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1-Adrenergic Receptors by Rab1 GTPase in Cardiac Myocytes
Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, Louisiana
Received October 17, 2005; accepted February 3, 2006
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Abstract |
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- and 
1-ARs in neonatal rat ventricular myocytes. Adenovirus-driven expression of Rab1 into myocytes selectively increased the cell-surface number of 1-AR, but not -AR, whereas the dominant-negative mutant Rab1N124I significantly reduced the cell-surface expression of -AR and 1-AR. Brefeldin A inhibited -AR and 1-AR export and antagonized the Rab1 effect on 1-AR expression. Manipulation of Rab1 function similarly influenced the transport of 1A- and 1B-ARs as well as 1- and 2-ARs. Fluorescent microscopy analysis demonstrated that expression of Rab1N124I and Rab1 small interfering RNA induced a marked accumulation of GFP-tagged 2-AR and 1B-AR in the ER. Consistent with the effects on receptor cell-surface targeting, Rab1 selectively enhanced ERK1/2 activation and hypertrophic growth in response to the 1-AR agonist phenylephrine but not to the -AR agonist isoproterenol. Rab1N124I inhibited both agonist-mediated ERK1/2 activation and hypertrophic growth in neonatal myocytes. These results demonstrate that the cell-surface targeting and signaling of - and 1-ARs require Rab1 and are differentially modulated by augmentation of Rab1 function. Our data provide strong evidence implicating the ER-to-Golgi traffic as a site for selective manipulation of distinct AR function in cardiac myocytes.
- and 1-adrenergic receptors (ARs) play a critical role in the regulation of cardiac growth and function both at normal and diseased conditions (Post et al., 1999
; Rockman et al., 2002). Three subtypes of -ARs (1-AR, 2-AR, and 3-AR) and at least two subtypes of 1-ARs (1A-AR and 1B-AR) have been identified in the mammalian hearts. These receptors belong to the seven transmembrane spanning receptor superfamily coupled to heterotrimeric G proteins. -ARs are coupled primarily to the stimulatory G protein Gs, whereas 1-ARs are coupled to the G protein Gq (Zhong and Minneman, 1999; Zhu et al., 2001; Xiang and Kobilka, 2003). The precise function of ARs is determined by their intracellular trafficking and targeting, which are highly coordinated by many regulatory factors at distinct organelles. ARs are synthesized in the ER and then transported to the plasma membrane through the Golgi apparatus, where the receptors are post-translationally modified to attain mature status (Wu et al., 2003; Duvernay et al., 2005). Once at the plasma membrane, ARs may undergo internalization to the endosome upon stimulation by their ligands. Receptor internalization involves phosphorylation by at least two kinases, protein kinase A and G protein receptor kinases and subsequent binding of the phosphorylated receptors to arrestins, which serves as adaptor proteins recruiting components of the transport machinery to the clathrin-coated pits and initiating formation of the early endosome (Krupnick and Benovic 1998; Baillie et al., 2003). The internalized receptors in the early endosome may be sorted to the lysosome for degradation or to the recycling endosome for return to the plasma membrane (Hertel and Staehelin, 1983; Morrison et al., 1996). The balance of intracellular trafficking (i.e., export, endocytosis, recycling, and degradation) dictates the level of receptor at the plasma membrane and, in turn, will influence the magnitude of the cellular response to a given signal.
Compared with the extensive studies on the events of the endocytic pathway, the molecular mechanisms underlying the transport processes of ARs from the ER through the Golgi to the cell surface in cardiac myocytes and its role in the regulation of receptor function and in the development of cardiac disease remain largely unknown. Several studies have demonstrated that homo- and heterodimerization of ARs may be required for their export from the ER and subsequent transport to the cell surface (Xu et al., 2003; Salahpour et al., 2004; Zhou et al., 2006). For example, heterodimerization of 1B-AR or 2-AR with 1D-AR enhances the cell-surface expression of 1D-AR (Hague et al., 2004; Uberti et al., 2005). In addition, N-linked glycosylation of the receptors may also play important roles in targeting receptors to the cell surface as well as other specific transport pathways (Duvernay et al., 2005).
Rab proteins are Ras-like small GTPases that coordinate protein transport in almost every discrete step of the secretory and endocytic pathways (Plutner et al., 1991; Martinez and Goud, 1998; Takai et al., 2001). To date, 63 Rab GTPases have been identified in mammalian cells each with unique subcellular localization and mediating protein transport at specific steps (Takai et al., 2001). For example, Rab5 specifically mediates the transport of G protein-coupled receptors from the plasma membrane to the early endosome and Rab4 is involved in the recycling of internalized receptors from the endosome to the plasma membrane (Takai et al., 2001; Seachrist and Ferguson, 2003). Rab1 is localized in the ER and Golgi and exclusively regulates antegrade protein transport specifically from the ER to the Golgi and between the Golgi compartments (Plutner et al., 1991; Tisdale et al., 1992; Allan et al., 2000). We previously demonstrated that 2-AR transport from the ER through the Golgi to the cell surface in HEK293T cells is dependent on Rab1, whereas 2B-AR transport to the cell surface is independent of Rab1 (Wu et al., 2003). In this report, we determined the role of Rab1 in the cell surface targeting and signaling of distinct endogenous AR subtypes in cardiomyocytes. Our results demonstrate that cell surface expression and function of -AR and 1-AR are similarly attenuated by inhibiting Rab1 function but are differentially augmented by enhancing Rab1 function. These data indicate that the function of distinct ARs can be selectively modulated through manipulating their export trafficking in the early secretary pathway in cardiac myocytes.
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Materials and Methods |
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). Human 2-AR tagged with green fluorescent protein (GFP) at its carboxyl terminus was generated as described previously (Wu et al., 2003). Human GFP-tagged 1B-AR was a kind gift of Dr. Kenneth P. Minneman (Emory University School of Medicine, Atlanta, GA) (Hague et al., 2004).
Isolation, Culture and Adenoviral Infection of Neonatal Rat Ventricular Myocytes. Neonatal ventricular myocytes were isolated from the hearts of 1- to 2-day-old Sprague-Dawley rats as described previously (Filipeanu et al., 2004; Li et al., 2005). The dominant-negative mutant Rab1N124I (a guanine nucleotide binding-deficient) was generated using QuikChange site-directed mutagenesis (Stratagene, La Jolla, CA). Adenoviruses expressing Rab1 and Rab1N124I tagged with the FLAG epitope at their amino termini were generated as described previously (Filipeanu et al., 2004). Isolated neonatal cardiac myocytes were cultured in DMEM and infected with control parent adenovirus or adenovirus expressing Rab1 or its dominant-negative mutant Rab1N124I at a multiplicity of infection (m.o.i.) of 20. After 48 h of infection, expression of Rab1 was determined by Western blotting using a FLAG high-affinity monoclonal antibody. Fluorescent microscopic analyses after immunostaining with anti-FLAG antibodies revealed that greater than 95% of the cardiomyocytes were infected (Filipeanu et al., 2004). To determine whether adenoviral expression of Rab1 could induce cell death, cardiac myocytes viability was measured using Calcein-AM retention and de-esterification as described previously (Yakovlev et al., 2000). The data indicate that adenoviral expression of wild-type Rab1 or Rab1N124I in neonatal cardiomyocytes did not significantly influence cell viability (wild-type Rab1-infected cells, 86 ± 7%; Rab1N124I-infected cells, 93 ± 2% relative to control virus-infected cells; n = 4, p > 0.05).
Measurement of Cell Surface Receptors. Cell-surface expression of -AR and 1-AR in neonatal cardiomyocytes was measured by intact cell ligand binding as described previously (McLean et al., 1999; Ricci et al., 1999; Calls et al., 2000) with modifications. Myocytes were cultured on 12-well plates at a density of 5 x 105 cells/well and infected for 48 h. The myocytes were then incubated with the ligand [3H]CGP12177 at a concentration of 20 nM for 2 h or with [7-methoxy-3H]prazosin at a concentration of 10 nM for 90 min at room temperature. To measure expression of individual AR subtypes, myocytes were preincubated for 30 min with the AR subtype-selective antagonists: ICI 118,551 (2-AR), atenolol (1-AR), niguldipine (1A-AR), or CEC (1B-AR) (10 µM). Nonspecific binding was determined in the presence of alprenolol (-AR) or phentolamine (-AR) (20 µM) and accounted for less than 10% of the total binding. The cells were washed twice with ice-cold phosphate-buffered saline and digested with 1 ml of 1 M NaOH. All ligand binding assays were performed in triplicate. The radioactivity was counted by liquid scintillation spectrometry.
Measurement of ERK1/2 Activation. Activation of ERK1/2 was measured as described previously (Wu et al., 2003; Filipeanu et al., 2004). Myocytes were cultured on six-well plates at a density of 1 x 106 cells/well and infected for 48 h. Myocytes were stimulated with PE (10 µM) or ISO (10 µM) for 8 min with or without pretreatment with the AR antagonists ICI 115,881 or atenolol (100 nM) for 30 min. The reaction was terminated by the addition of 600 µl of 1 x SDS gel loading buffer. After solubilizing the cells, 30 µl of total cell lysates was separated by 10% SDS-polyacrylamide gel electrophoresis. ERK1/2 activation was determined by immunoblotting to measure their phosphorylation with phospho-specific antibodies. The membranes were stripped and reprobed with anti-ERK1/2 antibodies to determine the total amount of kinases and to confirm equal loading of proteins. The signal was detected using ECL Plus (PerkinElmer Life and Analytical Sciences) and a Fuji Film luminescent image analyzer (LAS-1000 Plus) and quantitated using the Image Gauge program (version 3.4).
[3H]Leucine Incorporation. Protein synthesis rate was determined as described previously (Thaik et al., 1995; van Kesteren et al., 1997). In brief, neonatal cardiomyocytes were plated in 12-well dishes at a density of 5 x 105/well in DMEM supplemented with 10% fetal bovine serum. After infection with the desired construct, the myocytes were made quiescent by incubation in DMEM without fetal bovine serum for 48 h. The cardiomyocytes were then incubated with [3H]leucine (1 µCi) for 24 h at 37°C in the presence or absence of the AR agonists ISO (10 µM) or PE (10 µM) with or without ICI 115,881 or atenolol. The reaction was terminated by aspirating the medium. The cardiomyocytes were washed twice with 1 ml of 5% trichloroacetic acid followed by an extraction with 1 ml of 5% trichloroacetic acid for 1 h in ice to remove nonincorporated [3H]leucine. The cells were digested with 1 ml of 1 M NaOH for 6 h. The lysate was transferred to scintillation vials, neutralized with 1 ml of 1 M HCl, and counted by liquid scintillation spectrometry in 5 ml of Ecoscint A scintillation solution. Because Rab1 influences protein synthesis (Filipeanu et al., 2004), the effect of Rab1 on AR agonist-stimulated protein synthesis was calculated using the following formula: {[3H]leucine incorporation in presence of agonist and Rab1] - [[3H]leucine incorporation in presence of Rab1]}/{[3H]leucine incorporation in presence of agonist and control adenovirus] - [[3H]leucine incorporation in presence of control adenovirus]}.
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; Filipeanu et al., 2004). One microgram of GFP-tagged AR with or without 1 µg of pDsRed2-ER construct were diluted into 125 µl of serum-free Opti-MEM in a tube. In another tube, 5 µl of LipofectAMINE was diluted into 125 µl of serum-free Opti-MEM. Five minutes later both solutions were mixed and incubated for another 20 min. The transfection mixture was added to culture dishes containing 0.8 ml of fresh Dulbecco's modified Eagle's medium and 10% fetal bovine serum without antibiotics. After transfection 36 to 48 h, the myocytes were fixed with a mixture of 4% paraformaldehyde and 4% sucrose in phosphate-buffered saline for 15 min. The coverslips were mounted, and fluorescence was detected with a Leica DMRA2 epifluorescence microscope (Duvernay et al., 2004; Filipeanu et al., 2004). Images were deconvoluted using Slide-Book software and the nearest-neighbors deconvolution algorithm (Intelligent Imaging Innovations, Denver, CO) as described previously (Filipeanu et al., 2004). Based on the GFP signal, approximately 5% myocytes were transfected by this plasmid transfection protocol.
Double-Stranded Small Interfering RNA. siRNA targeting the sequence at positions 136 to 156 of human Rab1 and a control nonsilencing siRNA were purchased from QIAGEN Inc. (Valencia, CA). Control and Rab1 siRNA were delivered into neonatal cardiomyocytes using LipofectAMINE 2000 reagent as described previously (Wu et al., 2003). In brief, 8 µl of LipofectAMINE 2000 and 6 µl of 20 µM siRNA were added separately to 100 µl of Opti-MEM. After incubation for 5 min, both solutions were mixed for 20 min. The transfection mixture was then added to the adenovirus-infected cardiac myocytes. The cells were incubated with the transfection mixture for 8 h and then medium was changed to standard culture medium. After 36 to 48 h, the cells were processed for fluorescence microscopy as described above.
Statistical Analysis. Differences were evaluated using Student's t test, and p < 0.05 was considered as statistically significant. Data are expressed as the mean ± S.E.
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-AR and 1-AR Expression at the Cell Surface in Cardiomyocytes. To determine whether Rab1 modulated export trafficking of endogenous ARs, we first determined the effect of transient expression of Rab1 and its dominant-negative mutant Rab1N124I on the cell-surface expression of -AR and 1-AR in primary cultures of neonatal rat ventricular myocytes. Myocytes were infected with control, Rab1, or dominant-negative mutant, Rab1N124I, adenoviruses (Fig. 1A), and the cell-surface expression of -AR and 1-AR was quantitated by ligand binding in intact myocytes using [3H]CGP12177 and [3H]prazosin, respectively. Cell surface expression of total -AR and total 1-AR was significantly attenuated by 60% and 58%, respectively, in cardiomyocytes infected with Rab1N124I adenovirus compared with cells infected with control adenovirus (Fig. 1B). It is noteworthy that, in contrast to Rab1N124I, adenoviral expression of Rab1 produced different effects on the cell surface expression of -AR and 1-AR. Whereas cell-surface expression of -AR was not altered by Rab1, the cell-surface expression of 1-AR was significantly augmented by 52% in cardiomyocytes infected with the Rab1 adenovirus compared with cells infected with control adenovirus (Fig. 1B). These data indicate that the levels of -AR and 1-AR expression at the cell surface depend on the normal Rab1 function and that augmentation of Rab1 function by overexpressing wild-type Rab1 may selectively facilitate the cell-surface targeting of endogenous 1-AR in neonatal cardiomyocytes.
Rab1 GTPase regulates protein transport exclusively from the ER to the Golgi. To further determine the role of ER-to-Golgi transport in the cell-surface targeting of ARs, we determined whether BFA treatment could also modulate cell-surface expression of -AR and 1-AR. BFA is a fungal metabolite that disrupts the structures of the Golgi and blocks protein transport from the ER to the Golgi (Klausner et al., 1992; Yoo et al., 2002). Similar to the effect induced by adenoviral expression of Rab1N124I, BFA treatment reduced cell-surface numbers of -AR and 1-AR (Fig. 2A). Because expression of wild-type Rab1 selectively increased the cell-surface expression of 1-AR, we determined whether BFA treatment could antagonize the effect of Rab1 on 1-AR transport. Rab1-mediated increase in 1-AR expression at the cell surface was significantly blocked by BFA treatment (Fig. 2B). Furthermore, 1-AR expression at the cell surface was attenuated by BFA treatment in myocytes infected with Rab1N124. These data further indicate that the ER-to-Golgi transport plays an important role in the cell-surface targeting of -AR and 1-AR.
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-AR and 1-AR each has multiple subtypes in cardiomyocytes, we sought to define -AR and 1-AR subtypes, whose transport from the ER to the cell surface is regulated by Rab1. Cell surface expression of 1-AR, 2-AR, 1A-AR, and 1B-AR, predominant -AR and 1-AR subtypes in cardiomyocytes, was determined by ligand binding in the presence of the AR subtype-selective antagonists atenolol, ICI 118,551, niguldipine, and CEC, respectively. Cell-surface expression of 1-AR and 2-AR was markedly inhibited by adenovirus-mediated expression of Rab1N124I but was not altered by wild-type Rab1 (Fig. 3A). Similar to 1-AR and 2-AR, cell-surface expression of 1A-AR and 1B-AR was also inhibited by Rab1N124I (Fig. 3). In contrast to 1-AR and 2-AR, cell surface expression of 1A-AR and 1B-AR was increased by adenoviral expression of Rab1 (Fig. 3B). These data are consistent with the effects of Rab1 on total -AR and total 1-AR expression at the cell surface and indicate that Rab1 regulation of export trafficking from the ER to the cell surface is indistinguishable between 1-AR and 2-AR and between 1A-AR and 1B-AR.
We then determined the effect of Rab1 on the subcellular localization of the ARs. To this end, GFP-tagged 2-AR and 1B-AR were transiently transfected into neonatal cardiomyocytes after infection with control or Rab1N124I adenoviruses. The subcellular distribution of the GFP-tagged receptors at steady state was revealed by fluorescent microscopy. As anticipated, 2-AR-GFP and 1B-AR-GFP were mainly localized at the cell surface in myocytes infected with control adenovirus. In contrast, 2-AR-GFP and 1B-AR-GFP were accumulated in the perinuclear regions and unable to transport to the cell surface in myocytes infected with Rab1N124I (Fig. 4A). These receptors were strongly colocalized with the ER marker pDsRed2 (Fig. 4B), consistent with the Rab1 function in regulating protein transport form the ER to the Golgi.
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2-AR and 1B-AR. Our previous data have demonstrated that transient transfection of Rab1 siRNA selectively reduced the expression of endogenous Rab1 (Wu et al., 2003). Consistent with the Rab1N124I effect, transfection of Rab1 siRNA induced an accumulation of both 2-AR and 1B-AR in the perinuclear regions compared with that in myocytes transfected with control siRNA (Fig. 4C). These data strongly indicate that normal Rab1 level is required for the transport of 2-AR and 1B-AR to the cell surface.
Modulation of -AR and 1-AR Signaling by Rab1 in Neonatal Cardiomyocytes. To determine whether Rab1 is capable of regulating AR signaling through modifying export trafficking of the receptors, we determined the effect of Rab1 on AR-mediated ERK1/2 activation. Neonatal myocytes infected with control, Rab1, and Rab1N124I adenovirus were stimulated with the nonselective -AR agonist ISO in the absence or presence of atenolol or ICI 118,551. ERK1/2 activation in response to stimulation with the AR agonists was evaluated by measuring their phosphorylation. ISO-mediated ERK1/2 activation in the absence or presence of the antagonists was similarly inhibited in cardiomyocytes infected with Rab1N124I but was not altered in cardiomyocytes infected with Rab1 compared with control adenovirus-infected myocytes (Fig. 5). In contrast, PE-mediated ERK1/2 activation was attenuated by Rab1N124I and augmented by Rab1 (Fig. 5), suggesting that augmentation of Rab1 function by overexpressing Rab1 may selectively regulate AR signaling. These data are consistent with Rab1 effects on the cell surface expression of the receptors and indicate that Rab1 modulates not only AR traffic but also their signal transduction.
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-AR - and 1-AR-Mediated Hyper-trophic Response in Neonatal Cardiomyocytes. Our preceding data indicate that adenovirus-mediated expression of Rab1 selectively regulates the cell surface expression and signaling of ARs. We then determined whether manipulation of Rab1 function could influence hypertrophic growth by measuring total protein synthesis and sarcomeric organization in response to the agonists ISO and PE in cardiomyocytes. PE effects on protein synthesis were significantly increased by 54% in cardiomyocytes expressing Rab1 compared with cardiomyocytes infected with control adenovirus (Fig. 6A). In contrast, Rab1 had no influence on the protein synthesis in response to stimulation with ISO in the absence or presence of 1- and 2-AR antagonists (Fig. 6A). Total protein synthesis obtained from myocytes infected with parent adenoviral vector were close to that obtained from noninfected myocytes (data not shown), suggesting that influence of Rab1 infection on the protein synthesis could not be attributed to nonspecific effects of adenoviral infection.
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Discussion |
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- and 1-ARs in neonatal ventricular myocytes. Overexpression of Rab1 significantly increased the total cell-surface number of 1-AR as measured by intact cell ligand binding. In contrast, Rab1 expression had no significant influence on the total cell surface number of -AR. Consistent with total AR expression, the cell-surface numbers of 1A-AR and 1B-AR subtypes were largely increased, whereas Rab1 did not alter the cell-surface numbers of 1-AR and 2-AR subtypes. These data strongly indicate that increased Rab1 function facilitates the cell-surface targeting of 1-AR subtypes but not -AR subtypes. These data also suggest a novel way to selectively increase cell-surface expression of endogenous 1-AR.
There are several possibilities regarding the selective regulation of -AR and 1-AR export to the cell surface by Rab1. First, the ER-to-Golgi transport of -AR and 1-AR may be mediated through distinct ER-derived vesicles, which are differentially regulated by Rab1. Consistent with this possibility, different proteins or isoforms have been reported to segregate into distinct vesicles (Muniz et al., 2001). Second, transport of -AR and 1-AR to the cell surface in cardiomyocytes may be mediated through distinct pathways, in which the level of endogenous Rab1 is a rate-limiting factor for the ER-to-Golgi transport of 1-AR, but not -AR. However, we cannot exclude the possibility that Rab1 also facilitated the ER-to-Golgi transport of -AR, but did not significantly alter -AR expression at the cell surface, because the Golgi-to-cell surface transport is a rate-limiting step for -AR export. Third, structural differences in distinct ARs may contribute to their regulation by Rab1. We demonstrated previously that the transport of 2B-AR from the ER to the cell surface in HEK293T cells is independent of Rab1 (Wu et al., 2003). Our present results indicate that the transport of endogenous 1-AR and -AR to the cell surface is dependent on Rab1 and that increased Rab1 function selectively facilitates the cell-surface targeting of 1-AR subtypes. These data indicate that distinct ARs have different sensitivities to Rab1 manipulation. These ARs are structurally different, particularly at their C termini (C-terminal amino acid residues: 1A-AR, 136; 1B-AR, 174; 1-AR, 96; 2-AR, 88; 2B-AR, 20). However, whether the C termini of ARs are indeed responsible for selective regulation by Rab1 is currently under investigation.
The present work demonstrated that Rab1-mediated ER-to Golgi transport is required for cell surface targeting of all AR subtypes examined (i.e., 1A-, 1B-, 1-, and 2-ARs). The cell-surface number of total -AR and total 1-AR as well as individual 1A-AR, 1B-AR, 1-AR, and 2-AR as measured by radioligand binding was significantly attenuated by adenoviral expression of the dominant-negative mutant Rab1N124I in cardiomyocytes. The subcellular localization of GFP-conjugated ARs reflects the effect of Rab1 on the newly synthesized receptors, in that GFP-receptors were delivered into the cell after Rab1 infection. It is noteworthy that Rab1 is one of the most extensively studied and best characterized Rab GTPases, which localize to the ER and the Golgi and regulate protein transport between these two organelles. Therefore, the influence of Rab1 on the cell surface receptor expression is presumably through modulating the ER-to-Golgi transport of the receptor.
Microscopic analysis of subcellular localization of GFP-conjugated receptors, which were delivered into cardiomyocytes after Rab1 infection, indicated expression of Rab1N124I induced an accumulation of 1B-AR and 2-AR in the ER. A similar behavior was observed when endogenous Rab1 was depleted using siRNA. Furthermore, BFA treatment, which impairs Golgi function and blocks the ER-to-Golgi protein transport (Klausner et al., 1992; Yoo et al., 2002), inhibited the transport of 1-AR and -AR to the cell surface and attenuated Rab1-induced enhancement of 1-AR expression at the cell surface. These data suggest that -AR and 1-AR transport from the ER to the cell surface is mediated through the Rab1-dependend pathway and that Rab1 is involved in the transport from the ER to the Golgi.
Another important finding is that the functional response to -AR and 1-AR stimulation can be modulated by manipulating their transport along the early secretory pathway. Adenoviral expression of Rab1 markedly and selectively augmented ERK1/2 activation in response to stimulation with the 1-AR agonist PE, but not to the -AR agonist ISO in cardiomyocytes. Rab1N124I almost abolished the ERK1/2 activation by both 1-AR and -AR agonists. These data indicate that Rab1 can differentially regulate signaling of 1-AR and -AR in cardiomyocytes, which was due to the influence of Rab1 on the transport of the receptors from the ER to the cell surface.
Present results also demonstrated that cardiomyocyte growth in response to the AR agonists can be controlled by manipulating the AR transport in the early secretary pathway. Consistent with the effect of Rab1 on the cell surface expression and signaling of -AR and 1-AR, Rab1 selectively promoted PE-mediated hypertrophy, as measured by changes in total protein synthesis in neonatal cardiomyocytes, and Rab1N124I attenuated both PE and ISO-stimulated cardiomyocyte hypertrophy. We previously demonstrated that, similar to 1-AR, expression of Rab1 and Rab1N124I produced opposing effects on hypertrophic response to angiotensin II (Filipeanu et al., 2004). These data indicate that cardiomyocyte growth can be manipulated by controlling the transport of G protein-coupled receptors at the level of the ER and the Golgi compartment. It is note-worthy that the inhibitory effect of Rab1N124I on the agonist-promoted hypertrophic response (e.g., protein synthesis) and ERK1/2 activation was much more potent than on the cell-surface receptor number as determined by radioligand binding. It is unlikely that Rab1 GTPase, as a transport coordinator, is directly regulating signaling pathways. The differential influences of Rab1N124I on receptor cell-surface expression and agonist-activated signaling may suggest that the minimal amount of ARs at the cell surface is required for the agonist activation of the signaling pathways. When receptor expression at the cell surface is attenuated to a certain level by Rab1 expression, receptor-mediated signaling is almost totally blocked. If this turns out to be true, increased receptor expression would proportionally augment receptor-mediated signaling. Indeed, our data indicate that wild-type Rab1 expression increased receptor expression at the cell surface, agonist-mediated ERK1/2 activation and agonist-stimulated protein synthesis at the similar magnitudes. It is also possible that Rab1 regulates the export trafficking of other signaling molecules involved in the signal transduction systems of ARs.
We have shown that transgenic overexpression of Rab1 in the myocardium induces cardiac hypertrophy with progression to heart failure (Wu et al., 2001). However, the molecular mechanism responsible for Rab1-induced cardiomyocyte hypertrophy remains unknown. We have demonstrated that Rab1 promoted AT1R- and 1-AR-mediated signaling and cardiomyocyte growth. These data suggest that one of the possible molecular mechanisms underlying Rab1-induced hypertrophy in transgenic mouse hearts is that Rab1 overexpression activates signal transduction pathways of Gq-coupled AT1R and 1-AR. This possibility is supported by the abilities of AT1R and 1-AR activation to induce cardiomyocyte hypertrophic growth both in vivo animal hearts and in vitro cultured cardiomyocytes (Knowlton et al., 1993; Sadoshima and Izumo, 1993; Milano et al., 1994; Paradis et al., 2000).
In summary, we have shown for the first time a differential role of Rab1 in the transport and function of ARs, which are crucial for cardiac function under both normal and diseased conditions. Therefore, defining the functional role of export machinery (e.g., Rab1 GTPase) in cardiomyocytes by modifying the transport of selective G protein-coupled receptors in the early secretory pathway may provide a novel insight into understanding the regulation of these clinically important targets.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: AR, adrenergic receptor; ER, endoplasmic reticulum; BFA, brefeldin A; PE, phenylephrine; ISO, isoproterenol; ICI 118,551, (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol; CEC, chloroethylclonidine; GFP, green fluorescent protein; DMEM, Dulbecco's modified Eagle's medium; AT1R, angiotensin II type 1A receptor; ERK, extracellular signal-regulated kinase; CGP12177, 4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one; siRNA, small interfering RNA; WT, wild type.
Address correspondence to: Dr. Guangyu Wu, Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, 1901 Perdido St., New Orleans, LA 70112. E-mail: gwu{at}lsuhsc.edu
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