly or indirectly, could cause the promotion of the heterodimerization or crosstalk of AT₁R and β₂-AR, which leads to the stimulation of Aβ production. It is also worth noting that there are many GPCR family members in the brain, including serotonin receptors, dopamine receptors and opioid receptors,¹¹⁾ and that there are many medicines targeting these GPCRs including antidepressant and antipsychotic drugs.²⁷⁾ However, at present it is not known whether these medicines affect Aβ accumulation through modulating the heterodimerization or crosstalk of the target GPCR with AT₁R or β₂-AR.

Further study on the interaction of AT₁R and β₂-AR will be needed regarding the development of AD or the effect of the medicine targeting GPCR family members on Aβ accumulation.

Acknowledgments

This work was supported by Grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Grant-in-Aid for Young Scientists (19K16503), Grant-in-Aid for Scientific Research (C) (26430057) (19K07846) and Grant-in-Aid for Strategic Medical Science Research (S1491001); and funds from the Daiko Foundation and the Hirose International Scholarship Foundation.

Conflict of Interest

The authors declare no conflict of interest.

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